CN112771061A

CN112771061A - CD73 inhibitor and pharmaceutical application thereof

Info

Publication number: CN112771061A
Application number: CN201980059602.5A
Authority: CN
Inventors: 吕佳声; 顾家敏; 吴冬冬; 陈刚; 孙成勇; 吉祥; 王林; 周峰; 张秀春; 孔宪起
Original assignee: Risen Suzhou Pharma Tech Co Ltd
Current assignee: Risen Suzhou Pharma Tech Co Ltd
Priority date: 2018-09-11
Filing date: 2019-09-10
Publication date: 2021-05-07
Anticipated expiration: 2039-09-10
Also published as: JP7192130B2; WO2020051686A1; KR20210068036A; CN110885352B; JP2021527139A; CA3111450A1; CN110885352A; CN112771061B; BR112021004435A2

Abstract

The present invention provides CD73 (also referred to as extracellular-5' -nucleotidase) inhibitor compounds, as well as compositions and uses thereof in the treatment or prevention of CD73 related or related diseases, disorders and conditions, including cancer related and immune related disorders. CD73 inhibitorThe preparation compound comprises a compound with a structure shown in a formula I and pharmaceutically acceptable ester or salt thereof.

Description

CD73 inhibitor and pharmaceutical application thereof

Cross Reference to Related Applications

This application claims the benefit of priority from U.S. provisional application No.62/773,267 filed on day 11, month 30, 2018 and chinese patent application No.201811057145.x filed on day 11, month 9, 2018, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to compounds and compositions that inhibit CD73 (extracellular-5' -nucleotidase) and their use for the treatment and/or prevention of CD73 related or associated diseases, disorders, and conditions, including cancer-related and immune-related disorders.

Background

Extracellular nucleotidases are a group of extracellular enzymes located on the cell surface. Members of the extracellular nucleotidase family include extracellular-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), extracellular-nucleoside triphosphate diphosphohydrolases (E-