Modulators of extracellular 5 '-nucleotidase and uses thereof

Abstract

Described herein are compounds that modulate the conversion of AMP to adenosine by an extracellular 5 '-nucleotidase, as well as compositions containing the compounds and methods of synthesizing the compounds. Also provided are uses of such compounds and compositions for the treatment and / or prevention of a variety of diseases, disorders and conditions, including cancer and immune-related disorders, mediated by extracellular 5 '-nucleotidase.

Description

[0001] This application is a divisional application of the Chinese invention patent application (application number: 202210014893.X; application date: January 6, 2017).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 276,564, filed on January 8, 2016, and U.S. Provisional Application No. 62 / 324,077, filed on April 18, 2016, each of which is incorporated herein by reference in its entirety for all purposes.

[0004] STATEMENT OF RIGHTS TO INVENTS RESULTING FROM FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0005] not applicable

[0006] Reference to a "Sequence Listing", table or computer program listing appendix submitted on a CD-ROM

[0007] not applicable

[0008] field

[0009] Provided herein are compounds and compositions, for example, for inhibiting adenosine by extracellular 5′-nucleotidase (also known as CD73), and pharmaceutical compositions comprising the same. Also provided herein are methods, for example, for treating or preventing a disease, disorder or condition mediated by inhibition of adenosine by extracellular 5′-nucleotidase, or a symptom thereof. Background Art

[0010] Purinergic signaling is an extracellular signaling mediated by purine nucleotides and nucleosides such as ATP and adenosine, involving the activation of purinergic receptors in cells and / or nearby cells, leading to the regulation of cell function. Most cells have the ability to release nucleotides, which usually occurs through regulated exocytosis (see Praetorius, HA.; Leipziger, J. (1 March 2010) Ann Rev Physiology 72 (1).: 377-393). The released nucleotides can then be hydrolyzed outside the cell by a variety of cell membrane-bound enzymes called ectonucleases.

[0011] Extracellular nucleotides catalyze the conversion of ATP to adenosine, which is an endogenous regulator that affects multiple systems including the immune system, cardiovascular system, central nervous system, and respiratory system. Adenosine can also promote fibrosis in various tissues. In the first step of adenosine production, extracellular nucleoside triphosphate diphosphonate hydrolase 1 (ENTPD1), also known as CD39 (cluster of differentiation 39), hydrolyzes ATP to ADP, which is then hydrolyzed to AMP. In the next step, AMP is converted to adenosine by extracellular 5′-nucleotidase (NT5E or 5NT), also known as CD73 (cluster of differentiation 73).

[0012] The enzymatic activities of CD39 and CD73 play a key role in calibrating the duration, magnitude, and chemistry of purinergic signals delivered to various cells, such as immune cells. Alterations in the activities of these enzymes can alter the course or determine the outcome of several pathophysiological events, including cancer, autoimmune diseases, infection, atherosclerosis, and ischemia-reperfusion injury, suggesting that these extracellular enzymes represent novel therapeutic targets for the management of a variety of diseases.

[0013] Inhibition of CD73 with monoclonal antibodies, siRNA or small molecules delays tumor growth and metastasis (Stagg, J. (2010) PNAS USA 107: 1547-52). For example, anti-CD73 antibody treatment has been shown to inhibit breast tumor growth and metastasis in animal models (Stagg, J. (January 26, 2010) PNAS USA, 107 (4): 1547-52). In addition, antibodies that specifically bind to CD73 have been evaluated for the treatment of bleeding disorders (such as hemophilia) (USP 9,090,697). Recently, attempts have been made to develop therapeutically useful small molecule inhibitors of CD73. For example, Bhattarai et al. ((2015) J Med Chem 58:6248-63) have studied derivatives and analogs of α,β-methylene-ADP (AOPCP), which is one of the most metabolically stable, potent and selective CD73 inhibitors known, and purine CD73 derivatives have been reported in the patent literature (WO2015 / 164573). However, the development of small molecules has been hampered by, for example, less than ideal metabolic stability.

[0014] Given the role that CD73 plays in cancer and a wide variety of other diseases, disorders, and conditions, and the lack of CD73 inhibitors currently available to medical practitioners, there is a need for new CD73 inhibitors and compositions and methods related thereto. Summary of the invention

[0015] The present invention relates to compounds that regulate the conversion of AMP to adenosine by extracellular 5′-nucleotidase (NT5E or 5NT; also known as CD73), and compositions (e.g., pharmaceutical compositions) comprising the compounds. These compounds, including their synthesis methods and compositions, are described in detail below.

[0016] The present invention also relates to the use of these 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 associated with the treatment of a variety of diseases, including cancer, fibrosis, neurological and neurodegenerative diseases (e.g., depression and Parkinson's disease), ischemic diseases of the brain and heart, immune-related diseases and diseases with inflammatory components [see, for example, Sorrentino et al. (2013) OncoImmunol, 2: e22448, doi: 10.4161 / onci.22448; and Regateiro et al. (2012) Clin. Exp. Immunol, 171: 1-7]. In specific embodiments, the compounds described herein act to inhibit the immunosuppressive activity and / or anti-inflammatory activity of CD73, and can be used as therapeutic or prophylactic treatments when such inhibition is required. Unless otherwise specified, when describing the uses of the compounds of the present invention herein, it should be understood that these compounds can be in the form of compositions (e.g., pharmaceutical compositions),

[0017] As used herein, the terms "CD73 inhibitors," "CD73 blockers," "inhibitors of adenosine by extracellular 5'-nucleotidase," "NT5E inhibitors," "5NT inhibitors," and all other relevant art-accepted terms refer to compounds that are capable of directly or indirectly modulating the CD73 receptor in in vitro assays, in vivo models, and / or other means of indicating therapeutic efficacy. These terms also refer to compounds that exhibit at least some therapeutic benefit in human subjects.

[0018] Although it is believed that the compounds of the present invention achieve their activity by inhibiting CD73, a precise understanding of the mechanism of action of the compounds is not required for the practice of the present invention. For example, the compounds may also achieve their activity at least in part by modulating (e.g., inhibiting) other components of the purinergic signaling pathway (e.g., CD39). The purinergic signaling system consists of transporters, enzymes, and receptors responsible for the synthesis, release, action, and extracellular inactivation of (primarily) ATP and its extracellular breakdown product adenosine (Sperlagh, B et al. (December 2012) Neuropsychopharmacologia Hungarica 14(4): 231-38). Because inhibition of CD73 results in a decrease in adenosine, CD73 inhibitors can be used to treat diseases or conditions mediated by adenosine and its effects on adenosine receptors (including A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A110, A120, A130, A140, A150, A160, A170, A180, A190, A210, A220, A230, A240, A250, A260, A270, A280, A290, A310, A320, A330, A340, A350, A360, A370, A380, A390, A410, A420, A430, A590, A590, A60 2A , A2B and A3), [see Yegutkin, GG (May 2008) Biochimica Biophysica Acta 1783(5):673-94],

[0019] For the purposes of this disclosure, the purinergic signaling process can be described as comprising the following components. Purinergic receptors (P1, P2X and P2Y) are the first component, which are membrane receptors that mediate various physiological functions (e.g., intestinal smooth muscle relaxation) in response to the release of ATP or adenosine; in general, all cells have the ability to release nucleotides into the extracellular environment by regulating exocytosis. Nucleoside transporters (NTs), as the second component, are membrane transporters that transport nucleoside substrates (e.g., adenosine) across the cell membrane; the extracellular concentration of adenosine can be regulated by NTs, possibly connecting the receptor signaling to the feedback loop form of the transporter function. As previously mentioned, ectonucleotidases (CD73 and CD39) hydrolyze the nucleotides released into the extracellular environment and comprise additional components. Another component of the purinergic signaling process includes pan-connexins; in particular, the pan-connexin-1 channel (PANX1) is a component of the P2X / P2Y purinergic signaling pathway and a key factor in pathophysiological ATP release.

[0020] In a specific aspect, the present invention provides a compound of formula (I):

[0021]

[0022] or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein each R 1 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R 2 R 2 )-OC(O)-OR 3 The group consisting of two R 1 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; each R 3 are 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 selected from the group consisting of O, CH2 and S; A is selected from the group consisting of:

[0023]

[0024] Each of which is optionally replaced by 1 to 5 R 6 Substituents are substituted, and wherein the subscript n is an integer from 0 to 3; Z is selected from CH2, CHR6 NR 6 and O; each R 6 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 linked together to form a 5- to 6-membered ring containing at least one heteroatom as a ring vertex; and Het is selected from the group consisting of:

[0025]

[0026] The wavy line indicates the point of attachment to the rest of the compound, and the R a Selected from H, NH2, NHR 7 、NHC(O)R 7 NR 7 R 7 , R 7 , OH, SR 7 OR 7 The group composed of b Selected from H, halogen, NH2, NHR 7 NR 7 R 7 , R 7 , OH and OR 7 The group consisting of each R c and R d are independently selected from H, halogen, haloalkyl, NH2, NHR 7 NR 7 R 7 , R 7 , OH and OR 7 The group consisting of each R e and R f are independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; and

[0027] Each R 7 are independently selected from optionally substituted C1-C 10 alkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted 4-7 membered cycloheteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl, and optionally, two R attached to the nitrogen atom 7 The groups are linked together to form a 4-7 membered heterocyclic ring.

[0028] Excluded from the above are compounds where X, A and Het combine to give

[0029]

[0030] Where R g H or two R g The groups combine to form an acetonide; and

[0031] (i)R c and R e is hydrogen and R a is -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

[0032] (ii) R c is hydrogen, R a is -NH2, and R e is bromine, chlorine, aminomethyl or thioethyl; or

[0033] (iii) R c is hydrogen, R a is benzylamino, and R e For bromine.

[0034] In some embodiments, the present invention contemplates a method for 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 a method for treating or preventing cancer in a subject by administering to the subject an effective amount of a CD73 inhibitor to reverse or prevent the progression of CD73-mediated immunosuppression. In some embodiments, CD73-mediated immunosuppression is mediated by antigen presenting cells (APCs).

[0035] Examples of cancers that can be treated using the compounds and compositions described herein include, but are not limited to: prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal carcinoma), mesothelioma, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), adrenal gland cancer, thyroid cancer, kidney cancer or bone cancer; 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. Candidate cancers for treatment with the compounds and compositions of the present invention are discussed further below.

[0036] The present invention contemplates methods of treating a subject who has received a bone marrow transplant or a peripheral blood stem cell transplant 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 a malignancy after transplantation, increase the time to relapse-free survival after transplantation, and / or increase the long-term survival rate after transplantation.

[0037] 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 disease 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.

[0038] In other embodiments, the present invention contemplates methods of treating and / or preventing immune-related diseases, disorders, and conditions; diseases with an inflammatory component; and diseases related to the above with at least one CD73 inhibitor of the present invention. Examples of immune-related diseases, disorders, and conditions are described below.

[0039] Other diseases, disorders and conditions that can be treated or prevented, in whole or in part, by modulating CD73 activity are candidate indications for the CD73 inhibitor compounds of the invention.

[0040] The present invention further contemplates a combination of a CD73 inhibitor as described herein with one or more additional agents. One or more additional agents may have some CD73-modulating activity and / or they may act by different mechanisms of action, and in some embodiments, such agents include radiation (e.g., localized radiotherapy or systemic radiotherapy) and / or other treatment modalities of a non-pharmacological nature. When a combination therapy is used, one or more CD73 inhibitors and one or more additional agents may be in the form of a single composition or multiple compositions, and the treatment modality may be administered simultaneously, sequentially, or by some other regimen. For example, the present invention contemplates a treatment regimen in which a radiation phase is followed by a chemotherapy phase. The combination therapy may have an additive or synergistic effect. Other benefits of the combination therapy are described below.

[0041] In some embodiments, the present invention also includes the use of the CD73 inhibitors described herein in combination with bone marrow transplantation, peripheral blood stem cell transplantation, or other types of transplantation therapy.

[0042] In specific embodiments, the present invention contemplates the use of inhibitors of CD73 function as described herein in combination with immune checkpoint inhibitors, which have been shown to be a promising approach in the treatment of human cancer by blocking immune checkpoints (which lead to the amplification of antigen-specific T cell responses). Immune checkpoints (ligands and receptors), some of which are selectively upregulated in various types of tumor cells, are candidates for blockade, examples of which 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 receptor. Immune checkpoint inhibitors and combination therapies thereof are discussed in detail elsewhere herein.

[0043] In other embodiments, 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, such therapeutic agents including, but not limited to, alkylating agents (e.g., nitrogen mustards, such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines, such as thiotepa; mesylate esters, such as busulfan; nucleoside analogs (e.g., gemcitabine); nitrosoureas, such as carmustine, lomustine, and streptozotocin; topoisomerase 1 inhibitors (e.g., idarucizumab); irinotecan); platinum complexes such as cisplatin and carboplatin; bioreductive alkylating agents such as mitomycin, procarbazine, dacarbazine, and tetrachlorohydroxylamine; DNA strand breakers such as bleomycin; topoisomerase II inhibitors such as amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide; DNA minor groove binders such as plicamycin; antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, Cytarabine, and floxuridine; purine antagonists, such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparaginase; and ribonucleotide reductase inhibitors, such as hydroxyurea); tubulin-interactive agents (e.g., vincristine, estramustine, vinblastine, docetaxel, epothilone derivatives, and paclitaxel); hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbestrol; chlortrianisen; idenestrol; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol Ketones and; and androgens such as testosterone, testosterone propionate, fluoxymesterone, methyltestosterone and); adrenocortical steroids (e.g., prednisone, dexamethasone, methylprednisolone and prednisolone); luteinizing hormone-releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); and anti-hormone antigens (e.g., tamoxifen, anti-androgen drugs such as flutamide; and anti-adrenergic drugs such as mitotane and aminoglutamine). The present invention also contemplates the use of CD73 inhibitors in combination with other agents known in the art (e.g., arsenic trioxide) and other chemotherapeutic agents that may be developed in the future.

[0044] In some embodiments of the methods for 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 that observed by administration of either agent alone. In other embodiments involving methods for treating cancer, administration of a therapeutically effective amount of a CD73 inhibitor in combination with at least one chemotherapeutic agent results in a decrease in tumor size or a reduction in tumor growth that is greater than that observed by administration of either agent alone.

[0045] In other embodiments, the present invention contemplates a method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one signal transduction inhibitor (STI). In a specific embodiment, the at least one STI is selected from the group consisting of bcr / abl kinase inhibitors, epidermal growth factor (EGF) receptor inhibitors, her-2 / neu receptor inhibitors, and farnesyl transferase inhibitors (FTIs), and other candidate STI agents are presented elsewhere herein.

[0046] 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 that obtained by administering the CD73 inhibitor, chemotherapeutic agent, or radiation therapy alone.

[0047] In other embodiments, the present invention provides methods 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 immunomodulator that is not a CD73 inhibitor.

[0048] The present invention contemplates embodiments including methods 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 and a therapeutically effective amount of an anti-infective agent, e.g., one or more antimicrobial agents,

[0049] In another embodiment, treatment of infectious diseases is achieved by administering a vaccine in combination with administering a therapeutically effective amount of a CD73 inhibitor of the present invention. In some embodiments, the vaccine is an antiviral vaccine, including, for example, an anti-HIV vaccine. In other embodiments, the vaccine is effective against tuberculosis or malaria. In other embodiments, the vaccine is a tumor vaccine (e.g., a vaccine effective against melanoma); the tumor vaccine may comprise genetically modified tumor cells or genetically modified cell lines, including genetically modified tumor cells or genetically modified cell lines that have been transfected to express granulocyte-macrophage stimulating factor (GM-CSF). In a specific embodiment, the vaccine comprises one or more immunogenic peptides and / or dendritic cells.

[0050] In some embodiments, in certain embodiments involving treatment of infection by administering a CD73 inhibitor and at least one additional therapeutic agent, the symptoms of infection observed after administration of the CD73 inhibitor and the additional therapeutic agent are better than those observed after administration of either agent alone. In some embodiments, the observed symptoms of infection can be a decrease in viral load, CD4 + Increased T-cell counts, decreased opportunistic infections, increased survival time, eradication of chronic infections, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A simplified representation of extracellular purinergic signaling is depicted. DETAILED DESCRIPTION OF THE INVENTION

[0054] Before further describing the present invention, it is to be understood that the present invention is not limited to the particular embodiments described herein and it is to be further understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0055] When providing a range of values, it should be understood that unless the context clearly specifies otherwise, the intermediate values ​​(to one tenth of the lower limit unit) between the upper and lower limits of the range and any other provisions or intermediate values ​​within the stated range are included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the present invention. Subject to any specifically excluded restrictions within the described range, in the case where the described range includes one or two limits, the scope excluding any one or two of those included limits is also included in the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0056] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for specific terminology such as "only," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0057] The publications discussed herein are disclosed only prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0058] General

[0059] The number of subjects diagnosed with cancer and the number of deaths attributable to cancer continue to increase. Traditional treatments, including chemotherapy and radiation therapy, are often unaffordable to patients and become less effective as cancers (e.g., tumors) progress and bypass such treatments. Recent experimental evidence suggests that CD73 inhibitors may represent an important new therapeutic modality for the treatment of cancers (e.g., breast cancer).

[0060] Promising data also support the role of inhibitors of CD73 function to inhibit the anti-inflammatory activity of CD73 and / or the immunosuppressive activity of CD73, and therefore CD73 inhibitors may be useful in the treatment of, for example, immunosuppressive diseases such as HIV and AID. Inhibition of CD73 may also be an important therapeutic strategy for patients with neurological or neuropsychiatric disorders or diseases such as depression.

[0061] The present invention particularly relates to small molecule compounds having CD73 inhibitory activity and compositions thereof, as well as methods of using the compounds and compositions to treat and prevent the diseases, disorders and conditions described herein.

[0062] definition

[0063] Unless otherwise stated, the following terms are intended to have the meanings set forth below, with other terms defined elsewhere throughout the specification.

[0064] Unless otherwise indicated, the term "alkyl", by itself or as part of another substituent, refers to a radical having the specified number of carbon atoms (i.e., C 1-8 refers to a straight or branched hydrocarbon group of 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.

[0065] The term "cycloalkyl" refers to a hydrocarbon ring (e.g., C 3-6 "Cycloalkyl" may also refer to bicyclic hydrocarbon rings and polycyclic hydrocarbon rings, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc.

[0066] The term "cycloheteroalkyl" refers to a cycloalkyl ring having a specified number of ring vertices (or members) and having 1-5 heteroatoms selected from N, O or S replacing 1-5 carbon vertices, and wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The cycloheteroalkyl can be a monocyclic, bicyclic or polycyclic system. Non-limiting examples of cycloheteroalkyl include: pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolinone, 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 similar groups. Cycloheteroalkyl can be connected to the rest of the molecule through a ring carbon or heteroatom. When 'optionally substituted' is used to describe the term "cycloheteroalkyl" or "cycloheteroalkyl-alkyl", it refers to those wherein the cycloheteroalkyl or alkyl portion is optionally substituted, the substitution being as defined for the alkyl portion. For example, an optionally substituted cycloheteroalkyl-alkyl may be optionally substituted on either or both of the cycloheteroalkyl and alkyl portions, as described in the definition of alkyl substituents below.

[0067] As used herein, a wavy line that intersects a single, double, or triple bond in any chemical structure depicted herein is Indicates that a single bond, double bond or triple bond is connected to the point of the rest of the molecule. In addition, a bond extending to the center of a ring (e.g., a benzene ring) is meant to indicate a connection at any available ring vertex. It will be appreciated by those skilled in the art that multiple substituents shown as being connected to a ring will occupy the ring vertices that provide a stable compound and are otherwise spatially compatible. For a divalent component, the expression means to include either direction (forward or reverse). For example, the group "-C(O)NH-" means to include a bond in either orientation:-C(O)NH- or-NHC(O)-, and similarly, "-O-CH2CH2-" is intended to include-O-CH2CH2- and-CH2CH2-O-.

[0068] The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the rest of the molecule via an oxygen atom, a hydrogen atom or a sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl moieties may be the same or different and may be attached to the 3-7 membered ring via the nitrogen atom to which they are attached. Thus, with dialkylamino or -NR a R b The groups represented include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and similar groups

[0069] The terms "arylalkyl" and "heteroarylalkyl" are used in their traditional sense to refer to those in which the aryl or heteroaryl group is attached to the rest of the molecule via a C1-C4 alkylene linker. An example of an "arylalkyl" is phenylmethyl (or benzyl). Similarly, an example of a "heteroarylalkyl" is, for example, 3-pyridylpropyl. When "optionally substituted" is used to describe the term "arylalkyl" or "heteroarylalkyl", it means that the aryl or heteroaryl portion is optionally substituted as defined below, and the alkyl portion is optionally substituted as defined below.

[0070] Unless otherwise indicated, the terms "halo" or "halogen", by themselves or as part of another substituent, mean fluorine, chlorine, bromine or iodine. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C 1-4 "Haloalkyl" is intended to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0071] Unless otherwise indicated, the term "aryl" refers to a polyunsaturated, usually aromatic, hydrocarbon radical which may be a single ring or multiple rings (up to three) fused together or linked covalently. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl.

[0072] The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O or S, wherein the nitrogen and sulfur atoms are optionally oxidized and one or more nitrogen atoms are optionally quaternized. The heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridinyl, benzothiaxolyl, benzofuranyl, benzothiophenyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like. Substituents for the heteroaryl ring may be selected from the group of acceptable substituents described below.

[0073] The above terms (e.g., "alkyl", "aryl" and "heteroaryl"), in some embodiments, may be optionally substituted. Selected substituents for various types of groups are provided below,

[0074] The optional substituents of the alkyl group (including those commonly referred to as alkylene, alkenyl, alkynyl and cycloalkyl) may be a number from zero to (2m'+1) of various groups selected from the group consisting of halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C( 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", -CN and -NO2, wherein m' is the total number of carbon atoms in the group and R', R" and R'" each independently represent hydrogen, unsubstituted C 1-8 Alkyl, unsubstituted aryl, aryl substituted with 1 to 3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thiooxy, or unsubstituted aryl-C 1-4 Alkyl. When R' and R" are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include 1-pyrrolidinyl and 4-morpholinyl.

[0075] Similarly, the optional substituents for aryl and heteroaryl groups are variable and are typically selected from: -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)alkyl, the number of which ranges from zero to the total number of open valences on the aromatic ring system; and R', R" and R'"' are independently selected from: hydrogen, C 1-8 Alkyl, C 1-8 Haloalkyl, C 3-6 Cycloalkyl, C 2-8 Alkenyl and C 2-8 Other suitable substituents include the above-mentioned aryl substituents attached to the ring atoms via an alkylene group of 1 to 4 carbon atoms.

[0076] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by groups of the formula -TC(O)-(CH2) q -U-substituents shown in the substitution, wherein T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer of 0-2. Alternatively, two substituents on the adjacent atoms of an aryl or heteroaryl ring can be optionally substituted by the formula -A-(CH2)rB-, wherein A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer of 1 to 3. One of the single bonds of the new ring so formed can be optionally substituted by a double bond. Alternatively, two substituents on the adjacent atoms of an aryl or heteroaryl ring can be optionally substituted by the substituent of the formula -(CH2)sX-(CH2)t-, wherein s and t are independently an integer 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 C 1-6 alkyl.

[0077] As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0078] The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular substituents present on the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganic salts, potassium salts, sodium salts, zinc salts, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral forms of these compounds with a sufficient amount of the desired acid, which can be solvent-free 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, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from 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-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and amino acids such as arginine and organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge, SM et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functional groups, so that the compounds can be converted into base or acid addition salts.

[0079] 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 differs from its various salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention the salts are otherwise equivalent to the parent form of the compound.

[0080] In addition to salt forms, the present invention provides compounds in the form of prodrugs. Prodrugs of compounds described herein are those compounds that are susceptible to chemical changes under physiological conditions to provide compounds of the present invention. In addition, prodrugs can be converted into compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, when prodrugs are placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent, prodrugs can be slowly converted into compounds of the present invention.

[0081] Some compounds of the present invention can exist in non-solvated form as well as solvated form, including hydrated form. Generally speaking, solvated form is equivalent to non-solvated form, and is intended to be included within the scope of the present invention. Some compounds of the present invention can exist in polycrystalline or amorphous form. Generally, all physical forms are equivalent for the purposes contemplated by the present invention, and are intended to be within the scope of the present invention.

[0082] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., individual enantiomers) are intended to be included within the scope of the present invention. When a stereochemical description is shown, it is meant to refer to a compound in which one isomer is present and substantially free of another isomer, and "substantially free" of another isomer means at least an 80 / 20 ratio of the two isomers, more preferably 90 / 10 or 95 / 5 or more. In some embodiments, the content of one isomer is at least 99%.

[0083] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that make up these compounds. An unnatural proportion of an isotope can be defined as the amount from that found in nature to 100% consisting of the atom in question. For example, the compounds may incorporate radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or carbon-14 ( 14 C) or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C). Such isotopic variations may provide additional utility to those described elsewhere in this application. For example, isotopic variants of the compounds of the invention may find other uses, including but not limited to as diagnostic and / or imaging agents, or as cytotoxic / radiotoxic therapeutic agents. In addition, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic characteristics, which may help improve safety, tolerability or efficacy during treatment. All isotopic variants of the compounds of the invention, whether radioactive or not, are intended to be included within the scope of the invention.

[0084] The terms "patient" or "subject" are used interchangeably and may refer to either a human or a non-human animal (eg, a mammal).

[0085] When used with respect to, for example, a subject, a cell, a tissue, an organ, or a biological fluid, the terms "administering," "administering," and the like refer to contacting, for example, a CD73 inhibitor, a pharmaceutical composition thereof, or a diagnostic agent with a subject, a cell, a tissue, an organ, or a biological fluid. In the case of cells, administration includes contacting an agent with a cell (e.g., in vitro or ex vivo), as well as contacting an agent with a fluid, wherein the fluid is in contact with a cell.

[0086] The terms "treat," "therapeutic," "therapy," and the like refer to a course of action (e.g., administration of a CD73 inhibitor or a pharmaceutical composition comprising the same) initiated after a disease, disorder or condition, or symptoms thereof, is diagnosed, observed, etc., in order to temporarily or permanently eliminate, alleviate, suppress, relieve, or ameliorate at least one of the underlying causes of the disease afflicting the subject, or at least one of the symptoms associated with the disease, disorder, condition afflicting the subject. Thus, treatment includes inhibiting active disease (e.g., arresting the development or further development of a disease, disorder or condition, or clinical symptoms associated therewith).

[0087] As used herein, the term "in need of treatment" refers to the judgment made by a physician or other health care provider that a subject requires or will benefit from treatment, such judgment being based on a variety of factors within the physician's or health care provider's expertise.

[0088] The terms "prevention," "prophylactic," "prophylaxis," and the like refer to a course of action (such as administration of a CD73 inhibitor or a pharmaceutical composition comprising the same) that is initiated in some manner (e.g., prior to the onset of a disease, disorder or condition, or symptoms thereof) to temporarily or permanently prevent, suppress, inhibit, or reduce a subject's risk of developing a disease, disorder or condition, etc. (as determined by the absence of clinical symptoms) or delay its onset, usually in the case of a subject predisposed to a particular disease, disorder or condition. In certain instances, the terms also refer to slowing the progression of a disease, disorder or condition or inhibiting its development into a harmful or otherwise undesirable state.

[0089] As used herein, the term "in need of prevention" refers to the judgment made by a physician or other caregiver that a subject requires or will benefit from preventive care, such judgment being based on a variety of factors within the physician's or caregiver's area of ​​expertise.

[0090] The phrase "therapeutically effective amount" refers to an amount of an agent administered to a subject alone or as part of a pharmaceutical composition and in a single dose or as part of a series of doses, which, when administered to a subject, is capable of having any detectable positive effect on any symptom, aspect or characteristic of a disease, disorder or condition. A therapeutically effective amount can be determined by measuring relevant physiological effects, and can be adjusted based on dosing regimens and diagnostic assays of the subject's condition, among other things. For example, measuring serum levels of a CD73 inhibitor (or, for example, a metabolite thereof) at a specific time after administration can indicate whether a therapeutically effective amount has been used.

[0091] The phrase "an amount sufficient to effect a change" means that there is a detectable difference between the level of an indicator measured before (e.g., baseline level) and after administration of a particular therapy. Indicators include any objective parameter (e.g., serum concentration) or subjective parameter (e.g., a subject's sense of well-being).

[0092] The term "small molecule" refers to a chemical 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.

[0093] The term "ligand" refers to a peptide, polypeptide, membrane-associated or membrane-bound molecule or a complex thereof that can act as a receptor agonist or antagonist. Ligands include natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and binding compositions derived from antibodies and small molecules. The term also includes agents that are neither agonists nor antagonists but can bind to receptors without significantly affecting their biological properties (such as signal transduction or adhesion). In addition, the term includes membrane-bound ligands that have been changed into soluble forms of membrane-bound ligands by, for example, chemical or recombinant methods. A ligand or receptor may be completely intracellular, that is, it may be present in the cytoplasm, the nucleus, or some other intracellular compartments. The complex of a ligand and a receptor is referred to as a "ligand-receptor complex".

[0094] The terms "inhibitor" and "antagonist" or "activator" and "agonist" refer to inhibiting or activating molecules, respectively, for example, for activating, for example, ligands, receptors, cofactors, genes, cells, tissues or organs. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize or downregulate, for example, genes, proteins, ligands, receptors or cells. Activators are molecules that increase, activate, promote, enhance activation, sensitize or upregulate, for example, genes, proteins, ligands, receptors or cells. Inhibitors can also be defined as molecules that reduce, block or inactivate constitutive activity. "Agonists" are molecules that interact with targets to cause or promote increased activation of targets, and "antagonists" are molecules that act opposite to agonists. Antagonists prevent, reduce, inhibit or neutralize the activity of agonists, and antagonists can also prevent, inhibit or reduce the constitutive activity of targets, such as target receptors, even in the absence of identified agonists.

[0095] The terms "modulation", "modulation method" 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 can act alone, or cofactors such as proteins, metal ions or small molecules can be used. Examples of modulators include small molecule compounds and other bioorganic molecules. Many small molecule compound libraries (e.g., combinatorial libraries) are commercially available and can serve as a starting point for identifying modulators. Those skilled in the art are able to develop one or more assays (e.g., biochemical or cell-based assays) in which these compound libraries can be screened to identify one or more compounds with desired properties; thereafter, skilled medicinal chemists are able to optimize such one or more compounds by, for example, synthesizing and evaluating analogs and derivatives thereof. Synthetic and / or molecular modeling studies can also be used to identify activators.

[0096] The "activity" of a molecule can describe or refer 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; to modulate the activity of other molecules, etc. The term "proliferative activity" includes activities that promote normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis and angiogenesis that are required or specifically associated with normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis and angiogenesis.

[0097] As used herein, "comparable", "comparable activity", "activity comparable to...", "comparable effect", "effect comparable to...", etc. are relative terms that can be observed quantitatively and / or qualitatively. The meaning of the terms generally depends on the context in which they are used. For example, from a qualitative point of view, two drugs that activate a receptor may be considered to have comparable effects, but if the activity of one drug is only able to reach 20% of the activity of the other drug as determined in an assay method accepted in the art (e.g., a dose-response assay method) or in an animal model accepted in the art, then from a quantitative point of view, the two drugs may be considered to lack comparable effects. When comparing one result to another result (e.g., one result is compared to a reference standard), "comparable" often (although not always) means that 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, if a result deviates from the reference standard by less than 15%, less than 10%, or less than 5%, then its result is comparable to the reference standard. By way of example and not limitation, activity or effect may refer to potency, stability, solubility, or immunogenicity.

[0098] "Substantially pure" means that the component accounts for more than about 50% of the total content of the composition, and typically more than about 60% of the total content. More typically, "substantially pure" means that at least 75%, at least 85%, at least 90% or more of all components in the composition are the component of interest. In some cases, the component of interest will account for more than about 90% or more than about 95% of the total content of the composition.

[0099] When referring to ligand / receptor, antibody / antigen or other binding pairs, the term "specific binding" or "selective binding" refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, a specified ligand binds to a specific receptor and does not significantly bind to other proteins present in the sample. The antibody or binding composition derived from the antigen binding site of the antibody in the contemplated method has an affinity for its antigen or a variant or mutant thereof that is at least two times, at least ten times, at least 20 times, or at least 100 times greater than that for any other antibody or binding composition derived therefrom. In a specific embodiment, the antibody will have an affinity greater than about 10 as determined by, for example, Scatchard analysis (Munsen et al., 1980 Analyt. Biochem. 107: 220-239). 9 Liter / mol affinity.

[0100] The term "response", such as a response of a cell, tissue, organ or organism, includes a change in biochemical or physiological behavior, such as a change in concentration, density, adhesion or migration within a biological compartment, a change in the rate of gene expression or a differentiation state, wherein the change is associated with activation, stimulation or treatment, or is associated with internal mechanisms such as genetic programming. In some cases, the terms "activation", "stimulation", etc. refer to cell activation regulated by internal mechanisms as well as external or environmental factors; while the terms "inhibition", "downregulation", etc. refer to the opposite effect.

[0101] The terms "polypeptide", "peptide" and "protein" used interchangeably herein refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids and polypeptides with modified polypeptide backbones. The term includes fusion proteins, including but 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; immunolabeled proteins, and the like.

[0102] As used herein, the terms "variant" and "homolog" are used interchangeably to refer to an amino acid or DNA sequence similar to a reference amino acid or nucleic acid sequence, respectively. The term encompasses naturally occurring variants and non-naturally occurring variants. Naturally occurring variants include homologs (polypeptides and nucleic acids with different amino acid or nucleotide sequences between different species) and allelic variants (polypeptides and nucleic acids with different amino acid or nucleotide sequences between different species). Therefore, variants and homologs include naturally occurring DNA sequences and proteins encoded thereby and their isoforms, as well as splice variants of proteins or genes. The term also encompasses nucleic acid sequences that are different from one or more bases of a naturally occurring DNA sequence, but due to the degeneracy of the genetic code, the nucleic acid sequence is still translated into an amino acid sequence corresponding to a naturally occurring protein. Non-naturally occurring variants and homologs include polypeptides and nucleic acids that contain amino acid or nucleotide sequence changes, respectively, wherein the sequence changes (e.g., mutant proteins) are artificially introduced; for example, changes are produced in the laboratory by human intervention ("manually"). Therefore, non-naturally occurring variants and homologs may also refer to those that differ from naturally occurring sequences by one or more conservative substitutions and / or tags and / or conjugates.

[0103] As used herein, the term "mutant protein" refers broadly to a mutated recombinant protein. These proteins typically carry single or multiple amino acid substitutions and are typically derived from cloned genes that have been subjected to site-directed or random mutagenesis, or from fully synthetic genes.

[0104] The terms "DNA", "nucleic acid", "nucleic acid molecule", "polynucleotide" and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, whether 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.

[0105] Extracellular 5'-nucleotidase and its inhibitors

[0106] Human CD73 (also known as ecto-5′-nucleotidase; NT5E; or 5NT) is a 574 amino acid residue protein (Accession No. AAH6593). Eukaryotic CD73 functions as a non-covalent homodimer with two domains, wherein the N- and C-terminal domains are connected by a hinge region that enables the enzyme to undergo large domain movements and switch between open and closed conformations (Knapp, K et al., (2012) Structure 20: 2161-73).

[0107] As used herein, the terms "CD73 inhibitor," "CD73 blocker," "adenosine by extracellular 5′-nucleotidase inhibition," "NT5E inhibitor," "5NT inhibitor" and all other relevant art-recognized terms refer to compounds that are capable of directly or indirectly modulating the CD73 receptor in in vitro assays, in vivo models and / or other means of indicating therapeutic efficacy. The terms also relate to compounds that show at least some therapeutic benefit in human subjects. CD73 inhibitors can be competitive, non-competitive or irreversible CD73 inhibitors. "Competitive CD73 inhibitors" are compounds that reversibly inhibit CD73 enzyme activity at the catalytic site; "non-competitive CD73 inhibitors" are compounds that reversibly inhibit CD73 enzyme activity at non-catalytic sites; and "irreversible CD73 inhibitors" are compounds that irreversibly eliminate CD73 enzyme activity by forming a covalent bond with the enzyme (or other stable means of inhibiting enzyme function).

[0108] CD73 inhibitors can modulate purinergic signaling, which is an 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, leading to the regulation of cellular function. The enzymatic activity of CD73 plays a crucial role in calibrating the duration, magnitude, and chemical nature of purinergic signals delivered to various cells (e.g., immune cells). Changes in the activity of these enzymes can alter the course of disease or determine the outcome of several pathophysiological events (including cancer, autoimmune and inflammatory diseases, infection, atherosclerosis, and ischemia-reperfusion injury), indicating that these extracellular enzymes represent new therapeutic targets for the management of a variety of diseases.

[0109] Studies using tissues that overexpress CD73 and using CD73 knockout mice provide evidence that CD73 inhibitors have potential utility for melanoma, lung cancer, prostate cancer, and breast cancer (see, e.g., Sadej R. (2006) Melanoma Res 16: 213-22). Because high expression levels of CD73 are associated with tumor angiogenesis, invasiveness, resistance to chemotherapy, and metastasis, CD73 inhibitors can be used to control tumor progression and metastasis. Other potential utilities are discussed elsewhere herein.

[0110] As described above, although the compounds of the present invention are believed to exert their activity by inhibiting CD73, it is not necessary to precisely understand the mechanism of action of the compounds to implement the present invention. For example, the compounds may also exert their activity at least in part by regulating (e.g., inhibiting) other components of the purinergic signaling pathway (e.g., CD39). The purinergic signaling system consists of transporters, enzymes, and receptors responsible for the synthesis, release, action, and extracellular inactivation of (primary) ATP and its extracellular breakdown product adenosine (Sperlagh, B et al. (December 2012) Neuropsychopharmacologia Hungarica 14 (4): 231-38). Figure 1 A simplified representation of extracellular purinergic signaling is described (see, e.g., North RA (October 2002) Physiological Reviews 82(4): 1013-67). As noted therein, there are several potential opportunities for modulating the signaling process. However, it will be apparent to those skilled in the art that some of these opportunities are more tractable than others.

[0111] Identification of CD73 inhibitors with desirable properties

[0112] In part, the present invention relates to identifying CD73 inhibitors having at least one property or characteristic of therapeutic relevance. Candidate inhibitors can be identified by using, for example, art-accepted assays or models, examples of which will be apparent to those skilled in the art. Assays for determining the CD73 inhibitory activity of the compounds described herein are described in the Experimental Section.

[0113] Once identified, candidate inhibitors can be further evaluated using techniques that provide data on inhibitor characteristics (e.g., pharmacokinetic parameters). Comparison of candidate inhibitors to a reference standard (which may be the current "best" inhibitor) indicates the potential activity of these candidates.

[0114] CD73 inhibitors that can be used as reference or benchmark compounds include α,β-methylene-ADP (AOPCP) and its derivatives and analogs described by Bhattarai et al. ((2015) J Med Chem 58: 6248-63), and purine CD73 derivatives reported in PCT Publication No. 2015 / 164573. Other reference compounds subsequently identified by those skilled in the art can also be used to evaluate the activity of candidate CD73 inhibitors.

[0115] Compounds of the present invention

[0116] Provided herein are compounds of formula (I):

[0117]

[0118] or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein

[0119] Each R 1 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R 2 R 2 )-OC(O)-OR 3 The group consisting of two R 1 optionally combined to form a 5- to 7-membered ring;

[0120] Each R 2 independently selected from the group consisting of H and optionally substituted C1-C6 alkyl;

[0121] Each R 3 are independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl;

[0122] R 5 Selected from the group consisting of H and optionally substituted C1-C6 alkyl;

[0123] X is selected from the group consisting of O, CH2 and S;

[0124] A is selected from the group consisting of:

[0125]

[0126] Each of which is optionally replaced by 1 to 5 R 6 The substituent is substituted, and wherein the subscript n is an integer from 0 to 3;

[0127] Z is selected from CH2, CHR 6 NR 6 and O constitute the group;

[0128] Each R 6independently 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 linked together to form a 5- to 6-membered ring containing at least one heteroatom as a ring vertex; and

[0129] Het is selected from the group consisting of:

[0130]

[0131] where the wavy line indicates the point of attachment to the rest of the compound, and where:

[0132] R a Selected from H, NH2, NHR 7 、NHC(O)R 7 NR 7 R 7 , R 7 , OH, SR 7 OR 7 The group formed;

[0133] R b Selected from H, halogen, NH2, NHR 7 NR 7 R 7 , R 7 , OH and OR 7 The group formed;

[0134] R c and R d are independently selected from H, halogen, haloalkyl, NH2, NHR 7 NR 7 R 7 , R 7 , OH, OR 7 , SR 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 The group formed;

[0135] R e and Rf independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl;

[0136] Each X 1 is C1-C4 alkylene; and

[0137] Each R 7 are independently selected from the group consisting of: optionally substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-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 cycloheteroalkylC1-C4 alkyl, optionally substituted aryl, optionally substituted arylC1-C4 alkyl, optionally substituted arylC2-C4 alkenyl, optionally substituted arylC2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroarylC1-C4 alkyl, optionally substituted heteroarylC1-C4 alkenyl, optionally substituted heteroarylC2-C4 alkynyl, and optionally, two R attached to the nitrogen atom 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring optionally fused to an aromatic ring;

[0138] Provided that the compounds are not those compounds in which X, A and Het combine to give

[0139]

[0140] Where R g H or two R g The groups combine to form acetonide; and any of the following conditions

[0141] (i)R c and R e is hydrogen and R a is -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

[0142] (ii) R c is hydrogen, R a is -NH2, and R e is bromine, chlorine, aminomethyl or thioethyl; or

[0143] (iii) R c is hydrogen, Ra is benzylamino, and R e For bromine.

[0144] For the above formula, the term 'optionally substituted' is used with alkyl, cycloalkyl, cycloheteroalkyl, aryl and heteroaryl. In each of these groups, some selected optional substituents are as follows:

[0145] Alkyl: halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -CN and -NO2, R', R" and R"' each independently represent hydrogen, unsubstituted C 1-4 Alkyl, or C 1-4 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 can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include 1-pyrrolidinyl and 4-morpholinyl.

[0146] Cycloalkyl and cycloheteroalkyl: The substituents selected above for 'alkyl' are also useful for cycloalkyl and cycloheteroalkyl. In addition, each cycloalkyl and cycloheteroalkyl may be optionally substituted with oxygen (=O),

[0147] Aryl and heteroaryl: -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 perfluoro(C1-C4)alkyl, wherein R', R" and R"' are independently selected from hydrogen, C 1-4 Alkyl, C 1-4 Haloalkyl and C 3-6 Cycloalkyl.

[0148] In one selected group of embodiments, compounds of formula (I) are provided, wherein A has the formula:

[0149]

[0150] It is composed of 1 to 5 R 6 Optional substitution.

[0151] In another selected group of embodiments, compounds of formula (I) are provided, wherein A has a formula selected from the group consisting of:

[0152]

[0153] In some selected embodiments, any one of a1 to a16 can be independently combined with any one of b1 to 9 to provide alternative embodiments of formula (I).For example, the present disclosure provides 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; a6 / 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; a11 / 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; a14 / b5; a14 / b6; a14 / b7; a14 / b8; a14 / b9; a15 / b1; a15 / b2; a15 / b3; a15 / b4; a15 / b5; a15 / b6; a15 / b7; a15 / b8; a15 / b9; a16 / b1; a16 / b2; a16 / b3; a16 / b4; a16 / b5; a16 / b6; a16 / b7; a16 / b8; or a16 / b9.

[0154] In other selected embodiments, compounds of formula (I) are provided, wherein Het has the following formula:

[0155]

[0156] In some selected embodiments, R c Not for H.

[0157] In some other selected embodiments, there is provided a compound of formula (I) represented by one of the following sub-formulas:

[0158]

[0159] Each R g In a further selected embodiment of the above subformula, X is oxygen. In other selected embodiments of the above subformula, X is oxygen and R e In other selected embodiments of the above subformula, X is oxygen, R e is hydrogen, and each R g For hydrogen.

[0160] In another group of selected embodiments, compounds of formula (I) are provided, wherein Het is selected from:

[0161]

[0162] Where R a , R c and R e has the meanings provided with reference to the above formula (I). In some selected embodiments, R 5 is H, X is O, and each R 1 is H. In some other selected embodiments, R 5 is H, X is O, each R 1 For H, R e for H, and R a Selected from NH2, NHR 7 and N(R 7 )2. In some other selected embodiments, R 5 is H, X is O, each R 1 For H, R e For H, R c Not H, and R a NHR 7 .

[0163] Other selected embodiments of formula (I) are compounds having subformulas selected from the group consisting of:

[0164]

[0165] Where R 7 and R c has the meanings provided in formula (I), and some selected embodiments are as described herein.

[0166] In one set of embodiments, the present invention also provides compounds of the formula:

[0167]

[0168] or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein

[0169] Each R 1 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R 2 R 2 )-OC(O)-OR 3 The group consisting of two R 1 optionally combined to form a 5- to 7-membered ring;

[0170] Each R 2 independently selected from the group consisting of H and optionally substituted C1-C6 alkyl;

[0171] Each R 3 are independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl;

[0172] R 5 Selected from the group consisting of H and optionally substituted C1-C6 alkyl;

[0173] X is O;

[0174] A is selected from the group consisting of:

[0175]

[0176] and

[0177] Het is selected from the group consisting of:

[0178]

[0179] where the wavy line indicates the point of attachment to the rest of the compound, and where:

[0180] R a Selected from H, NH2, NHR 7 、NHC(O)R 7 NR 7 R 7 , R 7 , OH, SR7 OR 7 The group formed;

[0181] R b Selected from H, halogen, NH2, NHR 7 NR 7 R 7 , R 7 , OH and OR 7 The group formed;

[0182] R c and R d are independently selected from H, halogen, haloalkyl, NH2, NHR 7 NR 7 R 7 , R 7 , OH, OR 7 , SR 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 The group formed;

[0183] R e and R f independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl;

[0184] Each X 1 is C1-C4 alkylene; and

[0185] Each R 7 are independently selected from the group consisting of: optionally substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-C 10alkynyl, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkylC1-C4 alkyl, optionally substituted 4-7 membered cycloheteroalkyl, optionally substituted 4-7 membered cycloheteroalkylC1-C4 alkyl, optionally substituted aryl, optionally substituted arylC1-C4 alkyl, optionally substituted arylC2-C4 alkenyl, optionally substituted arylC2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroarylC1-C4 alkyl, optionally substituted heteroarylC1-C4 alkenyl, optionally substituted heteroarylC2-C4 alkynyl, and optionally, two R attached to the nitrogen atom 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring which is optionally fused to an aromatic ring.

[0186] In a selected group of embodiments, compounds of formula (IVa) are those wherein A is

[0187]

[0188] In another selected group of embodiments, the compounds of formula (IVa) are those, wherein Het is selected from the group consisting of:

[0189]

[0190] In another selected group of embodiments, the compound has the following formula:

[0191]

[0192] or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0193] In a selected group of embodiments, compounds of formula (IVb) are those wherein R a Selected from NH2, NHR 7 NR 7 R 7 , SR 7 OR 7 In a selected group of embodiments, the compounds of formula (Ib) are those wherein R c Selected from halogen, R 7 , OR 7 , SR 7 、SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X1 -SR 7 and -X 1 -SO2R 7 The group composed of.

[0194] In another selected group of embodiments, compounds of formula (IVb) are those wherein R e For H.

[0195] Synthesis method

[0196] Generally, the compounds provided herein can be prepared by conventional methods as described in the following examples.

[0197] Modifications that enhance inhibitor characteristics

[0198] It is often beneficial and sometimes necessary to improve one of the more physical properties of the therapeutic modalities disclosed herein and / or their modes of administration. Improvements in physical properties include, for example, methods of increasing aqueous solubility, bioavailability, serum half-life, and / or therapeutic half-life; and / or modulating biological activity.

[0199] Modifications known in the art include PEGylation, Fc-fusion, and albumin fusion. Although usually associated with macromolecular agents (e.g., polypeptides), such modifications have recently been evaluated with specific small molecules. For example, Chiang, M et al. (J. Am. Chem. Soc., 2014, 136 (9): 3370-73) describe small molecule agonists of adenosine 2a receptors coupled to immunoglobulin Fc domains. Small molecule-Fc conjugates retain effective Fc receptor and adenosine 2a receptor interactions and show superior properties compared to unconjugated small molecules. Covalent attachment of PEG molecules to small molecule therapeutics has also been described (Li, W et al., Advances in Polymer Science, 2013 38: 421-44).

[0200] Therapeutic and preventive uses

[0201] 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. Although specific uses are described in detail below, it should be understood that the present invention is not limited thereto. In addition, although general categories of specific diseases, disorders and conditions are listed below, some diseases, disorders and conditions may be members of more than one category, while others may not be members of any disclosed category.

[0202] Oncology related conditionsAccording to the invention, CD73 inhibitors can be used to treat or prevent proliferative disorders or conditions, including cancer, such as uterine cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, gastrointestinal cancer (such as esophageal cancer, oropharyngeal cancer, gastric cancer, small intestine cancer or large intestine cancer, colon cancer or rectal 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 cancer, thyroid cancer, brain cancer (such as glioma), ganglion cancer, cancer of the central nervous system (CNS) and cancer of the peripheral nervous system (PNS), as well as cancer of the hematopoietic system and cancer of the immune system (such as the 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 cancer, endothelial cell cancer, 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 cell 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 Sawaya et al. (2003) New Engl. J. Med. 349: 1501-09). In specific embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma or leukemia. Use of the term "cancer-related diseases, disorders and conditions" is intended to refer broadly to disorders that are directly or indirectly related to cancer and include, for example, angiogenesis and precancerous conditions such as dysplasia.

[0203] In certain embodiments, the cancer is metastatic or at risk of metastasizing, or may occur in disseminated tissues, including blood or bone marrow cancers (eg, leukemias). In some further embodiments, the compounds of the invention may be used to overcome T cell tolerance.

[0204] In some embodiments, the invention provides methods of treating a proliferative disorder, 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.

[0205] Immune-related diseases and diseases with an inflammatory componentAs used herein, terms such as "immune disease", "immune condition", "immune disorder", "inflammatory disease", "inflammatory condition", "inflammatory disorder" and the like are intended to broadly encompass any immune-related disease (e.g., autoimmune disease) or disease with an inflammatory component that can be treated with the CD73 inhibitors described herein to obtain some therapeutic benefit. Such diseases are often inseparable from other diseases, disorders and conditions. For example, "immune condition" can refer to proliferative conditions such as cancer, tumors, and angiogenesis; including infections (acute and chronic), tumors, and cancers that resist eradication by the immune system.

[0206] The CD73 inhibitors of the present invention can be used to increase or enhance the immune response; improve immunization, including improving vaccine efficacy; and increase inflammation. The compounds disclosed herein can be used to treat immunodeficiency associated with immunodeficiency diseases, immunosuppressive medical treatments, acute and / or chronic infections, and aging. CD73 inhibitors can also be used to stimulate the immune system of patients with iatrogenic-induced immunosuppression, including those who have received bone marrow transplants, chemotherapy, or radiation therapy.

[0207] In specific embodiments of the present disclosure, CD73 inhibitors are used to increase or enhance the immune response to an antigen by providing adjuvant activity. In specific 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. Also provided are therapeutic compositions comprising at least one antigenic agent or vaccine component and at least one CD73 inhibitor combination of the present invention, the antigenic agent or vaccine component including but not limited to viruses, bacteria and fungi or parts thereof, proteins, peptides, tumor-specific antigens and nucleic acid vaccines.

[0208] Microbial related diseases .By inhibiting the immunosuppressive and anti-inflammatory activities of CD73, 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 for which treatment with a CD73 inhibitor may be beneficial. Examples of such diseases and conditions include HIV and AIDS, staphylococcal and streptococcal infections (e.g., Staphylococcus aureus and Streptococcus sanguinis, respectively), Leishmania, Toxoplasma, Trichomonas, Giardia, Candida albicans, Bacillus anthracis, and Pseudomonas aeruginosa. The compounds of the present invention may be used to treat sepsis, reduce or inhibit bacterial growth, and reduce or inhibit inflammatory cytokines.

[0209] CNS-related and neurological disordersInhibition of CD73 may also be an important therapeutic strategy for patients with neurological, neuropsychiatric, neurodegenerative or other diseases, disorders and conditions that are somehow connected to the central nervous system, including disorders associated with impaired cognitive and motor function. Examples include Parkinson's disease, extrapyramidal syndrome (EPS), dystonia, akathisia, tardive dyskinesia, restless legs syndrome (RLS), epilepsy, periodic limb movements of 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.

[0210] Other conditions .Embodiments of the invention encompass administering to a subject a CD73 inhibitor described herein for the treatment or prevention of any other disorder that would benefit from at least some level of CD73 inhibition. These diseases, disorders, and conditions include, for example, cardiovascular disease (e.g., cardiac ischemia), gastrointestinal disease (e.g., Crohn's disease), metabolic disease (e.g., diabetes), liver disease (e.g., liver fibrosis, NASH, and NAFLD), lung disease (e.g., COPD and asthma), ophthalmologic disease (e.g., diabetic retinopathy), and renal disease (e.g., renal failure).

[0211] In some embodiments, the CD73 inhibitors of the present invention can be used to inhibit statin-induced adenosine production, or to reduce or decrease statin-induced increases in blood glucose in subjects taking statins (e.g., lovastatin and pravastatin).

[0212] Pharmaceutical composition

[0213] The CD73 inhibitors of the present invention may be in the form of a composition suitable for administration to a subject. Typically, such a composition is a "pharmaceutical composition" comprising one or more CD73 inhibitors and one or more pharmaceutically acceptable 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 implement the treatment and prevention methods and uses described herein.

[0214] 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 set forth herein. In addition, 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.

[0215] Pharmaceutical compositions containing active ingredients (e.g., CD73 function inhibitors) can be in a form suitable for oral use, such as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs. Pharmaceutical compositions for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions can contain one or more agents, such as sweeteners, flavoring agents, colorants and preservatives to provide pharmaceutically elegant and palatable preparations. Tablets, capsules, etc. contain active ingredients mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. These excipients can be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants, such as corn starch or alginic acid; binders, such as starch, gelatin or gum arabic, and lubricants, such as magnesium stearate, stearic acid or talc.

[0216] Tablets, capsules, etc. suitable for oral administration can be uncoated or coated by known technology to delay disintegration and absorption in the gastrointestinal tract and provide sustained action. For example, time-delay materials, such as glyceryl monostearate or glyceryl distearate, can be used. They can also be coated with technology known in the art to form osmotic therapeutic tablets for controlled release. Other agents include biodegradable or biocompatible particles or polymeric substances, such as polyesters, polyamino acids, hydrogels, polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide / glycolide copolymers, polylactide / glycolide copolymers or ethylene ethylene vinyl acetate copolymers to control the delivery of the composition used. For example, oral agents can be embedded in microcapsules prepared by coacervation technology or by interfacial polymerization, using hydroxymethylcellulose or gelatin microcapsules or poly (methyl methacrylate) microcapsules or colloidal drug delivery systems respectively. Colloidal dispersion systems include macromolecular 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.

[0217] Formulations for oral use may also be hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or 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.

[0218] Aqueous suspensions contain active materials mixed with excipients suitable for making them. Such excipients can be suspending agents, such as sodium carboxymethylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents, such as naturally occurring phospholipids (e.g., lecithin) or condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxides and long-chain fatty alcohols (e.g., heptadecanethenyloxycetanol) or condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate) or condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol anhydrides (e.g., polyvinyl sorbitan monooleate). Aqueous suspensions can also contain one or more preservatives.

[0219] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g., liquid paraffin). Oily suspensions may contain thickeners, such as beeswax, hard paraffin or cetyl alcohol. Sweeteners and flavoring agents such as those mentioned above may be added to provide a palatable oral preparation.

[0220] 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 herein.

[0221] The pharmaceutical composition of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture of these. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or tragacanth; naturally occurring 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.

[0222] The pharmaceutical composition generally comprises a therapeutically effective amount of the CD73 inhibitor contemplated by the present invention and one or more pharmaceutically and physiologically acceptable formulation agents. Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl paraben or ethyl paraben or propyl paraben), emulsifiers, suspending agents, dispersants, solvents, fillers, fillers, detergents, buffers, excipients, diluents and / or adjuvants. For example, a suitable carrier may be a physiological saline solution or citrate buffered saline, which may be supplemented with other substances commonly found in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is a further exemplary carrier. Those skilled in the art will readily recognize the various buffers that can be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. As an example, the buffer component can be a water-soluble substance 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), (N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS) and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0223] 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 a formulation can be stored in a ready-to-use form, a lyophilized form that requires reconstitution before use, a liquid form that requires dilution before use, or other acceptable form. In some embodiments, the pharmaceutical composition is in a disposable container (e.g., a disposable vial, ampoule, syringe, or autoinjector (similar to, for example, )), while other embodiments provide multi-use containers (e.g., multi-use bottles).

[0224] The formulation may also include a carrier to protect the composition from 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 used. Any drug delivery device may be used to deliver the CD73 inhibitor, including implants (e.g., implantable pumps) and catheter systems, slow injection pumps and devices, all of which are well known to those skilled in the art.

[0225] Depot injections, generally 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 oil-based and typically contain at least one formulation component described herein. Those of ordinary skill in the art are familiar with the possible formulations and uses of depot injections.

[0226] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension. The suspension may be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents as mentioned herein. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Acceptable diluents, solvents and dispersion media that may be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyols (e.g., glycerol, propyl glycol and liquid polyethylene glycol) and suitable mixtures thereof. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used to prepare injections. Prolonged absorption of a particular injectable formulation may be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin).

[0227] The present invention contemplates administration of CD73 inhibitors in the form of suppositories for rectal administration. Suppositories can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycol.

[0228] The CD73 inhibitors contemplated by the present invention may be in the form of any other suitable pharmaceutical composition (e.g., a spray for nasal or inhalation use) currently known or developed in the future.

[0229] Dosage

[0230] The present invention contemplates administration of CD73 inhibitors and compositions thereof in any appropriate manner. Suitable routes of administration include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intraventricular), nasal, vaginal, sublingual, intraocular, topical (e.g., transdermal), oral, and inhalation. Depot injections, generally administered subcutaneously or intramuscularly, can also be used to release the CD73 inhibitors disclosed herein over a defined period of time.

[0231] Particular embodiments of the invention contemplate oral administration.

[0232] Combination therapy

[0233] The present invention contemplates the use of CD73 inhibitors in combination with one or more active therapeutic agents (e.g., chemotherapeutic agents) or other preventive or therapeutic modalities (e.g., radiation). In such combination therapies, the various active agents typically have different complementary mechanisms of action. Such combination therapies may be particularly beneficial by allowing a dose reduction of one or more agents, thereby reducing or eliminating adverse effects associated with one or more agents. In addition, such combination therapies may have a synergistic therapeutic or preventive effect on the underlying disease, disorder, or condition.

[0234] As used herein, "combination" is meant to include therapies that can be administered separately, e.g., separately formulated for separate administration (e.g., as may be provided in a kit) as well as therapies that can be administered together in a single formulation (ie, "co-formulated").

[0235] In certain embodiments, the CD73 inhibitor is administered or applied sequentially, for example, one agent is administered before one or more other agents. In other embodiments, the CD73 inhibitor is administered simultaneously, for example, two or more drugs are administered at the same time or about the same time; the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, for the purposes of the present invention, they are considered to be administered in combination.

[0236] In this case, the CD73 inhibitor of the present invention can be used in combination with at least one other (active) agent in any suitable manner. In one embodiment, treatment with at least one active agent and at least one CD73 inhibitor of the present invention is maintained over a period of time. In another embodiment, when the treatment with the CD73 inhibitor of the present invention is at a constant dosing regimen, the treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable). In another embodiment, when the treatment with the CD73 inhibitor of the present invention is reduced (e.g., lower doses, less frequent dosing or shorter treatment regimen), the treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable). In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., when the subject is stable), and treatment with the CD73 inhibitor of the present invention is increased (e.g., higher doses, more frequent doses or longer treatment regimens). In another embodiment, treatment with at least one active agent is maintained and treatment with the CD73 inhibitor of the present invention is reduced or discontinued (e.g., lower doses, less frequent dosing or shorter treatment regimens). In yet another embodiment, treatment with at least one active agent and treatment with a CD73 inhibitor of the invention is reduced or discontinued (e.g., lower dose, less frequent dosing, or shorter treatment regimen).

[0237] Tumor-related diseases .The present invention provides methods for treating and / or preventing a proliferative disorder, cancer, tumor or precancerous disease, disorder or condition using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0238] In certain embodiments, the present invention provides a method for tumor inhibition of tumor growth, comprising administering a CD73 inhibitor described herein with a signal transduction inhibitor (STI) to achieve additive or synergistic inhibition 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. The signal transduction inhibitors (STI) 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) Akt family kinase inhibitors or Akt pathways (e.g., rapamycin); (v) cell cycle kinase inhibitors (e.g., flavopiridol); and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immunomodulation can also be used in combination with the CD73 inhibitors described herein to inhibit tumor growth in cancer patients.

[0239] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carloquinone, mitodopa and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trihydroxymethylmelamine; nitrogen mustards such as chiorambucil, naphthyl mustard, clofosamide, estramustine, ifosfamide, dichloromethyldiethylamine, dichloromethyldiethylamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trolofosamide, uracil mustard; nitroso Ureas, such as carmustine, chlorourea, fotemustine, lomustine, nimustine, laromustine; antibiotics, such as aclacinomycin, dactinomycin, authramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, aclarubicin, idarubicin, mexilomycin, mitoflash mycophenolic acid, mycophenolic acid, nogamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozocin, tuberculin, ubenimex, jinsistatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolate, methotrexate, pterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androstenone; hormones, such as caprotestosterone, drostanolone propionate, cyclothiocarb, mestanolidine, and testolactone; antiadreners, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; acetoglucuronolide; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclofenac; diaziquone; elformithine; elliptonium acetate; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophylloic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; piperobroman; gacytosine; Ara-C; cyclophosphamide; thiotepa; taxoids such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination compounds, Such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoroacetylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof. ;

[0240] Chemotherapeutic agents also include anti-hormonal agents for regulating or inhibiting the effects of hormones such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, troxifene, raloxifene, onapristone and toremifene; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the combination therapy comprises administration of hormones or related hormone agents.

[0241] Other treatment modalities that could be used in combination with CD73 inhibitors include radiation therapy, monoclonal antibodies directed against tumor antigens, complexes of monoclonal antibodies and toxins, T-cell adjuvants, bone marrow transplants, or antigen-presenting cells (e.g., dendritic cell therapy).

[0242] Immune checkpoint inhibitors .The present invention contemplates the use of the CD73 function inhibitors described herein in combination with immune checkpoint inhibitors.

[0243] The large number of genetic and epigenetic changes that are characteristic of all cancers provide a variety of antigens that the immune system can use to distinguish tumor cells from normal cells. In the case of T cells, the final amplitude (e.g., the level of cytokine production or proliferation) and quality (e.g., the type of immune response generated, such as the pattern of cytokine production) of the response initiated by antigen recognition generated by the T cell receptor (TCR) are regulated by the balance between co-stimulatory and inhibitory signals (immune checkpoints). Under normal physiological conditions, immune checkpoints are essential for preventing autoimmunity (i.e., maintaining self-tolerance) and protecting tissues from damage when the immune system responds to pathogenic infections. The expression of immune checkpoint proteins can be dysregulated by tumors as an important immune resistance mechanism.

[0244] 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, which can be divided into two categories based on their structural features: i) killer cell immunoglobulin-like receptors (KIRs) and ii) C-type lectin receptors (members of the type II transmembrane receptor family). Other less well-defined immune checkpoints have been described in the literature, 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].

[0245] The present invention contemplates the combination of inhibitors of CD73 function described herein with inhibitors of the above-mentioned immune checkpoint receptors and ligands, as well as immune checkpoint receptors and ligands to be described. Certain immune checkpoint modulators are currently available, while others are in late development. For example, when the fully humanized CTLA4 monoclonal antibody ipilimumab (YERVOY; Bristol-Myers Squibb) was approved for the treatment of melanoma in 2011, it became the first immune checkpoint inhibitor to receive regulatory approval in the United States. Fusion proteins comprising CTLA4 and antibodies (CTLA4-Ig; abatacept (ORENCIA; Bristol-Myers Squibb)) have been used to treat rheumatoid arthritis, and other fusion proteins have been shown to be effective in kidney transplant patients susceptible to Epstein-Barr virus. Antibodies to PD1 are in development (e.g., nivolumab (Bristol-Myers Squibb) and lambrolizumab (Merck)), and anti-PDL1 antibodies (e.g., MPDL3280A (Roche)) are being evaluated. Nivolumab has shown promise in patients with melanoma, lung cancer, and kidney cancer.

[0246] The present invention includes any of the pharmaceutically acceptable salts, acids or derivatives of the above.

[0247] Metabolic and cardiovascular disorders. The present invention provides methods of 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.

[0248] Examples of therapeutic agents that can be used to treat hypercholesterolemia (and atherosclerosis) include statins that inhibit enzymatic synthesis of cholesterol (e.g., CRESTOR, LESCOL, LIPITOR, MEVACOR, PRAVACOL, and ZOCOR); bile acid resins (e.g., COLESTID, LO-CHOLEST, PREVALITE, QUESTRAN, and WELCHOL), which chelate cholesterol and prevent its absorption; ezetimibe (ZETIA), which blocks cholesterol absorption; fibric acid (e.g., TRICOR), which reduces triglycerides and can modestly increase HDL; niacin (e.g., NIACOR), which modestly reduces LDL cholesterol and triglycerides; and / or combinations of the foregoing (e.g., VYTORIN (ezetimibe with simvastatin)). Alternative cholesterol therapeutics that can be used in combination with the CD73 inhibitors described herein include various supplements and herbs (e.g., garlic, polyunsaturated glycol, and guggul).

[0249] The present invention includes any of the pharmaceutically acceptable salts, acids or derivatives of the above.

[0250] Immune-related disorders and disorders with an inflammatory component. The present invention provides methods of treating and / or preventing immune-related diseases, disorders, and conditions; and diseases, disorders, and conditions with an inflammatory component using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0251] Examples of therapeutic agents useful in combination therapy are specific to the underlying disease, disorder or condition, and are known to those skilled in the art.

[0252] Microbial Diseases .The present invention provides methods for treating and / or preventing viral, bacterial, fungal and parasitic diseases, disorders and conditions, and disorders associated therewith, using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent (e.g., one or more other antiviral agents and / or one or more agents unrelated to viral therapy).

[0253] Such combination therapies include antiviral agents that target various stages of the viral life cycle and have different mechanisms of action, including but not limited to the following: viral uncoating inhibitors (e.g., amantadine and levantin); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine); drugs that target integrase; drugs that block the binding of transcription factors to viral DNA; drugs that affect translation (e.g., antisense molecules) (e.g., fomivirsen); drugs that modulate translation / ribonucleoside enzyme function; protease inhibitors; viral assembly regulators (e.g., rifampicin); antiretroviral drugs, 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 drugs that prevent the release of viral particles (e.g., zanamivir and oseltamivir). Treatment and / or prevention of certain viral infections (e.g., HIV) often requires a group ("cocktail") of antiviral agents.

[0254] Other antiviral agents used in combination with CD73 inhibitors include, but are not limited to, the following: abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, lipitor, boceprevirertet, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet, (http: / / en.wikipedia.org / wiki / Fusion_inhibitor), ganciclovir, ibatabine, imunovir, idoxuridine, imiquimod, indinavir ir), inosine, various interferons (such as peginterferon alfa-2a), lopinavir, lofetidine, maraviroc, moroxydine, methisazone, nelfinavir, nexavir, penciclovir, peramivir, pruconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramididine, saquinavir, stavudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, tromantine, Truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

[0255] The present invention contemplates the use of inhibitors of CD73 function described herein in combination with antiparasitic agents. These drugs include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, thiobisdichlorophen, oxamectin, triclosan, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox and nitroimidazole. Those skilled in the art are aware of other agents that can be used to treat parasitic diseases.

[0256] Embodiments of the present invention contemplate the use of CD73 inhibitors described herein in combination with agents useful for treating or preventing bacterial conditions. Antibacterial agents can be classified in various ways, including based on mechanism of action, based on chemical structure, and based on activity spectrum. 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 bacteriostatics, while agents such as aminoglycosides are bactericides. Another way to classify antibacterial agents is based on their targeting specificity; "narrow spectrum" agents target specific types of bacteria (e.g., Gram-positive bacteria such as Streptococcus), while "broad spectrum" agents have activity that targets a wider range of bacteria. Those skilled in the art know the types of antibacterial agents suitable for specific bacterial infections.

[0257] Embodiments of the present invention contemplate the use of a CD73 inhibitor described herein in combination with an agent useful for treating or preventing fungal conditions. 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).

[0258] The present invention includes pharmaceutically acceptable salts, acids or derivatives of the above-mentioned agents (and members of drug classes).

[0259] dose

[0260] The CD73 inhibitors of the present invention can be administered to a subject in an amount that depends on, for example, the target of administration (e.g., the desired resolution), the age, weight, sex, health and physical condition of the subject to whom the formulation is administered; the route of administration; and the nature of the disease, disorder, condition or its symptoms. The dosing regimen can also take into account the presence, nature and extent of any adverse effects associated with the administered agent, and the effective dose and dosage regimen can be readily determined from, for example, safety and dose escalation trials, in vivo studies (e.g., animal models) and other methods known to those skilled in the art.

[0261] Typically, dosing parameters specify a dose that is less than the amount that may be irreversibly toxic to the subject (maximum tolerated dose (MTD)) and not less than the amount required to produce a measurable effect on the subject. These amounts are determined by, for example, pharmacokinetic and pharmacodynamic parameters related to ADME, taking into account the route of administration and other factors.

[0262] The effective dose (ED) is the dose or amount of a pharmaceutical agent that produces a therapeutic response or desired effect in a portion of the subjects taking it. The "median effective dose" or ED50 of a pharmaceutical agent is the dose or amount of the pharmaceutical agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered. Although the ED50 is often used to measure the reasonable expectation of the effect of a drug, a dose that a clinician may consider appropriate after considering all relevant factors may not necessarily be the appropriate dose. Thus, in some cases, the effective amount is greater than the calculated ED50, in other cases, the effective amount is less than the calculated ED50, and in other cases, the effective amount is the same as the calculated ED50.

[0263] In addition, an effective dose of a CD73 inhibitor of the present invention can be an amount that produces a desired result relative to a healthy subject when administered to a subject in one or more doses. For example, for a subject experiencing a particular condition, an effective dose can be an amount that improves a diagnostic parameter, measurement, marker, etc. of the condition 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%, where 100% is defined as a diagnostic parameter, measurement, marker, etc. exhibited by a normal subject.

[0264] In certain embodiments, the CD73 inhibitors contemplated by the invention can be administered (e.g., orally) at a dosage level of about 0.01 mg / kg subject body weight / day to about 50 mg / kg subject body weight / day, or about 1 mg / kg subject body weight / day to about 25 mg / kg subject body weight / day, once or more per day to achieve the desired therapeutic effect.

[0265] For oral administration, the composition can be provided in the form of tablets, capsules, etc. containing 1.0-1000 mg of the active ingredient, specifically 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 mg of the active ingredient.

[0266] In certain embodiments, the desired dose of a CD73 inhibitor is contained in a "unit dosage form". The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined quantity of a CD73 inhibitor, alone or in combination with one or more additional agents, sufficient to produce the 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.

[0267] Reagent test kit

[0268] The present invention also contemplates kits containing CD73 inhibitors and pharmaceutical compositions thereof. As described below, the kit is generally in the form of a physical structure that contains various components and can be used, for example, to implement the above-mentioned methods.

[0269] 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., tablets or capsules) or in a form that requires, for example, reconstitution or dilution prior to administration (e.g., powder). When 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), a buffer, a pharmaceutically acceptable excipient, etc., packaged together with or separately from the CD73 inhibitor. When combined therapy is contemplated, the kit may contain several reagents individually, or they may already be combined in the kit. Each component of the kit may be contained in a separate container, and all of the various containers may be in a single package. The kit of the present invention may be designed to properly maintain the conditions required for the components contained therein (e.g., refrigeration or freezing).

[0270] The kit may include a label or package insert that includes identification information of the components therein and instructions for use (e.g., dosage parameters, clinical pharmacology of the active ingredient, including mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). The label or insert may include manufacturer information, such as a batch number and an expiration date. The label or package insert may, for example, be integrated into a physical structure that houses the components, be contained separately within a physical structure, or be attached to a component of the kit (e.g., an ampoule, tube, or vial).

[0271] The label or insert may additionally be included or incorporated into a computer-readable carrier such as a disk (e.g., hard disk, card, storage disk), an optical disk such as a CD or DVD-ROM / RAM, DVD, MP3, magnetic tape, or a combination of these, an electronic storage carrier such as RAM and ROM, or such as a magnetic / optical storage medium, FLASH medium, or a memory-type storage card. In some embodiments, the actual instructions are not in the kit, but a means for obtaining instructions from a remote source, such as via the Internet, is provided. Example

[0272] The following examples are presented to give those of ordinary skill in the art a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor do they represent that the experiments below have been completed or that they are all experiments that could be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but rather can be performed to generate data of the properties described therein, etc., and efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for.

[0273] 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, including the following: wt = wild type; bp = base pair; kb = kilobase; 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 = micromole; mM = millimole; M = mole; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = daily; BID = twice daily; 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 modification of Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0274] LC: Agilent 1100 sequence; Mass spectrometer: Agilent G6120BA, single quadrupole LC-MS method: Agilent Zorbax Eclipse Plus C18, 4.6×100 mm, 3.5 μM, 35° C., 1.5 mL / min flow rate, 2.5 min gradient wash from 0% to 100% B and 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

[0275] Fast column: ISCO Rf+

[0276] Reverse phase HPLC: ISCO-EZ; column: Kinetex 5μm EVO C18100 A; 250×21.2mm (Phenomenon)

[0277] Example 1

[0278] 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

[0279]

[0280] Step a: A mixture of 2,6-dichloropurine nucleoside (321 mg, 1 mmol), cyclopentylamine (103 μL, 1.05 mmol, 1.05 equiv) and triethylamine (146 μL, 1.05 mmol, 1.05 equiv) in anhydrous EtOH (3 mL) was stirred overnight at 60°C. The reaction mixture was evaporated and the crude product was used in the next step without purification. ESIMS [M+H] + , C 15 H 21 ClN5O4, calculated value 370.8, found value 370.2

[0281] Step b: The product of step a (370 mg, 1 mmol) was dissolved in trimethyl phosphate (5 mL) and cooled to 0 ° C (ice bath), and then a cold solution of trimethyl phosphate (2 mL) of methylenebis(phosphonic acid dichloride) (1.25 g, 5 mmol, 5 equivalents) was added dropwise. The reaction mixture was stirred at 0 ° C for 3 h, then slowly quenched with 0.5 M triethylammonium bicarbonate solution (7 mL) and stirred at 0 ° C for 15 min, and then stirred at room temperature for 2 h. The reaction mixture was purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 30% containing 0.1% TFA) to give the product (181 mg) as a white solid with a yield of 28%: 1 H NMR (400MHz, DMSO) δ8.45-8.32(m,2H),5.85(d,J=5.5Hz,1H),4.55-4.36(m,2H),4.23- 4.07(m,4H),2.26(t,J=20.5Hz,2H),2.04-1.85(m,2H),1.77-1.46(m,6H).ESIMS[M+H] + , C 16 H 25 ClN5O9P2, calculated value 528.8, found value 528.1

[0282] Example 2

[0283] 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

[0284]

[0285] The title compound was synthesized in a manner similar to Example 1, using 4-tert-butylbenzylamine in place of cyclopentylamine: 1 HNMR (400MHz, DMSO-d6) δ8.91(t,J=6.3Hz,1H),8.43(s,1H),7.33(d,J=8.2Hz,2H),7.26(d,J=8.2Hz,2H),5.86(d,J=5. 8Hz,1H),4.68-4.56(m,2H),4.52(t,J=5.4Hz,1H),4.23-4.03(m,4H),2.26(t,J=20.5Hz,2H),1.25(s,9H).ESIMS[M+H] + , C 22 H 31 ClN5O9P2, calculated value 606.1, found value 606.2.

[0286] Example 3

[0287] 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

[0288]

[0289] The title compound was synthesized in a manner similar to Example 1, substituting isopropylamine for cyclopentylamine: 1 H NMR (400MHz, DMSO-d6) δ8.40 (s, 1H), 8.23 ​​(d, J = 8.1Hz, 1H), 5.85 (d, J = 5.9Hz, 1H), 4.51 (t, J = 5.5Hz, 1H),4.36(s,1H),4.24-4.03(m,4H),2.25(t,J=20.5Hz,2H),1.21(dd,J=6.6,2.0Hz,5H).ESIMS[M+H] + , C 14 H 22 ClN5O9P2, calculated value 502.1, found value 502.1

[0290] Example 4

[0291] 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

[0292]

[0293] The title compound was synthesized by a method similar to Example 1, using cyclopropylamine instead of cyclopentylamine: 1 H NMR (400MHz, DMSO-d6) δ8.54(s,1H),8.42(s,1H),5.86(d,J=5.8Hz,1H),4.52(t,J=5.4Hz,1H) ,4.28-4.03(m,4H),2.97(s,1H),2.25(t,J=20.5Hz,2H),0.75(s,2H),0.64(s,3H).ESIMS[M+H] + , C 14 H 20 ClN5O9P2, calculated value 500.1, found value 500.1

[0294] Example 5

[0295] 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

[0296]

[0297] The title compound was synthesized by a method similar to Example 1, using neopentylamine instead of cyclopentylamine: 1 H NMR (400MHz, DMSO-d6) δ8.42(s,1H),8.32(t,J=6.4Hz,1H),5.85(d,J=5.7Hz,1H),4.52(t,J=5.4Hz,1H),4. 31-4.04(m,4H),3.82(d,J=7.0Hz,1H),3.42-3.17(m,2H),2.26(t,J=20.5Hz,2H),0.91(s,9H).ESIMS[M+H] + , C 16 H 26 ClN5O9P2, calculated value 530.1, found value 530.2

[0298] Example 6

[0299] 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

[0300]

[0301] The title compound was synthesized by a method similar to Example 1, using N-methylisopropylamine instead of cyclopentylamine: 1 HNMR(400MHz,DMSO-d6)δ8.42(s,1H),5.88(d,J=5.9Hz,1H),4.50(t,J=5.4Hz,1H),4.22-4.1 7(m,1H),4.11(d,J=6.4Hz,3H),3.03(s,3H),2.26(t,J=20.5Hz,2H),1.23(s,6H).ESIMS[M+H] + , C 15 H 24 ClN5O9P2, calculated value 516.1, found value 516.1

[0302] Example 7

[0303] 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

[0304]

[0305] The title compound was synthesized in a manner similar to Example 1, but using 6-chloropurine ribonucleoside and 3,5-di(trifluoromethyl)benzylamine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.67(s,1H),8.47(s,1H),8.26(s,1H),8.07(s,2H),7.99(s,1H),5.94(d,J=5.7Hz,1H), 4.88(s,2H),4.61(t,J=5.4Hz,1H),4.23(t,J=4.2Hz,1H),4.20-4.04(m,3H),2.25(t,J=20.5Hz,2H).ESIMS[MH] - , C 20 H 20 F6N5O9P2, calculated value 650.1, found value 650.2.

[0306] Example 8

[0307] 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

[0308]

[0309] The title compound was synthesized in a manner similar to Example 1, but using 6-chloropurine ribonucleoside and the corresponding amine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.60(s,1H),8.44(s,1H),8.24(s,1H),7.49(d,J=8.3Hz,2H),7.29(d,J=8.3Hz,2H),5.94(d,J=5. 7Hz,1H),4.67(s,2H),4.61(t,J=5.3Hz,1H),4.23(t,J=4.2Hz,1H),4.19-4.05(m,3H),2.25(t,J=20.5Hz,2H).ESIMS[MH] - , C 18 H 21 BrN5O9P2, calculated value 592.0, found value 592.1.

[0310] Example 9

[0311] 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

[0312]

[0313] The title compound was synthesized in a manner similar to Example 1, but using 6-chloropurine ribonucleoside and the corresponding amine in step a: 1 H NMR (400MHz, 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,2 H),4.60(t,J=5.4Hz,1H),4.23(t,J=4.1Hz,1H),4.20-4.05(m,3H),2.25(t,J=20.5Hz,2H),1.24(s,9H).ESIMS[MH] - , C 22 H 30 N5O9P2, calculated value 570.1, measured value 570.3.

[0314] Example 10

[0315] 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

[0316]

[0317] The title compound was synthesized by a method similar to Example 1, except that 6-chloropurine ribonucleoside and the corresponding amine were used in step a: 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). ESIMS [MH] - , C 24 H 26 N5O9P2, calculated value 590.1, measured value 590.2.

[0318] Embodiment 11

[0319] 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

[0320]

[0321] The title compound was synthesized by a method similar to Example 1, but using 6-chloropurine ribonucleoside and the corresponding amine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.68(s,1H),8.46(s,1H),8.25(s,1H),7.67(d,J=8.1Hz,2H),7.54(d,J=8.1Hz,2H),5.95(d,J=5. 8Hz,1H),4.79(s,2H),4.61(t,J=5.3Hz,1H),4.24(t,J=4.1Hz,1H),4.20-4.06(m,3H),2.25(t,J=20.5Hz,2H).ESIMS[MH] - , C 19 H 21F3N5O9P2, calculated value 582.1, found value 582.2.

[0322] Example 12

[0323] Synthesis of (((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((4-methylbenzyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

[0324]

[0325] The title compound was synthesized in a manner similar to Example 1, but using 6-chloropurine ribonucleoside and the corresponding amine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.61(s,1H),8.45(s,1H),8.26(s,1H),7.22(d,J=7.8Hz,2H),7.10(d,J=7.8Hz,2H),5.94(d,J= 5.7Hz,1H),4.67(s,2H),4.60(t,J=5.4Hz,1H),4.23(t,J=4.2Hz,1H),4.19-4.04(m,3H),2.31-2.18(m,5H).ESIMS[MH] - , C 19 H 24 N5O9P2, calculated value 528.1, measured value 528.2.

[0326] Example 13

[0327] 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

[0328]

[0329] The title compound was synthesized in a manner similar to Example 1, but using 6-chloropurine ribonucleoside and the corresponding amine in step a: 1H NMR (400MHz, DMSO-d6) δ8.61(s,1H),8.46(s,1H),8.26(s,1H),7.48(t,J=2.0Hz,1H),7.39(s,2H),5.95(d,J=5.7 Hz,1H),4.70(s,2H),4.61(t,J=5.4Hz,1H),4.24(t,J=4.2Hz,1H),4.20-4.05(m,3H),2.26(t,J=20.5Hz,2H).ESI MS[MH] - , C 18 H 20 Cl2N5O9P2, calculated value 582.1, found value 582.2.

[0330] Embodiment 14

[0331] 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

[0332]

[0333] 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) was added Pd(PPh3)4 (196 mg, 0.17 mmol, 10 mol%) and the reaction mixture was heated at 120°C overnight. LCMS indicated product formation. It was cooled to room temperature, diluted with water, extracted with ethyl acetate, dried (MgSO4), filtered and concentrated. The residue was purified by flash column to give the product (1 g). ESIMS [M+H] + , C 24 H 27 N5O7, calculated value 498.2, measured value 498.3

[0334] 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 rt for 1 h. Then, it was diluted with dichloromethane and filtered through a pad of silica. The filtrate was concentrated and purified with a flash column (ISCO, 40 g column, 0 to 20% methanol in dichloromethane, 20 min) to give the compound as an off-white solid (450 mg, 60%) ESIMS [M+H] + , C 18 H 21 N5O4, calculated value 372.2, measured value 372.2

[0335] Step c: The product of step b (150 mg, 0.4 mmol) was dissolved in trimethyl phosphate (3 mL) and cooled to 0 ° C (ice bath), and then an ice-cold solution of methylenebis(dichlorophosphine) (504 mg, 2 mmol, 5 equivalents) in trimethyl phosphate (1 mL) was added dropwise. The reaction mixture was stirred at 0 ° C for 3 h, then slowly quenched with 0.5 M triethylammonium bicarbonate solution (8 mL) and stirred at 0 ° C for 15 min, and then stirred at room temperature for 2 h. The reaction mixture was purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 30% containing 0.1% TFA) to give the product as a white solid: 1 H NMR (400MHz, DMSO-d6) δ8.48-8.32(m,2H),7.38-7.18(m,5H),5.92(d,J=6.0Hz,1H),4.71(s, 2H),4.55(t,J=5.5Hz,1H),4.19-3.98(m,4H),2,44(s,3H),2.23(t,J=20.5Hz,2H).ESIMS[MH] - , C 19 H 25 N5O9P2, calculated value 528.1, measured value 528.2.

[0336] Embodiment 15

[0337] 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

[0338]

[0339] Step a: 85°C, N 6 -benzyl-2-chloropurine ribonucleoside (783 mg, 2 mmol), vinylboronic acid pinacol ester (462 mg, 3 mmol, 1.5 eq.), K2CO3 (828 mg, 6 mmol, 3 eq.) and Pd(PPh3)4 in 1,2-dimethoxyethane:H2O (9:1, 10 mL) were stirred under N2 for 1 day. The reaction mixture 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 evaporated to give a yellow solid. The crude product was washed with MTBE (50 mL) and used directly in the next step (550 mg, 72%),

[0340] Step b: The title compound was synthesized by a method similar to Example 1. 1H NMR (400MHz, DMSO-d6) δ8.39(s,1H),7.38(d,J=7.0Hz,2H),7.29(t,J=7.6Hz,2H),7.25-7.15(m,1H),6.64(dd,J=17.2,10.4Hz,1H),6.39(dd,J=17.2 ,2.4Hz,1H),5.94(d,J=6.0Hz,1H),5.55(d,J=10.5Hz,1H),4.73(s,2H),4. 63(t,J=5.5Hz,1H),4.28-4.00(m,4H),2.25(t,J=20.4Hz,2H).ESIMS[M+H] + , C 20 H 26 N5O9P2, calculated value 542.1, measured value 542.2.

[0341] 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. The reaction mixture was vigorously stirred under H2 (balloon) for 2 h, and after filtration, the product was purified by RP18 HPLC (H2O+0.1%TFA / acetonitrile+0.1%TFA) to give a white solid (14 mg, 35%): 1 H NMR (400MHz, DMSO-d6) δ8.52-8.18(m,2H),7.33-7.27(m,2H),7.27-7.18(m,2H),7.15(t,J=7.2Hz,1H),5.86(d,J=6.0Hz,1H),4. 64(s,2H),4.55(t,J=5.5Hz,1H),4.19-3.98(m,4H),2.70-2.61(m,2H),2.16(t,J=20.5Hz,2H),1.16(t,J=7.6Hz,3H).ESIMS[M+H] + , C 20 H 27 N5O9P2, calculated value 544.1, measured value 544.2.

[0342] Example 16

[0343] Synthesis of (((((2R,3S,4R,5R)-5-(2-allyl-6-(benzylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

[0344]

[0345] The title compound was synthesized using a method similar to Example 15: 1 H NMR (400MHz, DMSO-d6) δ8.46(s,1H),8.37(s,1H),7.41-7.34(m,2H),7.30(t,J=7.5Hz,2H),7.25-7.18(m,1H),6.17-6.03(m,1H),5.92(d,J=6 .0Hz,1H),5.28-5.00(m,2H),4.70(s,2H),4.60(t,J=5.6Hz,1H),4.27-4.02(m,4H),3.49(d,J=6.8Hz,2H),2.24(t,J=20.5Hz,2H).ESIMS[M+H] + , C 21 H 28 N5O9P2, calculated value 556.1, measured value 556.3.

[0346] Embodiment 17

[0347] 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

[0348]

[0349] The title compound was synthesized by a method similar to that of Example 15, step c: 1 H NMR (400MHz, DMSO-d6) δ8.40(s,2H),8.29(s,1H),7.29(d,J=7.6Hz,2H),7.23(t,J=7.5Hz,2H),7.15(t,J=7.2Hz,1H),5.86(d,J=6.0Hz,1H),4.64 (s,2H),4.54(t,J=5.5Hz,1H),4.19-3.94(m,4H),2.71-2.55(m,2H),2.1 7(t,J=20.5Hz,2H),1.66(q,J=7.4Hz,2H),0.93-0.70(m,3H).ESIMS[M+H] + , C 21 H 30 N5O9P2, calculated value 558.1, measured value 558.2.

[0350] Embodiment 18

[0351] 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

[0352]

[0353] Step a: The known riboside (250 mg, 0.64 mmol) was dissolved in a 25% NaOMe solution in MeOH (2 mL) and stirred at 60 °C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with H2O (15 mL) and acetic acid to neutral pH. The product was collected by filtration (white solid, 180 mg, 73%). ESIMS [M+H] + , C 18 H 22 N5O5, calculated value 388.4, measured value 388.1.

[0354] Step b: The title compound was obtained by a method similar to Example 1 to give a white solid (37 mg, 14%): 1 HNMR (400MHz, DMSO) δ8.48 (s, 1H), 8.20 (s, 1H), 7.37-7.17 (m, 5H), 5.82 (d, J = 5.9Hz, 1H), 4.64(d,J=5.0Hz,3H),4.28-4.00(m,4H),3.80(s,3H),2.23(t,J=20.5Hz,2H).ESIMS[M+H] + , C 19 H 26 N5O 10 P2, calculated value 546.4, measured value 546.1.

[0355] Embodiment 19

[0356] 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

[0357]

[0358] Step a: The known riboside (250 mg, 0.64 mmol) was dissolved in a 40% MeNH2 solution in H2O (2 mL) and stirred at 60°C overnight. The reaction mixture was then concentrated under reduced pressure and the residue was diluted with H2O (15 mL). The product was collected by filtration (white solid, 210 mg, 85%), ESIMS [M+H] + , C18 H 23 N6O4, calculated value 387.4, measured value 387.3.

[0359] Step b: The title compound was obtained by a method similar to Example 1 to give a white solid (38 mg, 15%): 1 HNMR (400MHz, DMSO) δ8.08 (s, 1H), 7.42-7.19 (m, 5H), 5.79 (d, J = 6.1Hz, 1H), 4.75 -4.45(m,3H),4.24-4.02(m,4H),2.81(s,3H),2.22(t,J=20.4Hz,2H).ESIMS[MH] - , C 19 H 26 N6O9P2, calculated value 543.4, found value 543.2.

[0360] Embodiment 20

[0361] 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

[0362]

[0363] The title compound was synthesized in a similar manner to Example 19, but using dimethylamine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.15(s,1H),8.09(s,1H),7.36(d,J=7.2Hz,2H),7.29(t,J=7.5Hz,2H),7.21(t,J=7.2Hz,1H),5.81 (d,J=5.5Hz,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=20.4Hz,2H).ESIMS[M+H] + , C 20 H 29 N6O9P2, calculated value 559.1, found value 559.2.

[0364] Embodiment 21

[0365] 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

[0366]

[0367] The title compound was synthesized in a similar manner to Example 19, but using pyrrolidine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.15 (s, 2H), 7.42-7.14 (m, 5H), 5.82 (d, J = 5.5Hz, 1H), 4.71-4.51 (m, 3H), 4. 26(t,J=4.3Hz,1H),4.21-4.00(m,3H),3.46(s,4H),2.23(t,J=20.4Hz,2H),1.89(s,4H).ESIMS[M+H] + , C 22 H 31 N6O9P2, calculated value 585.1, measured value 585.2.

[0368] Embodiment 22

[0369] 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

[0370]

[0371] The title compound was synthesized in a similar manner to Example 19, but using piperidine in step a: 1 H NMR(400MHz, DMSO-d6)δ8.20(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=5.6Hz,1H),4 .66-4.52(m,3H),4.20(t,J=4.3Hz,1H),4.17-4.00(m,3H),3.74-3.62(m,4H),2.24(t,J=20.5Hz,2H),1.64-1.38(m,6H).ESIMS[MH] - , C 23 H 31 N6O9P2, calculated value 597.2, found value 597.3.

[0372] Embodiment 23

[0373] Synthesis of (((((2R,3S,4R,5R)-5-(6-(Benzylamino)-2-morpholin-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

[0374]

[0375] The title compound was synthesized in a similar manner to Example 19, but using morpholine in step a: 1 H NMR (400MHz, DMSO-d6) δ8.24-8.02(m,2H),7.37-7.17(m,5H),5.79(d,J=5.9Hz,1H), 4.72-4.51(m,3H),4.23-3.99(m,4H),3.61(s,8H),2.23(t,J=20.5Hz,2H).ESIMS[MH] - , C 22 H 29 N6O 10 P2, calculated value 599.2, measured value 599.3

[0376] Embodiment 24

[0377] 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

[0378]

[0379] Step a: A solution of compound X (5 g, 10.2 mmol) in MeOH (60 ml) was treated with ammonia 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. The mixture was concentrated and purified by preparative HPLC to give the desired product (750 mg, 20%).

[0380] Step b: The product of step a (0.36 g, 1 mmol), benzylamine (0.115 mL, 1.05 mmol, 1.05 equiv) and Et3N (0.15 mL, 1.1 mmol, 1.1 equiv) in anhydrous EtOH (3.3 mL) were stirred at 70° C. for 4 hours. The reaction mixture was then cooled to room temperature, concentrated and used without further purification.

[0381] Step c: The product of step b was dissolved in trimethyl phosphate (5 mL) and cooled to 0°C (ice bath), and then a cold solution of methylenebis(phosphonodichloride) (1.2 g, 15 mmol, 5 equivalents) in trimethyl phosphate (3 mL) was added dropwise. The reaction mixture was stirred at 0°C for 3 h, then slowly quenched with 0.5 M triethylammonium bicarbonate solution (6 mL) and stirred at 0°C for 15 min, then stirred at room temperature for 2 h. The reaction mixture was purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 40% containing 0.1% TFA) to give the product (38 mg) as a white solid, 6% yield: 1 H NMR (400MHz, DMSO-d6) δ8.53(s,1H),8.27(s,1H),7.37-7.17(m,5H),5.84(d,J=5.8Hz,1H),4.65(s,2H),4.56(t,J=5.5Hz ,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=6.8Hz,6H)..ESIMS[M+H] + , C 21 H 29 N5O9P2S, calculated value 590.1, measured value 590.2

[0382] Embodiment 25

[0383] 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

[0384]

[0385] Step a: The product of Example 24, step a (4.5 g, 12.5 mmol) in dichloromethane (50 mL) was treated with m-CPBA (2.2 g, 38.2 mmol) in batches. The reaction was stirred at room temperature until completion. The mixture was diluted with dichloromethane (200 mL), washed twice with aqueous NaHSO3, dried over Na2SO4, and concentrated. The residue was purified by prep-HPLC to give the desired product as a white solid (780 mg, 16%).

[0386] Step b: The product of step a (0.393 g, 1 mmol), benzylamine (0.115 mL, 1.05 mmol, 1.05 eq.) and Et3N (0.15 mL, 1.1 mmol, 1.1 eq.) in anhydrous EtOH (3.3 mL) were stirred at 70° C. for 4 hours. The reaction mixture was then cooled to room temperature, concentrated and used without further purification.

[0387] Step c: The product of step b was dissolved in trimethyl phosphate (4 mL) and cooled to 0°C (ice bath), and then a cold solution of methylenebis(phosphonodichloride) (1.2 g, 5 mmol, 5 equivalents) in trimethyl phosphate (2 mL) was added dropwise. The reaction mixture was stirred at 0°C for 3 h, then slowly quenched with 0.5 M triethylammonium bicarbonate solution (6 mL) and stirred at 0°C for 15 min, then stirred at room temperature for 2 h. The reaction mixture was purified by reverse phase HPLC (C18 column, 0 to 40% gradient of acetonitrile and water containing 0.1% TFA) to give the product (50 mg) as a white solid, 22% yield: 1 H NMR (400MHz, DMSO-d6) δ9.21(t,J=6.2Hz,1H),8.66(s,1H),7.42-7.15(m,5H),5.97(d,J=6.1Hz,1H),4.74-4.66(m,2H),4.60(dd,J=6.1, 5.0Hz,1H),4.26-4.22(m,1H),4.19-4.07(m,4H),3.78(p,J=6.8Hz,1H),2.26(t,J=20.5Hz,2H),1.12(dd,J=6.8,2.4Hz,6H).ESIMS[M+H] + , C 21 H 29 N5O 11 P2S, calculated value 622.1, measured value 622.2

[0388] Embodiment 26

[0389] 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

[0390]

[0391] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1H NMR(400MHz, DMSO-d6)δ8.17(s,1H),8.02-7.72(m,2H),6.15(d,J=4.3Hz,1H),4.30-4.09(m,4H),4.00-3.88(m,1H),2.24(t,J=20.5Hz,2H).ESIMS[M+H] + , C 11 H 17 FN5O9P2, calculated value 444.0, found value 444.1.

[0392] Embodiment 27

[0393] 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

[0394]

[0395] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1 H NMR (400MHz, DMSO-d6) δ8.45(s,1H),8.26(s,1H),7.92(s,2H),6.27(dd,J=17.2,2.8Hz,1H),5.50(dd d,J=52.5,4.5,2.8Hz,1H),4.64-4.52(m,1H),4.29-4.08(m,3H),2.25(t,J=20.4Hz,2H).ESIMS[M+H] + , C 11 H 16 FN5O8P2, calculated value 428.1, measured value 428.1.

[0396] Embodiment 28

[0397] 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

[0398]

[0399] The title compound was synthesized by a method similar to Example 1, using commercially available alcohol, 1H NMR (400MHz, DMSO-d6) δ8.28(d,J=2.2Hz,1H),7.92(s,2H),6.36(dd,J=14.3,4.6Hz,1H),5.26(dt,J=52.5,4.3Hz, 1H), 4.51 (dt, J=18.6, 4.7Hz, 1H), 4.19 (t, J=6.0Hz, 2H), 4.04 (t, J=5.0Hz, 1H), 2.26 (t, J=20.5Hz, 2H); MS: (ES) m / z C 11 H 15 Calculated value of ClFN5O8P2 [MH] - 460.1, measured value 460.1.

[0400] Embodiment 29

[0401] 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

[0402]

[0403] Step a: 2,6-Dichloropurine (3.6 g, 18.8 mmol) was dissolved in 90 mL of acetonitrile and treated with Cs2CO3 (7.5 g, 23 mmol, 1.2 eq.). The mixture was stirred at room temperature for 30 min. The known bromo derivative (8.75 g, 21 mmol, 1.1 eq.) was dissolved in 100 mL of acetonitrile and added dropwise to the mixture through an addition funnel. The mixture was stirred at room temperature overnight. The mixture was filtered on a silica pad and concentrated. The residue was absorbed with silica and purified by column chromatography (hexane / ethyl acetate) to give the product as a white solid (7.72 g), 77% yield, 1 HNMR (400MHz, chloroform-d) δ8.39 (d, J=3.0Hz, 1H), 8.10 (ddt, J=8.5, 3.1, 0.9Hz, 4H), 7.74-7.36 (m, 6H), 6.64 (dd, J=21.8, 2. 8Hz,1H),5.83-5.69(m,1H),5.40(ddd,J=49.9,2.8,0.8Hz,1H),4.89-4.77(m,2H),4.62(q,J=4.0Hz,1H).ESIMS[M+H] + , C 24 H 17 Cl2FN4O5, calculated value 531.1, found value 531.1.

[0404] Step b: The product of step a (9.0 g, 17 mmol), benzylamine (3 mL, 26 mmol, 1.5 eq.) and Et3N (5 mL, 34 mmol, 2.0 eq.) in anhydrous EtOH (60 mL) were stirred at 70 ° C for 4 hours. The reaction mixture was then cooled to room temperature, the product was collected by filtration, and used for further purification (white solid, 8.9 g, 87%), ESIMS [M+H] + , C 31 H 25 ClFN5O5, calculated value 602.2, found value 602.0.

[0405] The above product (10.2 g, 17 mmol) and K2CO3 (7 g, 51 mmol, 3 equiv) were dissolved in 170 mL methanol and stirred at room temperature for 4 hours. The reaction mixture was then filtered and concentrated on a silica gel pad. The reaction mixture was purified by column chromatography (dichloromethane / methanol) to give the product (5.3 g) as a white solid, 80% yield: 1 H NMR (400MHz, DMSO-d6) δ8.97(t,J=6.3Hz,1H),8.31(d,J=2.0Hz,1H),7.36-7.18(m,5H),6.34(dd,J=13.6,4.7Hz,1H),5.23(dt,J=52.6,4. 3Hz,1H),4.66(q,J=7.3,5.7Hz,2H),4.43(dt,J=19.0,4.8Hz,1H),3.84(q,J=4.9Hz,1H),3.65(tq,J=12.0,6.2,5.2Hz,2H).).ESIMS[M+H] + , C 17 H 18 ClFN5O3, calculated value 394.1, found value 394.1.

[0406] Step c: The product of step b (800 mg, 2 mmol) was dissolved in trimethyl phosphate (15 mL) and cooled to 0°C (ice bath), and then a cold solution of methylenebis(phosphonodichloride) (2.5 g, 10 mmol, 5 equivalents) in trimethyl phosphate (5 mL) was added dropwise. The reaction mixture was stirred at 0°C for 3 h, then slowly quenched with 0.5 M triethylammonium bicarbonate solution (15 mL) and stirred at 0°C for 15 min, then stirred at room temperature for 2 h. The reaction mixture was purified by reverse phase HPLC (C18 column, 0-40% gradient of acetonitrile and water, containing 0.1% TFA) to give the product (290 mg) as a white solid, 22% yield: 1H NMR (400MHz, DMSO-d6) δ8.99(t,J=6.3Hz,1H),8.30(d,J=2.2Hz,1H),7.40-7.18(m,5H),6.38(dd,J=14.3,4.6Hz,1H),5.45-5.04 (m,1H),4.65(t,J=5.5Hz,2H),4.54-4.42(m,1H),4.19(t,J=6.1Hz,2H),4.04(t,J=5.1Hz,1H),2.26(t,J=20.5Hz,2H).ESIMS[MH] - , C 18 H 21 ClFN5O8P2, calculated value 550.8, found value 550.2.

[0407] Embodiment 30

[0408] 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

[0409]

[0410] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400 MHz, 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, 4.5 Hz, 1H), 4.19 (t, J = 6.1 Hz, 2H), 4.05 (q, J = 5.0 Hz, 1H), 3.65 (s, 1H), 3.11 (s, 2H), 2.26 (t, J = 20.5 Hz, 2H). ESIMS [M+H] + , C 19 H 23 ClFN5O8P2, calculated value 566.1, found value 566.2

[0411] Example 31a

[0412] 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

[0413]

[0414] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.36(q,J=4.6Hz,1H),8.27(s,1H),6.45(brs,2H),6.37(dd,J=14.3,4.6Hz,1H),5.25(dt,J=52.4,4.3Hz,1H),4.50( dt,J=18.6,4.6Hz,1H),4.19(t,J=5.9Hz,2H),4.04(q,J=5.2Hz,1H),3.33(brs,1H),2.93(d,J=4.5Hz,3H),2.26(t,J=20.4Hz,2H).ESIMS[MH] - , C 12 H 17 ClFN5O8P2, calculated value 474.7, found value 474.1.

[0415] Example 31b

[0416] 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

[0417]

[0418] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1HNMR (400MHz, DMSO-d6) δ8.43(t,J=5.7Hz,1H),8.26(s,1H),7.28(brs,2H),6.37(dd,J=14.3,4.6Hz,1H),5.25(dt,J=52.4,4.3Hz,1H),4.50(dt,J=1 8.5,4.6Hz,1H),4.19(t,J=6.1Hz,2H),4.03(q,J=5.1Hz,1H),3.87(brs,1 H),3.45(m,1H),2.27(t,J=20.5Hz,2H),1.17(t,J=7.2Hz,3H).ESIMS[M+H] + , C 13 H 19 ClFN5O8P2, calculated value 490.7, found value 490.1.

[0419] Embodiment 32

[0420] 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

[0421]

[0422] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.27(m,2H),6.37(d,J=13.9Hz,1H),5.28(brs,2H),5.25(d,J=52.1Hz,1H),4.98(brs,1H),4.51(d,J =18.3Hz,1H),4.35(sept,J=7.9Hz,1H),4.19(m,2H),4.04(m,1H),2.26(t,J=20Hz,2H),1.21(dd,J=6.6,2.1Hz,6H).ESIMS[MH] - , C 14 H 21 ClFN5O8P2, calculated value 502.7, found value 502.2.

[0423] Embodiment 33

[0424] (((((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

[0425]

[0426] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.59(s,1H),8.28(d,J=2.1Hz,1H),6.38(dd,J=14.2,4.6Hz,1H),5.26(ddd,J=52.5,4.3,4.3Hz,1H),4.51 (dt,J=18.5,4.5Hz,1H),4.19(t,J=6.1Hz,2H),4.03(q,J=5.0Hz,1H),2.98(s,1H),2.36-2.15(m,2H),0.82-0.48(m,4H).ESIMS[MH] - , C 14 H 18 ClFN5O8P2, calculated value 500.03, found value 500.0.

[0427] Embodiment 34

[0428] 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

[0429]

[0430] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR(400MHz,DMSO-d6)δ8.54(s,1H),8.28(s,1H),6.37(dd,J=14.2,4.6Hz,1 H),5.25(ddd,J=52.5,4.3,4.3Hz,1H),4.54-4.47(m,1H),4.19(t,J=6.3Hz,2H ),4.05-4.01(m,1H),3.81-3.74(m,1H),3.30-3.27(m,1H),2.26(dd,J=20.5, 20.5Hz,2H),1.1-1.3(m,1H),0.48-0.37(m,2H),0.28-0.26(m,2H).ESIMS[MH] - , C 15 H 20 ClFN5O8P2, calculated value 514.1, found value 514.0.

[0431] Embodiment 35

[0432] 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

[0433]

[0434] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.41(d,J=7.8Hz,1H),8.27(s,1H),6.37(dd,J=14.4,4.6Hz,1H),5.25(dt,J=52.4,4.3Hz,1H),4.55-4. 37(m,2H),4.19(t,J=6.1Hz,2H),4.03(q,J=5.1Hz,1H),2.26(t,J=20.5Hz,2H),1.93(s,2H),1.64(d,J=62.5Hz,6H).ESIMS[M+H] + , C 16 H 23 ClFN5O8P2, calculated value 530.1, found value 530.2

[0435] Embodiment 36

[0436] 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

[0437]

[0438] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1HNMR(400MHz,DMSO-d6)δ8.66(d,J=6.2Hz,1H),8.31(s,1H),6.38(dd,J=14.3,4 .6Hz,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=6.1Hz,2H),4.04(q,J=5.0Hz,1H),3.89(dt,J=15.3,7.8Hz,2H), 3.73(q,J=7.8Hz,1H),3.61(dd,J=8.9,4.4Hz,1H),2.36-1.99(m,4H).ESIMS[MH] - , C 15 H 20 ClFN5O9P2, calculated value 530.04, found value 530.1.

[0439] Embodiment 37

[0440] 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

[0441]

[0442] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR(400MHz,DMSO-d6)δ8.65(d,J=6.8Hz,1H),8.31(s,1H),6.39(dd,J=14.2, 4.6Hz,1H),5.26(ddd,J=52.4,4.3,4.3Hz,1H),4.69-4.56(m,1H),4.51(dt,J= 18.6,4.6Hz,1H),4.20(t,J=6.1Hz,2H),4.04(q,J=5.0Hz,1H),3.89(dt,J=18. 6,7.9Hz,2H),3.74(q,J=7.8Hz,1H),3.67-3.54(m,1H),2.35-1.90(m,4H).ESI MS[MH] - , C 15 H 20 ClFN5O9P2, calculated value 530.04, found value 530.1.

[0443] Embodiment 38

[0444] 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

[0445]

[0446] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR(400MHz,DMSO-d6)δ8.47-8.34(m,1H),8.30(s,1H),6.37(dd,J=14.1,4 .8Hz,1H),5.25(ddd,J=52.4,4.3,4.3Hz,1H),4.92-4.65(m,1H),4.59-4.39( m,1H),4.19(t,J=6.2Hz,2H),4.03(q,J=5.1Hz,1H),3.89(d,J=11.3Hz,2H),3 .41(t,J=11.4Hz,2H),2.26(dd,J=20.5Hz,2H),1.92-1.45(m,4H).ESIMS[MH] - , C 16 H 22 ClFN5O9P2, calculated value 544.06, found value 544.1.

[0447] Embodiment 39

[0448] 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

[0449]

[0450] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1HNMR (400MHz, DMSO-d6) δ8.27(d,J=2.1Hz,1H),6.39(dd,J=14.1,4.7Hz,1H),5.26(dt,J=52.5,4.3Hz,1H),4.50(dt,J=18.5,4.6Hz,1H),4.19(t, J=5.9Hz,2H),4.05(q,J=5.3,4.1Hz,3H),3.60(t,J=6.8Hz,2H),2.27(t,J=20.5Hz,2H),2.01(p,J=6.7Hz,2H),1.92(q,J=6.7Hz,2H).ESIMS[M+H] + , C 15 H 21 ClFN5O8P2, calculated value 516.1, found value 516.1

[0451] Embodiment 40

[0452] 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

[0453]

[0454] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.30(d,J=2.2Hz,1H),6.39(dd,J=14.3,4.6Hz,1H),5.26(dt,J=52.4,4.3Hz,1H),4.50(dt,J=18.4,4. 6Hz,1H),4.19(t,J=6.0Hz,2H),4.04(q,J=5.0Hz,1H),3.88(m,2H),2.27(t,J=20.5Hz,2H),1.64(d,J=31.0Hz,8H).ESIMS[M+H] + , C 16 H 23 ClFN5O8P2, calculated value 530.1, found value 530.2

[0455] Embodiment 41

[0456] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-morpholin-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

[0457]

[0458] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.34(d,J=2.1Hz,1H),6.41(dd,J=13.9,4.6Hz,1H),5.27(dt,J=52.5,4.3Hz,1H),4.51(dt,J =18.5,4.6Hz,1H),4.19(t,J=5.8Hz,2H),4.04(q,J=5.1Hz,1H),3.79-3.67(m,5H),2.26(t,J=20.5Hz,2H).ESIMS[M+H] + , C 15 H 21 ClFN5O9P2, calculated value 532.1, found value 532.1

[0459] Embodiment 42

[0460] 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

[0461]

[0462] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.37(d,J=2.1Hz,1H),7.48(dt,J=9.9,4.7Hz,2H),7.43-7.28(m,2H),6.44(dd,J=13.8,4.7Hz,1H),5.41(s,2H),5.29(d t,J=52.6,4.4Hz,1H),4.98(s,2H),4.54(dt,J=18.7,4.7Hz,1H),4.21(t,J=5.9Hz,2H),4.05(q,J=4.9Hz,1H),2.27(t,J=20.5Hz,2H).ESIMS[M+H] + , C 19 H 21 ClFN5O8P2, calculated value 564.1, found value 564.1

[0463] Embodiment 43

[0464] 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

[0465]

[0466] The title compound was obtained by the same method as in Example 29, using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ9.01(t,J=6.2Hz,1H),8.32(d,J=2.1Hz,1H),7.64-7.08(m,4H),6.38(dd,J=14.3,4.6Hz,1H),5.26(dt,J=52.5,4.3 Hz,1H),4.64(q,J=7.3,5.4Hz,2H),4.51(dt,J=18.7,4.6Hz,1H),4.28-4.11(m,2H),4.04(q,J=5.1Hz,1H),2.27(t,J=20.5Hz,2H).ESIMS[MH] - , C 18 H 20 Cl2FN5O8P2, calculated value 584.0, found value 584.1.

[0467] Embodiment 44

[0468] 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

[0469]

[0470] The title compound was obtained by the same method as in Example 29, using the product of step a of Example 29 and the corresponding amine: 1 HNMR(400MHz, DMSO-d6)δ9.00(t,J=6.3Hz,1H),8.31(d,J=2.2Hz,1H),7.52-7.24(m,3H),7.23-7.01(m,2H),6.38(dd,J=14.3,4.6Hz, 1H),5.26(dt,J=52.4,4.3Hz,1H),4.72-4.55(m,2H),4.20(t,J=6.0Hz,3H),4.04(q,J=5.1Hz,1H),2.27(t,J=20.5Hz,2H).ESIMS[MH]- , C 18 H 20 ClF2N5O8P2, calculated value 568.0, found value 568.2.

[0471] Embodiment 45

[0472] 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

[0473]

[0474] The title compound was synthesized by a similar method to Example 29 to give a white solid (87.1 mg; 31%) using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ8.96(t,J=6.3Hz,1H),8.30(d,J=2.2Hz,1H),7.20(t,J =7.5Hz,1H),7.17-7.10(m,2H),7.04(d,J=7.4Hz,1H),6.38(dd,J=14.3,4.6Hz, 1H),5.25(dt,J=52.4,4.3Hz,1H),4.68-4.56(m,2H),4.51(dt,J=18.4,4.6Hz, 1H),4.19(t,J=6.0Hz,2H),4.03(q,J=5.1Hz,1H),2.35-2.17(m,2H).ESIMS[MH] - , C 19 H 22 ClFN5O8P2, calculated value 564.1, found value 564.2.

[0475] Embodiment 46

[0476] 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

[0477]

[0478] The title compound was synthesized by a similar method to Example 29 to give a white solid (65.1 mg; 23%) using the product of step a of Example 29 and the corresponding amine: 1H NMR(400MHz, DMSO-d6)δ9.02(t,J=6.3Hz,1H),8.32(d,J=2.2Hz,1H),7.42-7.32( m,1H),7.17(t,J=9.2Hz,2H),7.07(td,J=8.4,2.2Hz,1H),6.39(dd,J=14.4,4.6H z,1H),5.26(dt,J=52.5,4.3Hz,1H),4.74-4.60(m,2H),4.51(dt,J=18.5,4.7Hz, 1H),4.26-4.13(m,2H),4.04(q,J=5.0Hz,1H),2.27(t,J=20.5Hz,2H).ESIMS[MH] - , C 18 H 19 ClF2N5O8P2, calculated value 568.0, found value 568.2

[0479] Embodiment 47

[0480] 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

[0481]

[0482] The title compound was synthesized by a similar method to Example 29 to give a white solid (70.6 mg; 24%) using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ9.03(t,J=6.2Hz,1H),8.33(d,J=2.2Hz,1H),7.45-7.27(m,4H),6.39(dd,J=14.4,4.6Hz,1H),5.26(dt,J=52.4,4. 2Hz,1H),4.74-4.58(m,2H),4.51(dt,J=18.5,4.6Hz,1H),4.20(t,J=6.1Hz,2H),4.04(q,J=5.1Hz,1H),2.27(t,J=20.5Hz,2H).ESIMS[MH] - , C 18 H 19 Cl2FN5O8P2, calculated value 584.0, found value 584.0

[0483] Embodiment 48

[0484] 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

[0485]

[0486] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.99(t,J=6.1Hz,1H),8.35(s,1H),7.47(dd,J=6.0,3.3Hz,1H),7.35-7.22(m,3H),6.40(dd,J=14.2,4.6Hz,1H),5.27(dt, J=52.4,4.3Hz,1H),4.73(d,J=5.2Hz,2H),4.52(d,J=18.5Hz,1H),4.20(t,J=6.2Hz,2H),4.05(q,J=5.1Hz,1H),2.27(t,J=20.5Hz,2H).ESIMS[M+H] + , C 18 H 20 Cl2FN5O8P2, calculated value 586.0, found value 586.1

[0487] Embodiment 49

[0488] 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

[0489]

[0490] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1HNMR(400MHz,DMSO-d6)δ8.32(d,J=37.7Hz,1H),7.55-7.46(m,1H),7.31(bs,2H),7.15(bs,1H),6.41(d,J=14.4Hz,1H),5.61(bs,1H),5.26 (d,J=52.6Hz,1H),5.00(b,1H),4.49(bs,1H),4.17(bs,2H),4.03(bs,1H),3.70(bs,1H),3.18(bs,2H),2.25(t,J=20.4Hz,2H).ESIMS[M+H] + , C 19 H 24 Cl2N5O9P2, calculated value 600.0, found value 600.1.

[0491] Embodiment 50

[0492] 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

[0493]

[0494] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR(400MHz,DMSO-d6)δ9.13(s,1H),8.66(d,J=5.7Hz,2H),8.37(s,1H),7.65(d,J=5.6Hz,2H),6.40(dd,J=14.0,4.6Hz,1H),5.40 -5.08(m,1H),4.80(d,J=6.1Hz,2H),4.53(d,J=18.3Hz,1H),4.19(s,2H),4.04(d,J=5.2Hz,1H),2.25(t,J=20.4Hz,2H).ESIMS[M+H] + , C 17 H 20 ClFN6O8P2, calculated value 553.1, found value 553.2

[0495] Embodiment 51

[0496] 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

[0497]

[0498] The title compound was synthesized in a manner analogous to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 HNMR (400MHz, DMSO-d6) δ8.50(t,J=5.7Hz,1H),8.27(s,1H),7.38-7.13(m,5H),6.37(dd,J=14.4,4.7Hz,1H),5.25(dt,J=52.4,4.2Hz,1H),4.51(dt ,J=18.5,4.6Hz,1H),4.19(t,J=6.1Hz,2H),4.04(t,J=5.1Hz,1H),3.66(d,J=7.2Hz,2H),2.92(t,J=7.5Hz,2H),2.26(t,J=20.5Hz,2H).ESIMS[M+H] + , C 19 H 23 ClFN5O8P2, calculated value 566.1, found value 566.1

[0499] Embodiment 52

[0500] 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

[0501]

[0502] The title compound was synthesized in a similar manner to Example 29, substituting 6-chloro-2-methylpurine for 2,6-dichloropurine: 1 H NMR (400MHz, DMSO-d6) δ8.51(s,1H),8.23(s,1H),7.44-7.19(m,5H),6.44(dd,J=15.0,4.6Hz,1H),5.41-5.13(m,1H),4.72(s,2 H),4.53(dd,J=18.4,4.7Hz,1H),4.19(t,J=6.1Hz,2H),4.04(t,J=5.1Hz,1H),2.46(s,3H),2.26(t,J=20.5Hz,2H).ESIMS[M+H] + , C 19 H 24 FN5O8P2, calculated value 532.1, measured value 532.2

[0503] Embodiment 53

[0504] 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

[0505]

[0506] The title compound was synthesized in a similar manner to Example 29, substituting 6-chloro-2-methylpurine for 2,6-dichloropurine and cyclopentylamine for benzylamine: 1 H NMR (400MHz, DMSO-d6) δ8.56(s,1H),8.30(s,1H),6.45(dd,J=14.4,4.6Hz,1H),5.25(dt,J=52.5,4.3Hz,1H),4.53(dt,J=18.3 ,4.5Hz,1H),4.20(t,J=6.1Hz,2H),4.04(q,J=5.0Hz,1H),2.26(t,J=20.5Hz,2H),1.98(s,2H),1.82-1.46(m,6H).ESIMS[M+H] + , C 17 H 26 FN5O8P2, calculated value 510.1, measured value 510.2

[0507] Embodiment 54

[0508] 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

[0509]

[0510] The title compound was synthesized in a similar manner to Example 29, substituting 6-chloro-2-trifluoromethylpurine for 2,6-dichloropurine: 1H NMR(400MHz, DMSO-d6)δ9.11(d,J=6.3Hz,1H),8.49(d,J=2.1Hz,1H),7.39-7.35(m, 2H),7.34-7.27(m,2H),7.25-7.20(m,1H),6.48(dd,J=14.0,4.7Hz,1H),5.30(dt,J =52.4,4.3Hz,1H),5.20(bs,1H),4.70(t,J=5.7Hz,1H),4.56(dt,J=18.6,4.7Hz,1H ),4.21(t,J=6.2Hz,2H),4.06(q,J=5.1Hz,1H),2.26(t,J=20.5Hz,2H).ESIMS[M+H] + , C 19 H 21 F4N5O8P2, calculated value 586.1, found value 586.2.

[0511] Embodiment 55

[0512] 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

[0513]

[0514] The title compound was synthesized in a similar manner to Example 29, substituting 6-chloro-2-trifluoromethylpurine for 2,6-dichloropurine and cyclopentylamine for benzylamine: 1 H NMR(400MHz, DMSO-d6)δ8.59-8.40(m,2H),6.47(dd,J=13.9,4.7Hz,1H),5.30(dt,J=52.4,4.3Hz,1H),5.11(bs,1H),4.52(dd,J=28.1 ,14.1Hz,2H),4.21(t,J=6.0Hz,2H),4.06(q,J=5.2Hz,1H),2.26(t,J=20.4Hz,2H),2.08-1.90(m,2H),1.80-1.50(m,6H).ESIMS[M+H] + , C 17 H 23 F4N5O8P2, calculated value 564.2, measured value 564.1

[0515] Embodiment 56

[0516] 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

[0517] Step a

[0518]

[0519] Step a: The product of Example 29, step b (1) (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:H2O (10.3 mL). This mixture was degassed with N2 bubbling for 10 minutes. Pd(PPh3)4 (144 mg, 0.13 mmol) was then added and the resulting mixture was further degassed for 5 minutes then sealed and heated at 80°C overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with water and brine. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material consisted of a mixture of mono- and bis-debenzoylated products which were used directly in step b.

[0520] Step b: The product of 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 organics were washed with brine and then dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 0 to 10% gradient of MeOH and CH2Cl2) to give a white solid (41 mg, 8% over two steps). ESIMS [M+H] + , C 23 H 22 FN5O3, calculated value 436.2, measured value 436.3.

[0521] Step c: The title compound was obtained by the same method as in Example 1 to give a white solid: 1H NMR (400MHz, DMSO-d6) δ8.58 (s, 1H), 8.44-8.32 (m, 2H), 8.29 (d, J = 2.4Hz, 1H ),7.40-7.50(m,5H),7.31(dd,J=8.3,6.9Hz,2H),7.24-7.15(m,1H),6.59(d d,J=15.4,4.6Hz,1H),5.30(dt,J=52.4,4.1Hz,1H),4.82(s,2H),4.69-4.48 (m, 1H), 4.22 (d, J = 6.6Hz, 2H), 4.08 (q, J = 5.1Hz, 1H), 2.27 (t, J = 20.5Hz, 2H). ESIMS[MH] - , C 24 H 26 FN5O8P2, calculated value 592.1, measured value 592.2.

[0522] Embodiment 57

[0523] 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

[0524]

[0525] Step a: The product of step b (1) of Example 29 (391 mg, 0.659 mmol), potassium benzyl trifluoroborate (391 mg, 1.98 mmol) and cesium carbonate (1.07 g, 3.30 mmol) were suspended in 20:1 THF:H2O (6.5 mL). This mixture was degassed with N2 bubbling for 10 minutes. Pd(PPh3)2Cl2 (96 mg, 0.132 mmol) was then added and the resulting mixture was further degassed for 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 phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, EtOAc / hexanes) as a beige solid (174 mg, 40%).

[0526] Step b: The product of 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 hours and then partitioned between EtOAc and water. The organics were washed with brine and then dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 0 to 10% gradient of MeOH and CH2Cl2) to obtain a white solid (102 mg, 86%). ESIMS [M+H] + , C 24 H 24 FN5O3, calculated value 450.2, measured value 450.3.

[0527] Step c: The title compound was obtained by the same method as in Example 1 to give a white solid: 1 H NMR (400MHz, DMSO-d6) δ8.54(s,1H),8.22(s,1H),7.61-6.94(m,10H),6.44(dd,J=15.1,4.6Hz,1H),5.23(dt,J= 52.4, 4.1Hz, 1H), 4.82-4.40 (m, 3H), 4.18 (t, J = 6.5Hz, 2H), 4.03 (dd, J = 10.9, 5.9Hz, 3H), 2.26 (t, J = 20.5Hz, 2H). ESIMS[MH] - , C 25 H 28 FN5O8P2, calculated value 606.1, measured value 606.3.

[0528] Embodiment 58

[0529] 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

[0530]

[0531] Step a: The product of Example 35, Step a (10.0 g, 17.24 mmol), styrylboronic acid (3.83 g, 25.86 mmol) and sodium carbonate (5.44 mg, 51.72 mmol) were suspended in 3:1 THF:H2O (100 mL). This mixture was degassed with N2 bubbling for 10 minutes. Pd(PPh3)4 (1.99 g, 1.72 mmol) was then added and the resulting mixture was further degassed for 5 minutes 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 phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 5% to 50% EtOAc / hexanes) as a colorless solid (8.06 g, 72%).

[0532] Step b: To a suspension of the product of step a (8.06 g, 12.04 mmol), sodium periodate (15.5 g, 72.4 mmol) and 2,6-lutidine (2.80 mL, 24.1 mmol) in 2:1 THF:H2O (127.5 mL) was added potassium osmate dihydrate (100 mg, 0.30 mmol). The resulting thick suspension was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic phase was washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The title compound was purified by column chromatography (SiO2, EtOAc / hexanes) to give an off-white oil (6.74 g, 97%). ESIMS [M+H] + , C 30 H 28 FN5O6, calculated value 574.2, found value 574.4.

[0533] 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 uL, 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 organics were washed with brine, dried over MgSO4, and concentrated under reduced pressure to give the title compound which was used without further purification. ESI MS [M+H] + , C 35 H 39 FN6O5, calculated value 643.3, measured value 643.3.

[0534] 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 organics were washed with brine and then dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 0 to 100% gradient of MeOH and CH2Cl2) to obtain a white solid (151 mg, 40% in two steps). ESIMS [M+H] + , C 21 H 31 FN6O3, calculated value 435.2, measured value 435.3.

[0535] Step d: The title compound was obtained by the same method as in Example 1 to give a white solid: 1 H NMR (400MHz, DMSO-d6) δ9.40(s,1H),8.58-8.03(m,2H),6.45(dd,J=14.1,4.8Hz,1H),5.25(dt,J=52.5,4.3Hz,1H),4.59(d,J=16.3Hz ,2H),4.40(s,1H),4.20(t,J=6.1Hz,2H),4.04(q,J=5.1Hz,1H),3.61(s,1H),3.08(s,2H),2.24(t,J=20.4Hz,2H),2.06-1.35(m,10H). ESIMS[MH] - , C 22 H 35 FN6O8P2, calculated value 591.2, measured value 591.3.

[0536] Embodiment 59

[0537] 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

[0538]

[0539] Step a: To a solution of the product of Example 58, Step b (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 organics were washed with brine, dried over MgSO4, and concentrated under reduced pressure to give the title compound, which was used without further purification. ESIMS [M+H] + , C 30H 30 FN5O6, calculated value 576.2, measured value 576.3.

[0540] Step b: 1) TsCl (436 mg, 2.29 mmol) and triethylamine (400 uL, 2.87 mmol) were added to a solution of the product of step a in dichloromethane (10 mL) at 0°C. The reaction was heated to room temperature and stirred overnight. The reaction was diluted with EtOAc and washed with saturated NaHCO3, 10% citric acid, water and brine. The organics were dried over MgSO4 and concentrated under reduced pressure to give the crude title compound (1.20 g, 94%, two steps), which was used directly in the next step.

[0541] Step b: 2) Methanol (10 ml) was added to a flask containing crude tosylate (700 mg, 0.959 mmol) and potassium carbonate (662 mg, 4.8 mmol). The resulting suspension was stirred overnight and then diluted with EtOAc and washed with water and brine. The organics were dried over MgSO4 and concentrated under reduced pressure. The title compound (85 mg, 23%) was obtained by column chromatography (SiO2, 0 to 15% gradient of MeOH and CH2Cl2). ESIMS [M+H] + , C 17 H 24 FN5O4, calculated value 382.2, measured value 382.3.

[0542] Step c: The title compound was obtained by the same method as in Example 1 to give a white solid: 1 H NMR (400MHz, DMSO-d6) δ8.24(s,1H),6.46(dd,J=14.9,4.6Hz,2H),5.24(dt,J=52.5,4.2Hz,1H),4.54(dt,J=18.3,4.4Hz,2H),4.40( s,2H),4.20(t,J=6.1Hz,3H),4.04(t,J=5.0Hz,1H),3.37(s,5H),2.26(t,J=20.5Hz,2H),1.96(s,3H),1.81-1.41(m,10H).ESIMS[MH] - , C 18 H 28 FN5O9P2, calculated value 538.1, measured value 538.2.

[0543] Embodiment 60

[0544] 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

[0545]

[0546] Step a: To a solution of the product of Example 58, step b (330 mg, 0.58 mmol) in THF (6 mL) was added phenylmagnesium bromide (3.0 M / Et2O, 0.86 mL) at -78°C. The reaction was stirred at this temperature for 1 hour and then quenched with saturated NaHCO3. The crude reaction mixture was partitioned between EtOAc and water. The organics were washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The crude material consisted of isomeric mono-debenzoylated products, which were used directly in step b. ESIMS [M+H] + , C 29 H 30 FN5O5, calculated value 548.2, measured value 548.3.

[0547] Step b: The product of 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 partitioned between EtOAc and water. The organics were washed with brine and then dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 0 to 10% gradient of MeOH and CH2Cl2) to give a white solid (118 mg, 46% over two steps). ESIMS [M+H] + , C 22 H 26 FN5O4, calculated value 444.2, measured value 444.3.

[0548] Step c: The title compound was obtained by the same method as in Example 1 to give a white solid (1:1 mixture of diastereomers): 1 H NMR (400MHz, DMSO-d6) δ8.78-7.85(m,4H),7.49(s,4H),7.41-7.08(m,8H),6.47(dd,J=14.8,4.6Hz,2H),5.98-5.39(m,2H),5.24(dt,J=52.4,4.2H z,1H),5.07(s,1H),4.54(d,J=14.1Hz,0H),4.39-3.86(m,6H),2.26(t,J=20.5Hz,3H),1.99(d,J=34.0Hz,5H),1.65(d,J=52.4Hz,13H).ESIMS[MH]- , C 23 H 30 FN5O9P2, calculated value 600.2, measured value 600.3.

[0549] Embodiment 61

[0550] 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

[0551]

[0552] Step a: The product of step b (1) of Example 29 (750 mg, 1.24 mmol) was suspended in DMF (8.3 mL) and Et3N (260 uL) was added followed by phenylacetylene (205 uL). This mixture was degassed with N2 bubbling for 10 minutes. CuI (24 mg) and Pd(PPh3)2Cl2 (44 mg) were then added and the resulting mixture was heated to 80°C overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with 10% citric acid (aqueous), water, and brine. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, EtOAc / hexane) as a brown oil (762 mg, 92%).

[0553] Step b: The product of 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 organics were washed with brine and then dried (Na2SO4) and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 0 to 10% gradient of MeOH and CH2Cl2) to obtain a colorless oil. ESIMS [M+H] + , C 25 H 22 FN5O3, calculated value 460.2, measured value 460.2.

[0554] Step c: The title compound was obtained by the same method as in Example 1 to give a white solid: 1H NMR (400MHz, DMSO-d6) δ8.65 (s, 1H), 8.37 (d, J = 2.3Hz, 1H), 7.67-7.57 (m, 1H), 7 .47(td,J=5.2,2.1Hz,2H),7.39-7.29(m,4H),7.28-7.16(m,1H),6.50(dd,J=15. 2,4.4Hz,1H),5.28(dt,J=52.4,4.1Hz,1H),4.75(s,2H),4.52(d,J=18.1Hz,1H) ,4.20(d,J=6.4Hz,2H),4.06(q,J=5.0Hz,1H),2.28(t,J=20.5Hz,2H).ESIMS[MH] - , C 26 H 26 FN5O8P2, calculated value 616.1, found value 616.3.

[0555] Embodiment 62

[0556] 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

[0557]

[0558] Step a: To a solution of the product of step b of Example 61 (203 mg, 0.44 mmol) in ethanol (4.4 mL) was added palladium on carbon (10 wt% wet, 20 mg) under nitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogen and then 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. ESIMS [M+H] + , C 25 H 26 FN5O3, calculated value 464.2, measured value 464.4.

[0559] Step b: The title compound was obtained by the same method as in Example 1 to give a white solid: 1H NMR (400MHz, DMSO-d6) δ8.60-8.14(m,2H),7.58-6.91(m,11H),6.44(d,J=15.0Hz,1H),5.22(d,J=52.4Hz,1H),4.71(s,2H), 4.54(dt,J=18.4,4.4Hz,1H),4.19(t,J=6.2Hz,2H),4.11-3.96(m,1H),3.23-2.83(m,5H),2.26(t,J=20.5Hz,2H).ESIMS[MH] - , C 26 H 30 FN5O8P2, calculated value 620.2, measured value 620.2.

[0560] Embodiment 63

[0561] 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

[0562]

[0563] Step a: The product of Example 29, step b (1) (2.0 g, 3.32 mmol) was suspended in DMF (7.4 mL) and diisopropylamine (2.3 mL) was then added followed by trimethylsilyl-acetylene (703 uL, 4.98). This mixture was degassed with N2 bubbling for 10 minutes. CuI (125 mg, 0.66 mmol) and Pd(PPh3)2Cl2 (233 mg, 0.0.33 mmol) were then added and the resulting mixture was further degassed for 5 minutes and then sealed and heated at 80°C for 36 hours. After cooling to room temperature, the reaction was diluted with EtOAc and washed with saturated NH4Cl (aqueous), water, and brine. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was obtained as a beige solid (950 mg, 43%) by column chromatography (SiO2, 5% to 70% EtOAc / hexanes).

[0564] Step b: The product of 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 organics were washed with brine and then dried (Na2SO4) and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 0 to 10% gradient of MeOH and CH2Cl2) to obtain the title compound as a white solid (230 mg, 42%), ESIMS [M+H]+ , C 19 H 18 FN5O3, calculated value 384.1, measured value 384.2.

[0565] Step c: The title compound was obtained by the same method as in Example 1 to give a white solid: 1 H NMR(400MHz, DMSO-d6)δ8.65(s,1H),8.36(d,J=2.2Hz,1H),7.39-7.26(m,5H),7.28-7.17(m,1H),6.44(dd,J=14.8,4.5Hz ,1H),5.25(dt,J=52.5,4.1Hz,1H),4.69(s,2H),4.51(d,J=18.1Hz,1H),4.19(d,J=7.1Hz,2H),2.27(t,J=20.5Hz,2H).ESI MS[MH] - , C 20 H 22 FN5O8P2, calculated value 540.1, measured value 540.2.

[0566] Embodiment 64

[0567] 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

[0568]

[0569] Step a: Under 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 min. The product of Example 29, step b (1) (1.00 g, 1.88 mmol) was added and the mixture was stirred at room temperature for 2 h. 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%). ESIMS [M+H] + , C 17 H 17 ClFN4O4, calculated value 395.1, found value 395.1.

[0570] Step b: The product of step a (197 mg, 0.5 mmol) was dissolved in trimethyl phosphate (2.5 mL) and cooled to 0 ° C. A solution of trimethyl phosphate (1.5 mL) of methylenebis(dichlorophosphine) (624 mg, 2.5 mmol, 5 equivalents) was added dropwise. The reaction mixture was stirred at 0 ° C for 3 h and then quenched with 0.5 M triethylammonium bicarbonate solution (3.6 mL) at -20 ° C. The mixture was stirred at -20 ° C for 15 min, then at 0 ° C for 15 min, and then at room temperature for 15 min. The mixture was washed three times with ethyl acetate (10 mL). The aqueous layer was directly purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 50% containing 0.1% TFA) to obtain the desired product as a white solid (40.2 mg, 15%): 1 H NMR (400MHz, DMSO-d6) δ8.55(d,J=2.1Hz,1H),7.57-7.51(m,2H),7.46-7.35(m,3H),6.49(dd,J=13.6,4.7Hz,1H),5.61(s,2H),5.30( dt,J=52.4,4.4Hz,1H),4.53(dt,J=18.6,4.7Hz,1H),4.21(t,J=6.0Hz,2H),4.06(q,J=5.0Hz,1H),2.27(t,J=20.6Hz,2H).ESIMS[MH] - , C 18 H 19 ClFN4O9P2, calculated value 551.0, found value 551.2.

[0571] Embodiment 65

[0572] 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

[0573]

[0574] 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 Cs2CO3 (788 mg, 2.42 mmol, 1.3 eq.). 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 eq.) 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 on a silica pad and concentrated. The residue was adsorbed on silica and purified by column chromatography (hexane / ethyl acetate) to give the product as a white solid (480 mg) in 49% yield.

[0575] Step b: A mixture of the product of step a (480 mg, 0.9 mmol), benzylamine (97 mg, 0.9 mmol,) and Et3N (91 mg, 0.9 mmol,) in anhydrous EtOH (4 mL) was stirred at 65 ° C for 6 hours. Excess solvent was removed by vacuum. The residue was dried under high vacuum for 30 min. Methanol (4 mL) and K2CO3 (249 mg, 1.8 mmol) were added and stirred at room temperature for 1 h. LCMS showed that the reaction was complete. The filtrate was filtered and concentrated. The residue was purified by flash column to obtain the product in quantitative yield.

[0576] Step c: The product of step b (360 mg, 0.91 mmol) was dissolved in trimethyl phosphate (4 mL) and cooled to 0°C (ice bath), and then a cold solution of methylenebis(phosphonic acid dichloride) (801 g, 3.2 mmol, 3.5 equivalents) in trimethyl phosphate (2 mL) was added dropwise, and the reaction mixture was stirred at 0°C for 1 h, then slowly quenched with ice-cold 0.5 M triethylammonium bicarbonate solution (11 mL) and stirred at 0°C for 15 min, then stirred at room temperature for 1 h. The reaction mixture was purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 40% containing 0.1% TFA) to give the product as a white solid: 1 H NMR(400MHz, DMSO-d6)δ8.60(t,J=6.0Hz,1H),7.59-7.13(m,6H),6.72(s,1H),6.49(dd,J=15.7,4.5Hz,1H),5.45-5.04(m,1H) ,4.80-4.57(m,2H),4.42(dt,J=18.6,4.4Hz,1H),4.19-4,15(m,2H),3.98(q,J=5.0Hz,1H),2.26(t,J=20.5Hz,2H).ESIMS[MH] - , C 18 H22 ClFN4O8P2, calculated value 549.1, found value 549.2

[0577] Embodiment 66

[0578] 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

[0579]

[0580] The title compound was synthesized in a similar manner to Example 65, substituting cyclopentylamine for benzylamine: 1 H NMR (400MHz, DMSO-d6) δ7.90(d,J=7.2Hz,1H),7.24(s,1H),6.72(d,J=3.6Hz,1H),6.47(dd,J=15.9,4.4Hz,1H),5.15(dt,J=52.6,4.1Hz,1H),4.52-4. 35(m,2H),4.15(q,J=6.3,5.3Hz,2H),3.97(q,J=5.1Hz,1H),2.23(d,J=20. 5Hz,1H),1.98(d,J=10.6Hz,2H),1.72(s,2H),1.67-1.45(m,5H).ESIMS[MH] - , C 17 H 24 ClFN4O8P2, calculated value 527.1, found value 527.2

[0581] Embodiment 67

[0582] Synthesis of ((((1-(6-(Benzylamino)-9H-purin-9-yl)propan-2-yl)oxy)(hydroxy)phosphoryl)-methyl)phosphonic acid

[0583]

[0584] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1 H NMR (400MHz, DMSO-d6) δ8.79 (s, 1H), 8.31 (d, J = 15.8Hz, 2H), 7.46-7.13 (m, 5H), 4.92-4.62 (m,2H),4.49-4.25(m,2H),2.17(td,J=20.4,4.8Hz,2H),1.14(d,J=6.3Hz,3H).ESIMS[M+H] + , C16 H 22 N5O6P2, calculated value 442.1, found value 442.1.

[0585] Embodiment 68

[0586] Synthesis of (((2-(6-(Benzylamino)-9H-purin-9-yl)propoxy)(hydroxy)phosphoryl)-methyl)phosphonic acid

[0587]

[0588] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1 H NMR(400MHz,DMSO-d6)δ8.73(s,1H),8.35(s,1H),8.27(s,1H),7.41-7.18(m,5H),4.96-4.82(m,1 H),4.72(s,2H),4.39-4.19(m,2H),2.18(t,J=20.5,1.6Hz,2H),1.55(d,J=7.0Hz,3H).ESIMS[M+H] + , C 16 H 22 N5O6P2, calculated value 442.0, found value 442.1.

[0589] Embodiment 69

[0590] 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 di(carbonate)

[0591]

[0592] Methylene diphosphonic acid (20 mg, 0.03 mmol, the trifluoroacetate of Example 66) was dissolved in 0.5 mL of DMSO. Hunig's base (0.18 mL, 1 mmol, 30 equivalents) was added, followed by chloromethyl isopropyl carbonate (0.13 mL, 1 mmol, 30 equivalents). The reaction mixture was stirred at room temperature for 5 days. The reaction mixture was purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 40% containing 0.1% TFA) to give the product (3.6 mg) as a white solid in 14% yield. 1H NMR (400MHz, DMSO-d6) δ8.42(d,J=7.7Hz,1H),8.25(s,1H),6.37(dd,J=15.2,4. 4Hz,1H),5.67-5.43(m,4H),5.24(ddt,J=52.1,7.7,4.1Hz,1H),4.79(pd,J=6.2, 3.8Hz,2H),4.57-4.38(m,1H),4.37-4.19(m,2H),4.06(q,J=5.1Hz,1H),2.68(t, J=21.2Hz,2H),1.92(s,2H),1.80-1.47(m,6H),1.28-1.15(m,12H).).ESIMS[MH] - , C 26 H 39 ClFN5O 14 P2, calculated value 760.2, measured value 760.3

[0593] Embodiment 70

[0594] 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

[0595]

[0596] The title compound was synthesized by a method similar to Example 1. 1 H NMR(400MHz, DMSO-d6)δ8.42(s,1H),5.88(d,J=5.9Hz,1H),4.53-4.46(m,1H),4.19(dd,J=5.0 ,3.1Hz,1H),4.15-4.06(m,3H),3.17(brs,3H),2.26(t,J=20.5Hz,2H),1.94-1.53(m,9H),ESI MS[M+H] + , C 17 H 27 ClN5O9P2, calculated value 542.1, found value 542.2,

[0597] Embodiment 71

[0598] 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

[0599]

[0600] Step a: The alcohol (4.8 g, 11.1 mmol) was dissolved in anhydrous CH2Cl2 (100 mL) and Dess-Martin hyperiodide (5.6 g, 13.3 mmol, 1.2 equiv) was added. The reaction mixture was stirred at room temperature for 3 h and then quenched with 10% Na2S2O3 (20 mL) and saturated NaHCO3 (50 mL). The organic layer was separated, dried over MgSO4, filtered and evaporated. The crude aldehyde was purified by column chromatography (SiO2, CH2Cl2→CH2Cl2:MeOH, 9:1) to give a yellow solid (4.8 g, quantitative). ESIMS [M+H] + , C 20 H 21 ClN5O4, calculated value 430.1, found value 430.2

[0601] Step b: The product of step a (860 mg, 2.0 mmol) was dissolved in anhydrous THF (20 mL) and cooled to -78 °C. 3 M MeMgBr in Et2O (2 mL, 6 mmol, 3 eq) was added dropwise and 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. The mixture was quenched with saturated NH4Cl (10 mL), the organic layer was separated, dried over MgSO4, filtered and evaporated. The crude product was used without further purification. ESIMS [M+H] + , C 21 H 25 ClN5O4, calculated value 446.2, found value 446.3

[0602] Step c: The phosphorylation step was carried out in a similar manner to Example 1. 1 H NMR (400MHz, DMSO-d6) δ9.00-8.88(m,1H),8.51(s,1H),7.37-7.17(m,5H),5.81(d,J=7.0Hz,1H),4.70-4.51(m ,4H),4.32-4.25(m,1H),3.83(dd,J=5.3,2.6Hz,1H),2.22(t,J=20.5Hz,2H),1.26(d,J=6.4Hz,3H).ESIMS[M+H] + , C 19 H 25 ClN5O9P2, calculated value 564.1, found value 564.1.

[0603] Embodiment 72

[0604] Synthesis of [({1-[(2S,3S,4R,5R)-5-[6-(benzylamino)-2-chloro-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]propoxy}(hydroxy)phosphoryl)methyl]phosphonic acid

[0605]

[0606] The title compound was synthesized in a similar manner to Example 71. 1 H NMR(400MHz,DMSO-d6)δ8.97-8.91(m,1H),8.53(s,1H),7.38-7.20(m,5H), 5.79(d,J=7.4Hz,1H),4.65(d,J=6.3Hz,2H),4.62-4.56(m,1H),4.54-4.46( m,1H),4.34(d,J=5.5Hz,1H),3.89(dd,J=6.1,2.2Hz,1H),2.22(t,J=20.5Hz ,2H),1.69(s,1H),1.58(q,J=7.1Hz,1H),0.90(t,J=7.4Hz,3H).ESIMS[M+H] + , C 20 H 27 ClN5O9P2, calculated value 578.1, found value 578.2.

[0607] Embodiment 73

[0608] 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

[0609]

[0610] Step a: To β-D-ribofuranose, 2-C-methyl-, 1,2,3,5-tetrabenzoate (4.0 g, 6.89 mmol, 1 eq.) and 2,6-dichloropurine (1.43 g, 7.58 mmol, 1.1 eq.) 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 eq.), followed by the addition of trimethylsilyl trifluoromethanesulfonate (5.11 mL, 28.25 mmol, 4.1 eq.) dropwise over 5 min. The reaction mixture was stirred at 0°C for 15 min and heated at 65°C for 5 h. After cooling to room temperature, the reaction was diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (x2) and brine (x1). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was obtained by column chromatography (SiO2, 25% to 66% EtOAc / hexanes) as a white solid (1.30 g, 97%).

[0611] Step b: 1) The product of 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 anhydrous EtOH (6.7 mL). The mixture was stirred at 70° C. for 4 hours, and after cooling to room temperature, the mixture was concentrated under reduced pressure and the obtained material was used without further purification.

[0612] 2) The above product was dissolved in methanol (20 mL) and potassium carbonate (1.06 g, 7.63 mmol, 3.8 equivalents) was added. The mixture was stirred at room temperature for 2 hours, and the residue was adsorbed on celite and purified by column chromatography (SiO2, 0% to 10% DCM / MeOH) to a colorless oil (612 mg, 79%, two steps).

[0613] Step c: The title compound was synthesized by a method similar to Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.38(dd,J=18.2,8.1Hz,1H),8.26(d,J=10.0Hz,1H),5.86(s,1H),4.42(q,J=7.2Hz,1H),4.2 7(h,J=10.6,10.0Hz,2H),4.06(s,3H),2.28(t,J=20.4Hz,2H),1.93(d,J=16.3Hz,2H),1.78-1.43(m,6H).ESIMS[MH] - , C 17 H 25 ClN5O9P2, calculated value 540.1, found value 540.2.

[0614] Embodiment 74

[0615] 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

[0616]

[0617] Step a: 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(dichlorophosphine) (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 and anhydrous MeOH (30 mL) was added and slowly warmed to room temperature. The reaction mixture was neutralized with saturated NaHCO3 (80 mL) and diluted with water (150 mL) and EtOAc (150 mL). The organic layer was separated, dried over MgSO4, filtered and evaporated. The product was first purified by column chromatography (SiO2, EtOAc→EtOAc:MeOH, 8:2) and then by RP18 HPLC (H2O+0.1% TFA / acetonitrile+0.1% TFA) to give the desired product as a white solid (405 mg) in 11% yield. ESIMS[M+H] + , C 19 H 31 ClN5O9P2, calculated value 570.1, found value 570.2.

[0618] Step b: Sodium iodide (50 mg, 0.33 mmol) was added to a solution of the product of step a (75 mg, 0.13 mmol) in acetone (1 mL). The solution was heated at 65 °C for 6 h. The solution was evaporated; the residue was dissolved in water and purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 40% containing 0.1% TFA) to give the product (51 mg) as a white solid in 62% yield. 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=2.0Hz,1H),8.36(d,J=7.8Hz,1H),5.85(d,J=5.6Hz,1H),5.04(brs,1H),4.53(t,J=5.5Hz,1 H),4.47-4.34(m,1H),4.24-3.95(m,4H),3.58(d,J=11.3Hz,2H),2.37(dd,J=20.5,20.5Hz,2H),2.07-1.36(m,8H).ESIMS[M+H] + , C 17 H26 ClN5O9P2, calculated value 542.8, found value 542.2.

[0619] Embodiment 75

[0620] 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

[0621]

[0622] The alcohol (380 mg, 1 mmol) was dissolved in trimethyl phosphate (5 mL) and cooled to 0°C (ice bath), then a cold solution of methylenebis(dichlorophosphine) (375 mg, 1.5 mmol, 1.5 eq) in trimethyl phosphate (3 mL) was added dropwise and the reaction mixture was stirred at 0°C for 3 h. Solid phenol (470 mg, 5 mmol, 5 eq) was added and once dissolved, TEA (835 μL, 6 mmol, 6 eq) was added dropwise. The mixture was stirred at 0°C for 15 min and then stirred at room temperature overnight. It was diluted with H2O (15 mL) and the product was extracted with MTBE (2×10 mL). The combined organics were dried over MgSO4, filtered and evaporated, and the crude product was purified by column chromatography (SiO2, Hex→100% EtOAc) to give a white solid (80 mg, 10%). 1 H NMR (400MHz, DMSO-d6) δ8.38 (d, J=4.3Hz, 1H), 7.41-7.33 (m, 4H), 7.32-7.2 5(m,2H),7.25-7.11(m,9H),5.89(dd,J=5.3,3.2Hz,1H),5.63(dd,J=6.0,4 .4Hz,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).ESIMS[M+H] + , C 35 H 39 ClN5O9P2, calculated value 770.2, found value 770.3

[0623] Embodiment 76

[0624] 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

[0625]

[0626] The title compound was synthesized using a similar method to Example 75. 1 H NMR (400MHz, DMSO-d6) δ8.32 (s, 1H), 7.43-7.34 (m, 2H), 7.34-7.05 (m, 10H), 5.88 (t, J = 4.7Hz, 1H), 5.62 (s, 1H), 5 .43(s,1H),4.59-4.36(m,3H),4.26-4.10(m,2H),3.86-3.70(m,2H),3.04(s,3H),1.90-1.46(m,8H).ESIMS[M+H] + , C 35 H 36 Cl4N5O9P2, calculated value 872.1, found value 872.2.

[0627] Embodiment 77

[0628] 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

[0629]

[0630] The title compound was synthesized using a similar method to Example 75. 1 H NMR (400MHz, DMSO-d6) δ8.27(s,1H),7.65-7.57(m,2H),7.52-7.40(m,3H),7.32-7.04(m,4H),5.87(t,J=5.0Hz,1H),5.63(t,J=6.1Hz,1H ),5.43(dd,J=5.6,3.7Hz,1H),4.59-4.35(m,3H),4.30-4.08(m,2H),3.85(t,J=22.0Hz,2H),3.32(s,3H),1.89-1.45(m,8H).ESIMS[M+H] + , C 35 H 33Cl7N5O9P2, calculated value 974.0, found value 974.2.

[0631] Embodiment 78

[0632] 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

[0633]

[0634] The title compound was synthesized in a similar manner to Example 75. 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). ESIMS [M+H] + , C 41 H 45 ClN5O 15 P2, calculated value 944.2, measured value 944.3.

[0635] Embodiment 79

[0636] 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

[0637]

[0638] The title compound was synthesized in a similar manner to Example 29. 1H NMR(400MHz,DMSO-d6)δ8.92(d,J=8.3Hz,1H),8.28(s,1H),7.45(bs,2H),7 .11(td,J=9.1,1.4Hz,2H),6.34(dd,J=14.3,4.6Hz,1H),5.39(bs,1H),5.31 -5.12(m,1H),5.14(bs,1H),4.48(dt,J=18.5,4.5Hz,1H),4.17(s,3H),4.01 (d,J=5.2Hz,2H),2.24(t,J=20.4Hz,3H),1.51(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2

[0639] Embodiment 80

[0640] 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

[0641]

[0642] The title compound was synthesized in a similar manner to Example 29. 1 H NMR(400MHz,DMSO-d6)δ8.90(d,J=8.3Hz,1H),8.28(s,1H),7.42(d,J=7.6Hz ,2H),7.29(t,J=7.5Hz,2H),7.19(bs,1H),6.34(dd,J=14.8,4.4Hz,1H),5.3 9(bs,1H),5.23(d,J=52.6Hz,1H),4.49(d,J=19.2Hz,2H),4.17(bs,2H),4.0 1(d,J=5.1Hz,1H),2.24(t,J=20.6Hz,2H),1.52(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O8P2, calculated value 566.1, found value 566.1

[0643] Embodiment 81

[0644] 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

[0645]

[0646] The title compound was synthesized in a similar manner to Example 29. 1 H NMR (400MHz, DMSO-d6) δ8.91(d,J=8.3Hz,1H),8.28(s,1H),7.41(d,J=7.5Hz,2H),7.29(t,J=7.6Hz,2H),7.20(d,J=7.6Hz,1H),6.34(d,J=14.1Hz,1H ),5.39(bs,1H),5.21(d,J=52.5Hz,1H),4.47(d,J=18.3Hz,2H),4.17(s,2 H),4.01(s,1H),2.24(t,J=20.6Hz,2H),1.52(d,J=7.1Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O8P2, calculated value 566.1, found value 566.1

[0647] Embodiment 82

[0648] Synthesis of [({[(2R,3R,4S,5R)-5-[6-(cyclopentylamino)-2-[hydroxy(oxacyclohexane-4-yl)methyl]-9H-purin-9-yl]-4-fluoro-3-hydroxyoxacyclohexane-2-yl]methoxy}(hydroxy)phosphoryl)methyl]-phosphonic acid

[0649]

[0650] Step a: The product of step b of 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, prepared in THF) was added dropwise. The reaction mixture was warmed to room temperature and stirred at room temperature for 3 h. The reaction mixture was cooled to 0°C, methanol (50 mL) was added, and the mixture was stirred at room temperature for 14 h. The reaction mixture was dry loaded onto silica gel and purified by silica gel column chromatography (DCM containing 0-10% MeOH) to give the desired product as a white solid (273 mg, 35%).

[0651] Step b: The title compound was synthesized as a white solid (44 mg; 29%) in a similar manner to Example 29.1 H NMR(400MHz, DMSO-d6)δ6.60-6.40(m,1H),5.26(d,J=53.3Hz,1H),4.63-4.39(m,2H),4.30-4.13(m,2H),4.13-3.97(m ,1H),3.94-3.75(m,2H),3.38-3.13(m,2H),2.26(t,J=20.4Hz,2H),2.17-1.85(m,3H),1.85-1.22(m,12H).ESIMS[MH] - , C 23 H 35 FN5O9P2, calculated value 606.2, measured value 606.3.

[0652] Embodiment 83

[0653] 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

[0654]

[0655] This compound was obtained by a similar method to Example 29. 1 H NMR(400MHz, DMSO-d6)δ8.7(brs,2H),8.30(d,J=2.1Hz,1H),6.40(dd,J=14.3,4.6Hz,1H),6.09(brs,1H),5.25(dt,J=52.5,4.3Hz,1H), 4.53-4.43(m,1H),4.23-4.14(m,2H),4.09-3.98(m,1H),2.28(dd,J=20.5Hz,J=20.5Hz,2H),2.5(s,3H),1.96-1.44(m,9H).ESIMS[M+H] + , C 17 H 25 ClFN5O8P2, calculated value 544.8, calculated value: 544.2

[0656] Embodiment 84

[0657] 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

[0658]

[0659] Step a: 2-Chloropurine fluoronucleoside (579 mg, 1.5 mmol) was dissolved in trimethyl phosphate (7.5 mL) and cooled to 0°C (ice bath), then 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 h, then slowly quenched with methanol (7 mL) and stirred at 0°C for 30 min, then stirred at room temperature for 1 hour and stirred at 40°C for 4 h. The reaction mixture was concentrated under vacuum 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 was purified by column chromatography (0 to 10% gradient of methanol in dichloromethane) to give the desired product as a light yellow solid (701 mg, 80%). 1 H NMR (400MHz, DMSO-d6) δ8.32(dd,J=12.0,2.3Hz,1H),6.42(dd,J=15.5,4.4Hz,1H),6.15(t,J=4.8Hz,1H),5.43-5.07(m,1H),4.60-4.39(m,1H), 4.27(q,J=7.3,5.7Hz,2H),4.12-4.03(dq,J=9.6,5.3Hz,1H),3.69-3.5 9(m,9H),2.96-2.74(m,2H),2.50(s,3H),2.04-1.42(m,9H).ESIMS[M+H] + , C 20 H 31 ClFN5O8P2, calculated value 586.9, found value 586.2.

[0660] Step b: Sodium iodide (75 mg, 0.5 mmol) was added to a solution of the product of step a (58 mg, 0.1 mmol) in acetone (1 mL). The solution was heated at 60 ° C for 24 h. The solution was evaporated, the residue was dissolved in water and purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 30% containing 0.1% TFA) to give the product (42 mg) as a white solid in 65% yield. 1H NMR (400MHz, DMSO-d6) δ8.31(d,J=2.2Hz,1H),6.40(dd,J=14.6,4.6Hz,1H),5.25(dt,J=52.4,4.2Hz,1H),4.48(dt,J=18.3,4.4Hz,1H),4 .18(t,J=6.1Hz,2H),4.04(m,2H),3.58(d,J=11.2Hz,3H),2.5(s,3H).2.39(dd,J=20.4Hz,J=20.4Hz,2H),2.00-1.42(m,9H).ESIMS[M+H] + , C 18 H 27 ClFN5O8P2, calculated value 558.8, found value 558.2.

[0661] Embodiment 85

[0662] 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

[0663] as well as

[0664] 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

[0665]

[0666] Step a: To a solution of the product of 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 h. The solution was evaporated, the residue was dissolved in water and purified by reverse phase HPLC (C18 column, acetonitrile and water with a gradient of 0 to 30% containing 0.1% TFA) to give ((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-tetrahydrofuran-2-yl)methyl hydrogen ((dimethoxyphosphoryl)methyl)-phosphonate as a white solid (35 mg) in 20% yield. 1H NMR (400MHz, DMSO-d6) δ8.31(d,J=2.2Hz,1H),6.41(dd,J=14.9,4.5Hz,1H),5.25(dt,J=52.3,4.1Hz,1H),4.53-4.43(m,1H),4.24-4.12(m,2H), 4.08-4.02(m,1H),3.66(d,J=2.0Hz,3H),3.63(d,J=2.0Hz,3H),2.60(dd,J=20.8Hz,J=20.8Hz,2H),2.50(s,3H),2.01-1.55(m,9H).ESIMS[M+H] + , C 19 H 29 ClFN5O8P2, calculated value 572.9, found value 572.3.

[0667] 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 as a 1:1 mixture of diastereomers (52 mg), 30% yield. 1 H NMR(400MHz,DMSO-d6)δ8.34(d,J=2.3Hz,0.5H,1 st dia),8.30(d,J=2.3Hz,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=11.3Hz,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,2H),2.5(s,3H),1.97-1.52(m,9H).ESIMS[M+H] + , C 19 H 29 ClFN5O8P2, calculated value 572.9, found value 572.2.

[0668] Embodiment 86

[0669] 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

[0670]

[0671] This compound was obtained in a similar manner to Example 84 as a 1:1 mixture of diastereomers. 1 H NMR (400 MHz, DMSO-d6) δ8.34 (d, J = 2.3 Hz, 0.5H, first diastereomer), 8.29 (d, J = 2.3 Hz, 0.5H, second diastereomer), 6.52-6.32 (m, 1H), 6.07 (brs, 1H), 5.34-5.14 (m, 1H), 4.56-4.43 (m, 1H), 4.3 0-4.21 (m, 2H), 4.11-4.03 (m, 1H), 3.63 (d, J = 11.2 Hz, 1.5H, first diastereomer), 3.63 (d, J = 11.2 Hz, 1.5H, second diastereomer), 2.50 (s, 3H), 2.48-2.34 (m, 2H), 1.92-1.53 ​​(m, 9H). ESIMS [M+H] + , C 18 H 27 ClFN5O8P2, calculated value 558.3, found value 558.2.

[0672] Embodiment 87

[0673] 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}(hydroxy)phosphoryl)methyl]phosphonic acid

[0674]

[0675] 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 hexamethyldisilazide. The mixture was then heated to reflux and stirred for 3 hours. The mixture was then concentrated to dryness. The solid residue was then dissolved in 300 mL of acetonitrile, and the protected nucleoside (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 dissolved in ethyl acetate. The organics were washed with saturated NaHCO3 and brine. The organics were dried over MgSO4, filtered, and concentrated. The crude residue was purified by column chromatography (hexane / ethyl acetate) to give the desired compound (48 g, 108 mmol) in a total yield of 82%. 1 H NMR (400MHz, DMSO-d6) δ8.75 (s, 1H), 6.47 (d, J = 3.2Hz, 1H), 5.82 (dd, J = 5.3, 3.2Hz, 1H), 5.63 (t, J = 5.8Hz, 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), ESIMS[M+Na] + , C 16 H 16 Cl2N4NaO7, calculated value 469.0, found value 469.0.

[0676] Step b: The product of 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 eq) and triethylamine (7.2 mL, 51.8 mmol, 1.05 eq) were added and the reaction mixture was stirred at 0°C for 15 min and then at room temperature for 4 h. 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 crude product was used in the next step without purification. ESIMS [M+H] + , C 15 H 21 ClN5O4, calculated value 370.1, found value 370.2.

[0677] Step c: The phosphonation step was carried out in a similar manner to Example 1. 1H NMR (400MHz, DMSO-d6) δ8.68(d,J=7.2Hz,1H),8.24(s,1H),6.00(d,J=4.2Hz,1H),4.49(t,J=4.7Hz,1H),4.41(q,J=6.7Hz,1H) ,4.26(t,J=4.7Hz,1H),4.15-4.00(m,2H),3.94-3.84(m,1H),2.16(t,J=20.5Hz,2H),2.04-1.91(m,2H),1.79-1.45(m,6H).ESI MS[M+H] + , C 16 H 25 ClN5O9P2, calculated value 528.1, found value 528.2.

[0678] Embodiment 88

[0679] 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

[0680]

[0681] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ9.38-9.18(m,1H),8.35-8.16(m,1H),7.39-7.19(m,5H),6.07-5.94(m,1H),4.69(d,J=5.4Hz ,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=20.9Hz,2H).ESIMS[MH] - , C 18 H 22 ClN5O9P2, calculated value 548.1, found value 548.1.

[0682] Embodiment 89

[0683] 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

[0684]

[0685] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ9.26-8.95(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).ESIMS[MH] - , C 19 H 24 ClN5O9P2, calculated value 562.1, found value 562.2.

[0686] Embodiment 90

[0687] 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

[0688]

[0689] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ9.16 (d, J = 8.4Hz, 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, 1 H),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=20.8Hz,2H),1.71-1.37(m,4H).ESIMS[MH] - , C 19 H 24 ClN5O9P2, calculated value 562.1, found value 562.2.

[0690] Embodiment 91

[0691] 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

[0692]

[0693] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ9.41-9.19(m,1H),8.32-8.17(m,1H),7.43-7.30(m,4H),6.02(d,J=2.9Hz,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=20.4Hz,1H).ESIMS[MH] - , C 18 H 21 Cl2N5O9P2, calculated value 582.0, found value 582.0.

[0694] Embodiment 92

[0695] 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

[0696]

[0697] The title compound was synthesized in a similar manner to Example 87. 1H NMR(400MHz, DMSO-d6)δ9.28-9.15(m,1H),8.33(dd,J=1.5,0.7Hz,1H),7.43(t,J=7.8Hz, 1H),7.29(dd,J=7.8,5.6Hz,1H),7.23-7.08(m,2H),6.00(d,J=4.2Hz,1H),5.65-5.51(m, 1H),4.48(t,J=4.9Hz,1H),4.26(t,J=4.5Hz,1H),4.05(dq,J=10.1,5.9,5.2Hz,2H),3.88 (dt,J=11.3,6.0Hz,1H),2.29-2.08(t,J=20.4Hz,2H),1.53(d,J=6.8Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O9P2, calculated value 582.1, found value 582.1

[0698] Embodiment 93

[0699] 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

[0700]

[0701] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ9.23(d,J=7.6Hz,1H),8.34(s,1H),7.44(t,J=7.8Hz,1H),7. 30(q,J=7.0Hz,1H),7.18(dt,J=9.4,6.4Hz,2H),6.00(d,J=4.3Hz,1H),5.60(q,J=7.1 Hz,1H),4.51(t,J=4.6Hz,1H),4.26(t,J=4.6Hz,1H),4.05(tt,J=10.1,5.8Hz,2H),3. 88(dd,J=11.0,6.2Hz,1H),2.17(t,J=20.4Hz,2H),1.53(d,J=6.7Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O9P2, calculated value 582.1, found value 582.1

[0702] Embodiment 94

[0703] 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

[0704]

[0705] The title compound was synthesized in a similar manner to Example 87. 1 H NMR(400MHz,DMSO-d6)δ9.17(d,J=7.9Hz,1H),8.31(s,1H),7.50-7.30(m,1H),7.22( d,J=8.2Hz,2H),7.06(td,J=8.7,2.5Hz,1H),6.00(d,J=4.2Hz,1H),5.41(t,J=7.3Hz ,1H),4.48(t,J=4.7Hz,1H),4.26(t,J=4.8Hz,1H),4.05(dq,J=11.7,6.5Hz,2H),3.8 8(dt,J=11.2,6.2Hz,1H),2.17(t,J=20.5Hz,2H),1.53(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O9P2, calculated value 582.1, found value 582.1

[0706] Embodiment 95

[0707] 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

[0708]

[0709] The title compound was synthesized in a similar manner to Example 87. 1H NMR(400MHz, DMSO-d6)δ9.18(d,J=7.9Hz,1H),8.31(t,J=0.9Hz,1H),7.43-7.32( m,1H),7.23(d,J=8.8Hz,2H),7.07(t,J=8.6Hz,1H),6.00(d,J=4.3Hz,1H),5.42( t,J=7.3Hz,1H),4.51(t,J=4.5Hz,1H),4.26(t,J=4.7Hz,1H),4.11-3.98(m,2H), 3.88(t,J=8.6Hz,1H),2.17(t,J=20.5Hz,2H),1.52(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O9P2, calculated value 582.1, found value 582.1

[0710] Embodiment 96

[0711] 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

[0712]

[0713] The title compound was synthesized in a similar manner to Example 87. 1 H NMR(400MHz,DMSO-d6)δ9.16(d,J=7.9Hz,1H),8.30(d,J=1.2Hz,1H),7.42(dd ,J=8.4,5.4Hz,2H),7.15(td,J=8.9,1.2Hz,2H),6.00(d,J=4.2Hz,1H),5.40(t ,J=7.3Hz,1H),4.48(t,J=4.8Hz,1H),4.25(t,J=4.5Hz,1H),4.18-3.95(m,2H) ,3.95-3.82(m,1H),2.16(t,J=20.4Hz,2H),1.52(d,J=7.2Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O9P2, calculated value 582.1, found value 582.1

[0714] Embodiment 97

[0715] 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

[0716]

[0717] The title compound was synthesized in a similar manner to Example 87. 1 H NMR(400MHz,DMSO-d6)δ9.16(d,J=7.9Hz,1H),8.30(t,J=0.9Hz,1H),7.42(dt ,J=6.1,3.2Hz,2H),7.23-7.08(m,2H),6.00(d,J=4.3Hz,1H),5.40(t,J=7.2Hz ,1H),4.50(t,J=4.5Hz,1H),4.26(t,J=4.7Hz,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).ESIMS[M+H] + , C 19 H 24 ClFN5O9P2, calculated value 582.1, found value 582.1

[0718] Embodiment 98

[0719] 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

[0720]

[0721] The title compound was synthesized using a method similar to Example 87. 1H NMR (400MHz, DMSO-d6) δ9.29(s,1H),8.29(d,J=1.9Hz,1H),7.48(dd,J=5.9,3.1Hz,1H),7.43(d,J=5.9Hz,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=5.0Hz,1H),4.27(s,1H),4.06(s,2H),3.89(s,1H),2.16(t,J=20.6Hz,2H). ESIMS[M+H] + , C 18 H 22 Cl2N5O9P2, calculated value 584.0, found value 584.1

[0722] Embodiment 99

[0723] 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

[0724]

[0725] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ8.41(s,1H),7.49(d,J=7.4Hz,1H),7.38-7.24(m,2H),7.16(d,J=7.6Hz,1H),6.08(bs,1H),5.04(bs, 2H), 4.50 (d, J = 30.8Hz, 1H), 4.24 (d, J = 39.5Hz, 1H), 4.06 (s, 2H), 3.89 (s, 1H), 3.37 (d, J = 54.8Hz, 3H), 2.15 (t, J = 20.8Hz, 2H). ESIMS[M+H] + , C 19 H 24 Cl2N5O9P2, calculated value 598.0, found value 598.1

[0726] Embodiment 100

[0727] 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

[0728]

[0729] The title compound was synthesized in a similar manner to Example 87. 1 H NMR(400MHz,DMSO-d6)δ8.95(t,J=5.6Hz,1H),8.28-8.10(m,1H),7.48-7.39(m ,1H),7.33(d,J=6.4Hz,1H),7.29-7.18(m,2H),6.00(d,J=4.0Hz,1H),4.50(t,J =4.5Hz,1H),4.25(t,J=4.5Hz,1H),4.06(d,J=14.9Hz,2H),3.87(t,J=5.8Hz,1H ),3.69(q,J=6.8Hz,2H),3.10-3.00(m,2H),2.15(t,J=20.4Hz,2H).ESIMS[M+H] + , C 19 H 24 Cl2N5O9P2, calculated value 598.0, found value 598.2

[0730] Embodiment 101

[0731] 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

[0732]

[0733] The title compound was synthesized in a similar manner to Example 87. 1 H NMR(400MHz, DMSO-d6)δ8.35(s,1H),7.30(dd,J=20.4,7.5Hz,5H),6.07(bs,1H),4.99(bs,1H),4.53(b s,1H),4.28(bs,1H),4.05(s,3H),3.88(s,1H),3.37-3.24(m,3H),2.14(t,J=20.9Hz,3H).ESIMS[M+H] + , C 19 H 25ClN5O9P2, calculated value 564.1, found value 564.1

[0734] Embodiment 102

[0735] 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

[0736]

[0737] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ8.31 (s, 1H), 6.06 (s, 1H), 4.51 (d, J = 4.7Hz, 1H), 4.28 (d, J = 5.1Hz, 1H), 4.14-3.96(m,2H),3.88(s,1H),3.21(s,3H),2.14(t,J=19.9Hz,2H),1.67(bs,8H).ESIMS[M+H] + , C 17 H 27 ClN5O9P2, calculated value 542.1, found value 542.2

[0738] Embodiment 103

[0739] 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

[0740]

[0741] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ8.82(s,1H),8.16(s,1H),6.01(bs,1H),4.50(d,J=5.9Hz,1H),4.26(b s,1H),4.05(bs,2H),3.88(bs,1H),2.95(d,J=4.6Hz,3H),2.15(t,J=20.4Hz,2H).ESIMS[M+H] + , C 12 H 19 ClN5O9P2, calculated value 474.0, found value 474.2

[0742] Embodiment 104

[0743] 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

[0744]

[0745] The title compound was synthesized in a similar manner to Example 87. 1 H NMR (400MHz, DMSO-d6) δ9.82(d,J=9.3Hz,1H),8.51(s,1H),7.65(d,J=7.2Hz,2H),7.56-7.31(m,3H),6.33(p,J=8.8Hz,1H),6.04(d,J =4.2Hz,1H),4.50(t,J=4.4Hz,1H),4.26(t,J=4.6Hz,1H),4.16-4.00(m,2H),3.90(dd,J=10.6,5.8Hz,1H),2.18(t,J=20.5Hz,2H).ESI MS[MH] - , C 19 H 21 ClF3N5O9P2, calculated value 616.1, found value 616.2.

[0746] Embodiment 105

[0747] 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

[0748]

[0749] The title compound was synthesized in a similar manner to Example 87. 1H NMR (400MHz, DMSO-d6) δ8.95(d,J=6.4Hz,1H),8.26(s,1H),6.01(d,J=4.2Hz,1H),4.69-4.59(m,1H),4.50(t,J=4.2Hz,1H),4.26(t,J=4. 5Hz,1H),4.15-3.99(m,2H),3.95-3.81(m,3H),3.74(d,J=7.9Hz,1H),3.67-3.58(m,1H),2.35-2.06(m,3H),1.98-1.80(m,1H).ESIMS[MH] - , C 15 H 22 ClN5O 10 P2, calculated value 528.1, measured value 528.2.

[0750] Embodiment 106

[0751] 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)phosphonic acid

[0752]

[0753] The title compound was synthesized in a similar manner to Example 69. 1 H NMR(400MHz,DMSO-d6)δ8.71-8.60(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),4.5 1-4.37(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).ESIMS[MH] - , C 26 H 40 ClN5O 15 P2, calculated value 758.2, measured value 758.3.

[0754] Embodiment 107

[0755] 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)phosphonic acid

[0756]

[0757] The title compound was synthesized in a similar manner to Example 69. 1 H NMR(400MHz, DMSO-d6)δ9.17(d,J=7.9Hz,1H),8.32-8.27(m,1H),7.42-7.28(m,4H),7.26-7.20(m,1H),6.01(d,J=3.7Hz,1H),5.58-5.32 (m,6H),4.84-4.69(m,2H),4.49-4.37(m,1H),4.37-3.81(m,5H),2.72-2.52(m,1H),1.53(d,J=6.9Hz,3H),1.28-1.17(m,12H).ESIMS[MH] - , C 29 H 40 ClN5O 15 P2, calculated value 794.2, measured value 794.2.

[0758] Embodiment 108

[0759] 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

[0760]

[0761] Step a: 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.0 g, 5.3 mmol) was dissolved in anhydrous CH3CN (10 mL), cyclopentylamine (478 mg, 5.6 mmol, 1.05 eq) was added followed by TEA (779 μL, 5.6 mmol, 1.05 eq). The mixture was stirred at room temperature overnight, then anhydrous Cs2CO3 (3.4 g, 10.6 mmol, 2 eq) and bromide (2.2 g, 5.3 mmol) were added. The reaction mixture was stirred at room temperature overnight and then evaporated. The crude residue was dissolved in MeOH (20 mL) and anhydrous K2CO3 (2.2 g, 15.9 mmol, 3 eq) was added. The mixture was stirred at room temperature overnight, evaporated with silica gel and purified by column chromatography (SiO2, Hex→100% EtOAc) to first obtain product B (800 mg, 41%) and then product A (600 mg, 30%). B: ESIMS [M+H] + , C 15 H 20 ClFN5O3, calculated value 372.1, found value 372.2.

[0762] Step b: The phosphonation step is carried out in a similar manner to Example 1, using the product B of step a: 1 HNMR (400MHz, DMSO-d6) δ8.76(d,J=7.2Hz,1H),8.29(s,1H),6.52(d,J=6.5Hz,1H),5.50-5.29(m,1H),4.75(dt,J=18.7,7.5Hz,1H),4.43(h,J= 6.9Hz,1H),4.31-4.22(m,1H),4.18-4.05(m,1H),4.04-3.92(m,1H),2.20(t,J=20.5Hz,2H),2.05-1.93(m,2H),1.80-1.46(m,6H).ESIMS[M+H] + , C 16 H 24 ClFN5O8P2, calculated value 530.1, found value 530.2.

[0763] Embodiment 109

[0764] 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

[0765]

[0766] The title compound was synthesized by a method similar to Example 108. 1 H NMR(400MHz, DMSO-d6)δ9.19(d,J=8.1Hz,1H),8.33(s,1H),7.40(d,J=7.9Hz,2H), 7.33(t,J=7.5Hz,2H),7.23(t,J=7.4Hz,1H),),6.34(dd,J=14.3,4.6Hz,1H),5.39( bs,1H),5.31-5.12(m,1H),5.14(bs,1H),4.48(dt,J=18.5,4.5Hz,1H),4.17(s,3H ),4.01(d,J=5.2Hz,2H),2.24(t,J=20.4Hz,3H),1.51(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O8P2, calculated value 566.1, found value 566.1

[0767] Embodiment 110

[0768] 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

[0769]

[0770] The title compound was synthesized by a method similar to Example 108. 1 H NMR(400MHz,DMSO-d6)δ9.18(d,J=8.0Hz,1H),8.33(d,J=1.2Hz,1H),7.40( d,J=7.9Hz,2H),7.33(t,J=7.3Hz,2H),7.24(t,J=7.6Hz,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,1 H),3.94(bs,1H),2.17(t,J=20.5Hz,2H),1.53(d,J=7.1Hz,3H).ESIMS[M+H] + , C 19 H 24 ClFN5O8P2, calculated value 566.1, found value 566.2

[0771] Embodiment 111

[0772] 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

[0773]

[0774] The title compound was synthesized by a method similar to Example 108. 1 H NMR (400MHz, DMSO-d6) δ9.27 (d, J = 7.6Hz, 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=6.6Hz,1H),5.59(p,J=7.1Hz,1H),5 .49-5.26(m,1H),4.74(dt,J=18.4,7.6Hz,1H),4.30-4.17(m,1H),4.15-4.02(m, 1H),3.99-3.90(m,1H),2.17(t,J=20.5Hz,2H),1.54(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2.

[0775] Embodiment 112

[0776] 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

[0777]

[0778] The title compound was synthesized by a method similar to Example 108. 1H NMR (400MHz, DMSO-d6) δ9.37-9.16(m,1H),8.36(d,J=3.4Hz,1H),7.44(d,J=8.4Hz,1H ),7.29(t,J=7.0Hz,1H),7.18(dt,J=10.6,5.6Hz,2H),6.51(t,J=4.8Hz,1H),5.60(t, J=6.8Hz,1H),5.53-5.22(m,1H),4.84-4.64(m,1H),4.31-4.16(m,1H),4.16-4.00(m, 1H),3.94(p,J=3.7Hz,1H),2.18(t,J=20.4Hz,2H),1.54(d,J=7.2Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2

[0779] Embodiment 113

[0780] 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

[0781]

[0782] The title compound was synthesized by a method similar to Example 108. 1 H NMR(400MHz,DMSO-d6)δ9.21(d,J=7.8Hz,1H),8.33(s,1H),7.46-7.30(m,1 H),7.27-7.16(m,2H),7.12-7.00(m,1H),6.51(d,J=6.5Hz,1H),5.49-5.26( m,2H),4.74(dt,J=18.4,7.6Hz,1H),4.29-4.18(m,1H),4.14-4.02(m,1H),4 .00-3.89(m,1H),2.17(t,J=20.5Hz,2H),1.54(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2.

[0783] Embodiment 114

[0784] 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

[0785]

[0786] The title compound was synthesized by a method similar to Example 108. 1 H NMR(400MHz,DMSO-d6)δ9.21(d,J=8.0Hz,1H),8.34(s,1H),7.42-7.32(m,1 H),7.27-7.20(m,2H),7.12-7.03(m,1H),6.51(d,J=6.5Hz,1H),5.49-5.29( m,2H),4.74(dt,J=18.8,7.8Hz,1H),4.29-4.18(m,1H),4.15-4.03(m,1H),3 .99-3.90(m,1H),2.17(t,J=20.5Hz,2H),1.54(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2.

[0787] Embodiment 115

[0788] 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

[0789]

[0790] The title compound was synthesized by a method similar to Example 108. 1H NMR(400MHz, DMSO-d6)δ9.22(d,J=7.9Hz,1H),8.34(s,1H),7.45(dd,J=8.7, 5.6Hz,2H),7.17(t,J=8.9Hz,2H),6.52(d,J=6.5Hz,1H),5.53-5.27(m,2H),4 .75(dt,J=18.7,7.6Hz,1H),4.31-4.20(m,1H),4.17-4.04(m,1H),3.97(dd, J=7.5,3.9Hz,1H),2.19(t,J=20.5Hz,2H),1.54(d,J=7.0Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2

[0791] Embodiment 116

[0792] 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

[0793]

[0794] The title compound was synthesized by a method similar to Example 108. 1 H NMR(400MHz,DMSO-d6)δ9.21(d,J=7.9Hz,1H),8.34(s,1H),7.54-7.37(m,2H),7 .17(t,J=8.8Hz,2H),6.52(d,J=6.6Hz,1H),5.60-5.23(m,2H),4.76(dt,J=18.7 ,7.6Hz,1H),4.24(dt,J=7.4,4.8Hz,1H),4.09(dt,J=10.9,7.4Hz,1H),3.96(dt ,J=10.7,5.3Hz,1H),2.19(t,J=20.5Hz,2H),1.54(d,J=7.1Hz,3H).ESIMS[M+H] + , C 19 H 23 ClF2N5O8P2, calculated value 584.1, found value 584.2

[0795] Embodiment 117

[0796] Synthesis of [({[(2R,3S,4R,5R)-5-[5-chloro-7-(cyclopentylamino)-3H-imidazo[4,5-b]pyridin-3-yl]-3,4-dihydroxyoxacyclopentane-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid

[0797]

[0798] Step a: N,O-bis(trimethylsilyl)acetamide (0.523 mL, 2.14 mmol) was added dropwise to a solution of 5,7-dichloroimidazole [4,5-b] pyridine (376 mg, 2 mmol) in MeCN (14 mL) at room temperature, and the reaction mixture was heated at 85°C for 1 hour. The mixture was cooled to room temperature, and a solution of β-D-furanose 1,2,3,5-tetraacetate (726 mg, 2.28 mmol) in MeCN (7 mL) and trimethylsilyl trifluoromethanesulfonate (0.471 mL, 2.60 mmol) were added dropwise in sequence. The reaction mixture was heated at 85°C for 4 hours. The mixture was cooled and aqueous saturated sodium bicarbonate (50 mL) was added, followed by extraction with EtOAc (100 mL) three times, dried over sodium sulfate, and concentrated.

[0799] Step b: Dioxane (2 mL) and cyclopentylamine (0.987 mL, 10 mmol) were added to the residue. The mixture was heated at 100°C for 16 hours. The reaction mixture was loaded onto silica gel and purified by silica gel column chromatography (0-10% MeOH in DCM) to give the desired product as a brown solid (298 mg, 40%).

[0800] Step c: The title compound was synthesized as a white solid (10 mg; 6%) in a similar manner to Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.40(s,1H),7.12(d,J=7.3Hz,1H),6.40(s,1H),5.91(d,J=5.8Hz,1H),4.53(t,J=5.4Hz,1H),4.23- 4.18(m,1H),4.18-4.05(m,3H),2.26(t,J=20.5Hz,2H),2.04-1.91(m,2H),1.76-1.64(m,2H),1.64-1.48(m,4H).ESIMS[MH] - , C 17 H 24 ClN4O9P2, calculated value 525.1, found value 525.2

[0801] Embodiment 118

[0802] 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

[0803]

[0804] Step a: Sodium hydride (130 mg, 3.24 mmol, 60%, oil suspension) was added to a solution of 5,7-dichloroimidazole [4,5-b] pyridine (564 mg, 3 mmol) in MeCN (18 mL) at room temperature. 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 adding methanol (5 mL) and sodium bicarbonate (5 g), filtered through celite, and concentrated.

[0805] Step b: 1) Add dioxane (5 mL) and cyclopentylamine (1.48 mL, 15 mmol) to the residue. Heat the mixture at 100° C. for 20 hours. Cool the reaction mixture to room temperature.

[0806] 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 column chromatography (0-15% MeOH in DCM) to give the desired product as a brown solid (499 mg, 45%).

[0807] Step c: The title compound was synthesized as a white solid (26 mg; 10%) in a similar manner to Example 1. 1 H NMR(400MHz,DMSO-d6)δ8.24(d,J=2.3Hz,1H),7.19(br s,1H),6.42(dd,J=15.4,4.4Hz,1H),6.42(s,1H),5.23(dt,J=52.4,4.1Hz,1H),4.58-4.44(m,1H),4.19(t,J=6.1Hz ,2H),4.08-3.99(m,1H),2.27(t,J=20.5Hz,2H),2.04-1.90(m,2H),1.78-1.64(m,2H),1.64-1.47(m,4H).ESIMS[MH] - , C 17 H 23 ClFN4O8P2, calculated value 527.1, found value 527.2.

[0808] Embodiment 119

[0809] 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

[0810]

[0811] Step a: Ethyl (ethoxymethylene)cyanoacetate (50.5 g, 299.0 mmol) was dissolved in anhydrous EtOH (350 mL), followed by the addition of the hydrazine product (50 g, 328.9 mmol, 1.1 equivalents). The reaction mixture was stirred at reflux overnight and then evaporated. The solid residue was washed with MTBE to give a white solid (55.5 g, 63%), ESIMS [M+H] + , C 14 H 18 N3O3, calculated value 276.1, measured value 276.2.

[0812] Step b: Diethyl malonate (90 mL, 0.59 mole, 4 eq.) was dissolved in anhydrous EtOH (300 mL) and cooled to 0 °C (ice bath). 21% NaOEt in EtOH (220 mL, 0.59 mole, 4 eq.) was added dropwise (over 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 portionwise (over 2 min) and the reaction mixture was refluxed for 5 days and then evaporated. The residue was diluted with H2O (1.2 L) and neutralized with AcOH to pH ~5. The product was filtered off, washed with H2O (200 mL) and dried in vacuo (48.4 g, 96%). ESIMS [M+H] + , C 17 H 18 N3O5, calculated value 344.1, measured value 344.2.

[0813] Step c: The product of step b (48.4 g, 141.1 mmol) was dissolved in 15% aqueous NaOH (500 mL) and refluxed for 5 h. Cooled to 0 °C and slowly neutralized with AcOH to pH ~ 5. The white solid was filtered off, washed with H2O (100 mL) and dried under vacuum (38 g, quantitative). ESIMS [M+H] + , C 14 H 14 N3O3, calculated value 272.1, measured value 272.2.

[0814] Step d: The product of step c (38 g, 140.2 mmol) and phenylphosphonic dichloride (79.5 mL, 560.8 mmol, 4 equiv) were stirred at 170 °C for 7 h, then cooled to ~80 °C and poured into vigorously stirred ice. A brown viscous material precipitated and became solid after extensive stirring. The ice-cold mixture was neutralized with concentrated aqueous NH3 to pH ~7 and the product was extracted with CH2Cl2 (2×400 mL). The combined organics were dried over MgSO4, filtered and evaporated to give the product which was used without further purification (24 g, 55%), ESIMS [M+H] + , C 14 H 12 Cl2N3O, calculated value 308.0, found value 308.1.

[0815] Step e: The product of 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 gray solid was filtered off, washed with saturated NaHCO3 and then H2O and dried in vacuo. ESIMS [M+H] + , C6H4Cl2N3, calculated value 188.0, found value 188.1.

[0816] Step f: The product of step f is synthesized using a method similar to Example 87. 1 H NMR (400MHz, DMSO-d6) δ8.55(s,1H),7.72(s,1H),6.48(d,J=3.0Hz,1H),5.90-5.83(m,1H),5.67-5.61(m,1H),4.46-4.38(m,1 H),4.33(ddd,J=12.1,3.5,1.2Hz,1H),4.05(ddd,J=12.2,5.1,1.2Hz,1H),2.09(s,3H),2.06(s,3H),1.96(s,3H), ESIMS[M+H] + , C 17 H 18 Cl2N3O7, calculated value 446.0, found value 446.1.

[0817] Step g: The product of step g was synthesized by a method similar to that of Example 87. ESIMS [M+H] + , C 16 H 22 ClN4O4, calculated value 369.1, found value 369.2.

[0818] Step h: The title compound was synthesized in a similar manner to Example 87. 1H NMR (400MHz, DMSO-d6) δ8.27(s,1H),7.66(d,J=6.7Hz,1H),6.22(s,1H),6.08(d,J=4.2Hz,1H),4.51(t,J=4.7Hz,1H) ,4.26(t,J=5.1Hz,1H),4.17-3.83(m,4H),2.17(t,J=20.5Hz,2H),2.06-1.92(m,2H),1.77-1.45(m,6H), ESIMS[M+H] + , C 17 H 26 ClN4O9P2, calculated value 527.1, found value 527.2.

[0819] Embodiment 120

[0820] 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

[0821]

[0822] The title compound was synthesized by a method similar to Example 119. 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),8.20(d,J=7.2Hz,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,1 H),4.50(t,J=4.5Hz,1H),4.25(t,J=4.8Hz,1H),4.14-3.97(m,2H),3.93-3.81(m,1H),2.17(t,J=20.5Hz,2H),1.52(d,J=6.2Hz,3H).ESIMS[M+H] + , C 20 H 26 ClN4O9P2, calculated value 563.1, found value 563.2.

[0823] Embodiment 121

[0824] 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

[0825]

[0826] The title compound was synthesized by a method similar to Example 119. 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),8.20(d,J=7.2Hz,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=4.9Hz,1H),4.12-3.95(m,2H),3.91-3.79(m,1H),2.17(t,J=20.5Hz,2H),1.51(d,J=6.6Hz,3H).ESIMS[M+H] + , C 20 H 26 ClN4O9P2, calculated value 563.1, found value 563.2.

[0827] Embodiment 122

[0828] 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

[0829]

[0830] The title compound was synthesized by a method similar to Example 119. 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),8.23(d,J=6.9Hz,1H),7.42-7.34(m,1H),7.33-7.09(m,3H),6.06(d,J=4.3Hz,1H),5.97(s,1H),5.04(s, 1H),4.53-4.47(m,1H),4.25(t,J=4.7Hz,1H),4.13-3.97(m,2H),3.92-3.82(m,1H),2.16(d,J=20.5Hz,2H),1.56(d,J=6.4Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O9P2, calculated value 581.1, found value 581.2.

[0831] Embodiment 123

[0832] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)-methyl]phosphonic acid

[0833]

[0834] The title compound was synthesized by a method similar to Example 119. 1 H NMR (400MHz, DMSO-d6) δ8.36 (s, 1H), 8.18 (d, J = 7.2Hz, 1H), 7.46-7.39 (m, 2H), 7.19-7.10 (m, 2H), 6.13-5.99 (m, 2H), 4.89 (s, 1H), 4.5 3-4.46(m,1H),4.25(t,J=4.8Hz,1H),4.12-3.97(m,2H),3.92-3.81(m,1H),2.18(t,J=20.5Hz,2H),1.50(d,J=7.3Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O9P2, calculated value 581.1, found value 581.2.

[0835] Embodiment 124

[0836] 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

[0837]

[0838] Step a: To a mixture of 4,6-dichloro-1H-pyrazolo[3,4-b]pyridine (2.1 g, 11.1 mmol) and bromide (4.7 g, 11.1 mmol) in anhydrous CH3CN (50 mL) was added Cs2CO3 (4.3 g, 13.3 mmol, 1.2 equiv.), and the reaction mixture was stirred at room temperature overnight. Evaporation with silica gel and purification by column chromatography (SiO2, Hex→Hex:EtOAc, 2:8) gave a white solid (2.5 g, 42%). 1H NMR(400MHz, DMSO-d6)δ8.53(s,1H),8.05(d,J=8.4Hz,2H),7.91(d,J=8.4Hz,2H),7.75-7.65(m,2H),7.65-7.51(m,3H), 7.49-7.41(m,2H),6.96(d,J=6.6Hz,1H),6.45(dt,J=15.8,7.2Hz,1H),6.19-5.97(m,1H),4.78-4.53(m,3H).ESIMS[M+H] + , C 25 H 19 Cl2FN3O5, calculated value 530.1, found value 530.2.

[0839] Step b: A mixture of the product of step a (500 mg, 0.94 mmol), cyclopentylamine (84 mg, 0.99 mmol, 1.05 eq.), TEA (138 L, 0.99 mmol, 1.05 eq.) in anhydrous EtOH (5 mL) was placed in a pressure vial and heated at 110 ° C for 2 days. Cooled to room temperature and K2CO3 (262 mg, 1.9 mmol, 2 eq.) was added, and the reaction mixture was stirred overnight. Evaporated with silica gel and purified by column chromatography (SiO2, Hex→100% EtOAc) to give a white solid (170 mg, 49%). 1 H NMR (400MHz, DMSO-d6) δ8.28(s,1H),7.66(d,J=6.9Hz,1H),6.53(d,J=6.6Hz,1H),6.23(s,1H),5.80(d,J=5.7Hz,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).ESIMS[M+H] + , C 16 H 21 ClFN4O3, calculated value 371.1, found value 371.3.

[0840] Step c: The title compound was synthesized by a method similar to Example 1. 1H NMR (400MHz, DMSO-d6) δ8.30(s,1H),7.69(d,J=6.7Hz,1H),6.55(d,J=6.6Hz,1H),6.23(s,1H),5.48-5.25(m,1H),4.77(dt,J=1 8.1,7.6Hz,1H),4.28-4.18(m,1H),4.13-3.88(m,3H),2.17(t,J=20.5Hz,2H),2.07-1.93(m,2H),1.77-1.44(m,6H).ESIMS[M+H] + , C 17 H 25 ClFN4O8P2, calculated value 529.1, found value 529.1.

[0841] Embodiment 125

[0842] 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

[0843]

[0844] The title compound was synthesized by a method similar to Example 124. 1 H NMR (400MHz, DMSO-d6) δ8.40 (s, 1H), 8.23 ​​(d, J = 7.1Hz, 1H), 7.44-7.37 (m, 2H), 7.37-7.28(m,2H),7.26-7.16(m,1H),6.54(d,J=6.5Hz,1H),6.04(s,1H),5.36( dt,J=53.5,7.1Hz,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=20.5Hz,2H),1.52(d,J=6.4Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O8P2, calculated value 565.1, found value 565.2,

[0845] Embodiment 126

[0846] 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

[0847]

[0848] The title compound was synthesized by a method similar to Example 124. 1 H NMR (400MHz, DMSO-d6) δ8.41 (s, 1H), 8.22 (d, J = 7.1Hz, 1H), 7.43-7.36 (m, 2H), 7.36-7.27(m,2H),7.26-7.17(m,1H),6.54(d,J=6.5Hz,1H),6.04(s,1H),5.37( dt,J=53.7,7.2Hz,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=20.5Hz,2H),1.52(d,J=6.4Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O8P2, calculated value 565.1, found value 565.2.

[0849] Embodiment 127

[0850] 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

[0851]

[0852] Step a: To a mixture of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (14.8 g, 78.9 mmol) and bromide (40 g, 118.3 mmol, 1.5 equiv) in anhydrous CH3CN (600 mL) was added Cs2CO3 (38.6 g, 118.3 mmol, 1.5 equiv) and the reaction mixture was stirred at room temperature overnight. Evaporation with silica gel and purification by column chromatography (SiO2, Hex→Hex:EtOAc, 2:8) gave a white solid (13.8 g, 39%). ESIMS [M+H] + , C 17 H18 Cl2N3O7, calculated value 446.0, found value 446.1.

[0853] Step b and step a were carried out in a similar manner to Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=7.9Hz,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=6.0 Hz,1H),5.58(t,J=7.3Hz,1H),4.29(t,J=5.7Hz,1H),4.14-3.96(m,4H),2.24(t,J=20.5Hz,2H),1.51(d,J=6.9Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O9P2, calculated value 581.1, found value 581.2.

[0854] Embodiment 128

[0855] 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

[0856]

[0857] The title compound was synthesized using a method similar to Example 127: 1 H NMR (400MHz, DMSO-d6) δ8.34(d,J=8.1Hz,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 .0Hz,1H),5.45-5.33(m,1H),4.29(t,J=5.5Hz,1H),4.14-3.98(m,4H),2.24(d,J=20.5Hz,2H),1.51(d,J=6.8Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O9P2, calculated value 581.1, found value 581.2.

[0858] Embodiment 129

[0859] 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

[0860]

[0861] The title compound was synthesized by a method similar to Example 127. 1 H NMR (400MHz, DMSO-d6) δ8.32(d,J=8.0Hz,1H),7.46-7.34(m,3H),7.17-7.08(m,2H),6.76(s,1H),5.97(d,J=6.3Hz,1H) ,5.44-5.33(m,1H),4.29(t,J=5.8Hz,1H),4.14-3.97(m,4H),2.24(d,J=20.5Hz,2H),1.50(d,J=6.9Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O9P2, calculated value 581.1, found value 581.2.

[0862] Embodiment 130

[0863] 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

[0864]

[0865] The title compound was synthesized in a similar manner to Example 65. 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=8.4Hz,1H),7.39(d,J=7.6Hz,2H),7.35-7 .23(m,3H),7.21(t,J=7.2Hz,1H),6.78(s,1H),6.44(dd,J=15.5,4.6Hz,1H), 5.40(t,J=8.1Hz,1H),4.40(dt,J=18.8,4.6Hz,1H),4.13(d,J=6.7Hz,2H),3. 95(q,J=5.1Hz,1H),2.22(t,J=20.3Hz,2H),1.51(d,J=7.0Hz,3H).ESIMS[MH]- , C 209 H 23 ClFN4O8P2, calculated value 563.1, found value 563.2.

[0866] Embodiment 131

[0867] 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

[0868]

[0869] The title compound was synthesized in a similar manner to Example 65. 1 H NMR (400MHz, DMSO-d6) δ8.37(d,J=8.3Hz,1H),7.39(d,J=7.7Hz,2H),7.31(t,J=7.5Hz,2H),7.28-7.16(m,2H),6.79(s,1H),6.44(dd,J=15.8,4.5 Hz,1H),5.39(s,1H),4.39(dt,J=18.7,4.4Hz,1H),4.12(m,2H),3.95(q,J=5.1Hz,1H),2.23(t,J=20.5Hz,2H),1.51(d,J=7.0Hz,3H).ESIMS[M+H] + , C 20 H 25 ClFN4O8P2, calculated value 565.1, found value 565.2.

[0870] Embodiment 132

[0871] Synthesis of [({[(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)-phosphoryl)methyl]phosphonic acid

[0872]

[0873] The title compound was synthesized in a similar manner to Example 65. 1H NMR(400MHz, DMSO-d6)δ8.40(d,J=8.0Hz,1H),7.40-7.16(m,4H),7.03(td,J=8.7 ,2.6Hz,1H),6.78(d,J=3.9Hz,1H),6.45(dd,J=15.7,4.4Hz,1H),5.43-5.34(m,1 H),5.11(dt,J=52.7,4.0Hz,1H),4.38(dq,J=18.7,4.5Hz,1H),4.18-4.06(m,1H) ,3.96(q,J=5.0Hz,1H),2.25(t,J=20.5Hz,2H),1.51(d,J=6.9Hz,3H).ESIMS[MH] - , C 20 H 22 ClF2N4O8P2, calculated value 581.1, found value 581.2.

[0874] Embodiment 133

[0875] 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

[0876]

[0877] The title compound was synthesized in a similar manner to Example 65. 1 H NMR(400MHz, DMSO-d6)δ8.37(d,J=8.1Hz,1H),7.42(dd,J=8.4,5.5Hz,2H),7.26(t, J=3.1Hz,1H),7.22-7.09(m,2H),6.77(d,J=3.6Hz,1H),6.44(dd,J=15.5,4.5Hz,1H) ,5.43-5.34(m,1H),5.14(dt,J=52.7,4.0Hz,1H),4.40(dt,J=18.7,4.3Hz,1H),4.2 0-4.02(m,2H),3.95(q,J=5.1Hz,1H),2.24(t,J=20.5Hz,2H),1.50(d,J=7.0Hz,3H).

[0878] ESIMS[MH] - , C 20 H 22 ClF2N4O8P2, calculated value 581.1, found value 581.2.

[0879] Embodiment 134

[0880] 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

[0881]

[0882] The title compound was synthesized in a similar manner to Example 65. 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=7.7Hz,1H),7.41(dd,J=8.6,5.5Hz,2H),7.26(s,1H),7.18-7.08(m,2H),6.77(s,1H),6.44(dd,J=15.6,4.3Hz,1H ),5.38(s,1H),5.24-4.96(m,1H),4.45-4.34(m,1H),4.13(s,2H),3.95(d ,J=5.2Hz,1H),2.23(t,J=20.4Hz,2H),1.50(d,J=7.1Hz,3H).ESIMS[M+H] + , C 20 H 24 ClF2N4O8P2, calculated value 583.1, found value 583.2.

[0883] Embodiment 135

[0884] 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

[0885]

[0886] The title compound was synthesized in a similar manner to Example 65. 1H NMR (400MHz, DMSO-d6) δ8.46(d,J=7.8Hz,1H),7.44(t,J=7.8Hz,1H),7.28(dt,J=11.3,4.5Hz, 2H),7.17(q,J=8.1,7.4Hz,2H),6.83(d,J=3.9Hz,1H),6.47(dd,J=15.7,4.5Hz,1H),5.60(t,J =7.4Hz,1H),5.13(dt,J=52.5,4.2Hz,1H),4.41(dt,J=18.8,4.4Hz,1H),4.14(td,J=12.0,10. 7,5.7Hz,2H),3.97(q,J=5.0Hz,1H),2.27(t,J=20.5Hz,2H),1.53(d,J=7.0Hz,3H).ESIMS[M+H] + , C 20 H 24 ClF2N4O8P2, calculated value 583.1, found value 583.2.

[0887] Embodiment 136

[0888] 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

[0889]

[0890] The title compound was synthesized in a similar manner to Example 65. 1 H NMR (400MHz, DMSO-d6) δ8.44(s,1H),7.43(t,J=7.8Hz,1H),7.28(s,2H),7.21-7.09(m,2H),6.44(dd,J=15.8,4.7Hz,1H),5.59(s,1H),5.1 4(d,J=52.8Hz,1H),4.40(d,J=18.9Hz,1H),4.13(s,2H),3.95(d,J=5.7Hz,1H),2.24(t,J=20.7Hz,2H),1.51(d,J=6.9Hz,3H).ESIMS[M+H] + , C 20 H 24 ClF2N4O8P2, calculated value 583.1, found value 583.2.

[0891] Embodiment 137

[0892] 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

[0893]

[0894] The title compound was synthesized in a similar manner to Example 65. 1 H NMR(400MHz,DMSO-d6)δ8.39(d,J=8.1Hz,1H),7.35(td,J=8.0,7.5,6.0Hz,1H),7.31- 7.17(m,3H),7.04(td,J=8.6,2.5Hz,1H),6.81-6.75(m,1H),6.45(dd,J=15.6,4.4Hz, 1H),5.44-5.35(m,1H),5.14(dt,J=52.8,4.1Hz,1H),4.40(dt,J=18.8,4.4Hz,1H),4. 19-4.09(m,1H),3.95(q,J=5.0Hz,1H),2.24(t,J=20.5Hz,2H),1.50(d,J=6.9Hz,3H). ESIMS[MH] - , C 20 H 22 ClF2N4O8P2, calculated value 581.1, found value 581.2.

[0895] Embodiment 138

[0896] Synthesis of [({[(2R,3S,4R,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-imidazo[4,5-c]pyridin-1-yl]-3,4-dihydroxyoxacyclopentane-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid

[0897]

[0898] 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-imidazole [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 hours. The reaction was cooled to 0 °C and poured into saturated NaHCO3 solution. The mixture was transferred to a separatory funnel and extracted with DCM (3x). The combined organics were dried over MgSO4 and concentrated to dryness. The crude material (1.4 g) was used without further purification. ESIMS [M+H] + , C 17 H 17 Cl2N3O7, calculated value 446.0, found value 446.1.

[0899] 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 at 80°C overnight. The reaction was cooled to room temperature and concentrated to dryness under reduced pressure. The crude product was redissolved in DCM and purified by column chromatography (SiO2, 0 to 15% MeOH / DCM) to give the desired product (352 mg). ESIMS [M+H] + , C 16 H 21 ClN4O4, calculated value 368.1, found value 369.2.

[0900] Step C: The title compound was synthesized by a method similar to Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.33(s,1H),7.05(s,1H),6.88(s,1H),5.77(d,J=5.9Hz,1H),4.43(br.s,1H),4.28(dd ,J=5.9,5.0Hz,1H),4.21-4.04(m,5H),2.28(t,J=20.5Hz,2H),2.05-1.77(m,2H),1.79-1.45(m,7H).ESIMS[MH] - , C 17 H 25 ClN4O9P2, calculated value 525.1, found value 525.2.

[0901] Embodiment 139

[0902] 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

[0903]

[0904] Step a: A solution of 4,6-dichloro-1H-imidazole [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 Cs2CO3 (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 organics were dried over MgSO4 and concentrated under reduced pressure. The crude product was used without further purification. ESIMS [M+H] + , C 25 H 18 Cl2FN3O5, calculated value 530.1, found value 530.2.

[0905] 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 at 80 °C overnight. The reaction was cooled to room temperature and concentrated to dryness under reduced pressure. The crude product was redissolved in DCM and purified by column chromatography (SiO2, 0 to 15% MeOH / DCM) to give the desired product. ESI MS [M+H] + , C 16 H 20 ClFN4O3, calculated value 371.1, found value 371.2.

[0906] Step c: The title compound was synthesized using a method similar to Example 1: 1 H NMR (400MHz, DMSO-d6) δ8.22(d,J=1.8Hz,1H),7.06(s,2H),6.98-6.82(m,1H),6.37(dd,J=15.9,4.4Hz,1H),5.21(dt,J=52.4,3.8Hz,1 H),4.53-4.33(m,2H),4.21(t,J=5.8Hz,2H),4.00(q,J=4.9Hz,1H),2.28(t,J=20.4Hz,2H),1.93(s,2H),1.74-1.47(m,7H).ESIMS[MH] - , C 17 H 24ClFN4O8P2, calculated value 527.1, found value 527.2.

[0907] Embodiment 140

[0908] 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

[0909]

[0910] The title compound was synthesized by a method similar to Example 139. 1 H NMR (400MHz, DMSO-d6) δ8.24(d,J=1.8Hz,1H),6.98(d,J=0.7Hz,1H),6.39(dd,J=15.4,4.4Hz,1H),5.80(p,J=7.7Hz,1H),5.41-5.03(m,1H),4 .43(ddd,J=19.9,5.4,3.5Hz,1H),4.31-4.14(m,2H),4.01(q,J=4.9Hz,1H),3.15(s,4H),2.29(t,J=20.5Hz,2H),1.94-1.47(m,9H).ESIMS[MH] - , C 18 H 26 ClFN4O8P2, calculated value 541.1, found value 541.2.

[0911] Table 1: Specific Examples (Efficacy: CD73 IC 50 :+ indicates >1μM, ++ indicates 100nM to 1μM, +++ indicates <100nM).

[0912]

[0913]

[0914]

[0915]

[0916]

[0917]

[0918]

[0919]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925]

[0926]

[0927]

[0928]

[0929]

[0930]

[0931]

[0932]

[0933]

[0934]

[0935]

[0936]

[0937]

[0938]

[0939]

[0940]

[0941]

[0942] Biological experiments

[0943] Materials and methods

[0944] The following general materials and methods were used where indicated or in the examples below:

[0945] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley & Sons, New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)).

[0946] The scientific literature describes 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 glycosylation of proteins (see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley & Sons, New York).

[0947] Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (see, e.g., GCG Wisconsin package (Accelrys, Inc., San Diego, CA); and DeCypher TM (TimeLogic, Inc., Crystal Bay, Nevada).

[0948] There is a wealth of assays and other experimental techniques in the literature that can be used as a basis for evaluating the compounds described herein.

[0949] Inhibits ecto-5'-nucleotidase activity Compounds were evaluated to determine their ecto-5′-nucleotidase (CD73) inhibitory activity. Briefly, CHO-K1 cells stably transfected with human CD73 were generated by LakePharma (Belmont, CA) using a molecular clone of human CD73 (http: / / www.uniprot.org / uniprot / P21589) and a mammalian transient expression vector (P21589.1). After antibiotic selection in CD OptiCHO cell culture medium (Invitrogen, Catalog #12681-011) containing 5 μg / mL puromycin and 200 μg / mL hygromycin B, suspension pools of CHO-CD73 cells were collected and frozen in 7.5% DMSO in cell culture medium without antibiotics.

[0950] On the day of the experiment, a vial of CHO-CD73 cells was thawed and suspended in an assay medium consisting of 20mM HEPES, pH 7.4, 137mM NaCl, 5.4mM KCl, 1.3mM CaCl2, 4.2mM NaHCO3, and 0.1% glucose. To test the ability of the compounds to inhibit CD73 enzymatic activity, 2 μL of 500 μM compound dissolved in DMSO (50x) was added to a 96-well polystyrene plate containing 58 μL of assay buffer. Next, 20 μL of CHO-CD73 cells in assay buffer were added to the assay plate, followed by 20 μL of 125 μM AMP (adenosine 5′-monophosphate monohydrate) in assay buffer. The final assay conditions consisted of 2500 cells per well in 2% DMSO and 25 μM AMP substrate. After incubation for 50 minutes (37°C and 5% CO2) and centrifugation at 225×g for 5 minutes, 80 μL of the supernatant was transferred to a 96-well Spectra Plate (PerkinElmer, cat# 6005640) pre-dispensed with 20 μL PiColorLock Gold colorimetric assay reagent (Thermo, catalog number 3030030). The amount of inorganic phosphate was determined by reading the absorbance at 620 nm on an EnVision Multilabel plate reader (PerkinElmer). The enzymatic activity of CD73 is based on the amount of phosphate produced. The percent activity was calculated based on control wells with DMSO and no cells. The IC of the compound was determined by a four-parameter nonlinear regression fit of the percent activity in GraphPad Prism software 50 value.

[0951] Pharmacodynamic and pharmacokinetic evaluation Pharmacodynamic assays can be based on the measurement of CD73-mediated adenosine serum levels. Adenosine levels can be determined by HPLC analysis, and serum compound levels can also be optionally determined in the same HPLC run.

[0952] Specific embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. After reading the foregoing description, changes in the disclosed embodiments may become apparent to those skilled in the art, and it is expected that such changes may be appropriately adopted by those skilled in the art. Therefore, the present invention is intended to be implemented in a manner different from that specifically described herein, and the present invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

[0953] All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A compound having the formula: or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Each R 1 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R 2 R 2 )-OC(O)-OR 3 The group consisting of two R 1 optionally combined to form a 5- to 7-membered ring; Each R 2 independently selected from the group consisting of H and optionally substituted C1-C6 alkyl; Each R 3 are independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl; R 5 Selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is O; A is selected from the group consisting of: and Het is selected from the group consisting of: where the wavy line indicates the point of attachment to the rest of the compound, and where: R a Selected from H, NH2, NHR 7 、NHC(O)R 7 NR 7 R 7 , R 7 , OH, SR 7 OR 7 The group formed; R b Selected from H, halogen, NH2, NHR 7 NR 7 R 7 , R 7 , OH and OR 7 The group formed; R c and R d are independently selected from H, halogen, haloalkyl, NH2, NHR 7 NR 7 R 7 , R 7 , OH, OR 7 , SR 7 、O2R 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 The group formed; R e and R f independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; Each X 1 is C1-C4 alkylene; and Each R 7 are independently selected from the group consisting of: optionally substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-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 cycloheteroalkylC1-C4 alkyl, optionally substituted aryl, optionally substituted arylC1-C4 alkyl, optionally substituted arylC2-C4 alkenyl, optionally substituted arylC2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroarylC1-C4 alkyl, optionally substituted heteroarylC1-C4 alkenyl, optionally substituted heteroarylC2-C4 alkynyl, and optionally, two R attached to the nitrogen atom 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring which is optionally fused to an aromatic ring.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein A is 3. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Het is selected from the group consisting of:

4. The compound of claim 1 having the formula: or a pharmaceutically acceptable salt, hydrate or solvate thereof.

5. The compound according to claim 4, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R a Selected from NH2, NHR 7 NR 7 R 7 , SR 7 OR 7 The group composed of.

6. The compound according to claim 4, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R c Selected from halogen, R 7 , OR 7 , SR 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 The group composed of.

7. The compound according to claim 4, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R e For H.

8. A compound having the formula: or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Each R 1 are independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl and -C(R 2 R 2 )-OC(O)-OR 3 The group consisting of two R 1 optionally combined to form a 5- to 7-membered ring; Each R 2 independently selected from the group consisting of H and optionally substituted C1-C6 alkyl; Each R 3 are independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl; R 5 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 selected from the group consisting of: Each of which is optionally replaced by 1 to 5 R 6 The substituent is substituted, and wherein the subscript n is an integer from 0 to 3; Z is selected from CH2, CHR 6 NR 6 and O constitute the group; Each R 6 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 linked together to form a 5- to 6-membered ring containing at least one heteroatom as a ring vertex; and Het is selected from the group consisting of: where the wavy line indicates the point of attachment to the rest of the compound, and where: R a Selected from H, NH2, NHR 7 、NHC(O)R 7 NR 7 R 7 , R 7 , OH, SR 7 OR 7 The group formed; R b Selected from H, halogen, NH2, NHR 7 NR 7 R 7 , R 7 , OH and OR 7 The group formed; R c and R d are independently selected from H, halogen, haloalkyl, NH2, NHR 7 NR 7 R 7 , R 7 , OH, OR 7 , SR 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 The group formed; R e and R f independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; Each X 1 is C1-C4 alkylene; and Each R 7 are independently selected from the group consisting of: optionally substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-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 cycloheteroalkylC1-C4 alkyl, optionally substituted aryl, optionally substituted arylC1-C4 alkyl, optionally substituted arylC2-C4 alkenyl, optionally substituted arylC2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroarylC1-C4 alkyl, optionally substituted heteroarylC1-C4 alkenyl, optionally substituted heteroarylC2-C4 alkynyl, and optionally, two R attached to the nitrogen atom 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring optionally fused to an aromatic ring; Provided that the compounds are not those compounds in which X, A and Het are combined to give, Where R g H or two R g The groups combine to form an acetonide; and any of the following: (i)R c and R e is hydrogen and R a is -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, R a is -NH2, and R e is bromine, chlorine, aminomethyl or thioethyl; or (iii) R c is hydrogen, R a is benzylamino, and R e is bromine; or (iv) R c is amino, R e is hydrogen, and R a is -NH2, dimethylamino, diethylamino, benzylamino or N-benzyl-N-methylamino; or (v)R c is chlorine, 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 For iodine, R e is hydrogen, and R a is -NH2, benzylamino or N-benzyl-N-methylamino; or (vii) R a is amino, R e is hydrogen, and R c It is piperazinyl, thioallyl or cyclohexylethylthio.

9. The compound of claim 8, wherein A is: It is composed of 1 to 5 R 6 Optional substitution.

10. The compound of claim 9, wherein A is selected from the group consisting of:

11. The compound of claim 8, wherein Het is:

12. The compound of claim 9, wherein Het has the formula:

13. The compound of claim 12, wherein R c Not hydrogen.

14. The compound of claim 13, having a residue selected from the group consisting of the following formula: Each R g Independently selected from the group consisting of H and C(O)-C1-C6 alkyl.

15. The compound of claim 14, wherein X is oxygen.

16. The compound of claim 15, wherein R e For hydrogen.

17. The compound of claim 16, wherein each R g For hydrogen.

18. The compound of claim 8, wherein R 5 For H.

19. The compound of claim 8, wherein R 5 is H, and X is O.

20. The compound of claim 8, wherein R 5 is H, X is O, and each R 1 For H.

21. The compound of claim 8, wherein Het is selected from:

22. The compound of claim 21, wherein R 5 is H, X is O, and each R 1 For H.

23. The compound of claim 21, wherein R 5 is H, X is O, each R 1 For H, R e for H, and R a Selected from NH2, NHR 7 and N(R 7 )2.

24. The compound of claim 21, wherein R 5 is H, X is O, each R 1 For H, R e For H, R c Not for H, and R a NHR 7 .

25. The compound of claim 14 having the formula:

26. The compound of claim 14 having the formula:

27. The compound of claim 25 having the formula:

28. The compound of claim 26 having the formula:

29. The compound of claim 8, selected from the compounds of Table 1.

30. A pharmaceutical composition comprising the compound of claim 8 and a pharmaceutically acceptable excipient.

31. A method of treating a disease, disorder or condition mediated at least in part by CD73, the method comprising administering to a subject in need thereof an effective amount of a compound of claim 8.

32. The method of claim 31, wherein the compound is administered in an amount effective to reverse or stop CD73-mediated immunosuppressive processes.

33. The method of claim 31, wherein the disease, disorder or condition is cancer.

34. The method of claim 33, wherein the cancer is prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, stomach cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal carcinoma), mesothelial lining cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), adrenal cancer, thyroid cancer, kidney or bone; or is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin or testicular seminoma.

35. The method of claim 33, wherein the cancer is selected from the group consisting of melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumor, lymphoma, ovarian cancer, and Kaposi's sarcoma.

36. The method of claim 31, wherein the disease, disorder or condition is an immune-related disease, disorder or condition 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, infection, Crohn's disease, ulcerative colitis, allergic contact dermatitis and other eczema, systemic sclerosis, and multiple sclerosis.

37. A combination comprising a compound as claimed in claim 8, and at least one additional therapeutic agent.

38. The combination of claim 37, wherein the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and / or inflammatory modulator, an anti-hypercholesterolemic agent, or an anti-infective agent.

39. The combination of claim 37, wherein the at least one additional therapeutic agent is an immune checkpoint inhibitor.

40. A kit comprising the compound of claim 8, and at least one additional therapeutic agent.

41. The kit of claim 40, wherein the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and / or inflammatory modulator, an anti-hypercholesterolemic agent, or an anti-infective agent.

42. The kit of claim 40, wherein the at least one additional therapeutic agent is an immune checkpoint inhibitor.

43. A method of treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound of claim 8 and an immune checkpoint inhibitor.

44. The method of claim 43, wherein the administering is performed before, during or after radiation therapy.

45. The method of claim 43, wherein the compound and the immune checkpoint inhibitor are administered in combination.

46. ​​The method of claim 43, wherein the compound and the immune checkpoint inhibitor are administered sequentially.

47. The method of claim 43, wherein the compound is administered after the immune checkpoint inhibitor.

48. The method of claim 43, wherein the compound is administered prior to the immune checkpoint inhibitor.

49. Any one of the composition of claim 39, the kit of claim 42, and the method of claim 43, wherein the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, and lalizumab.

50. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a compound of claim 29 and an immune checkpoint inhibitor.

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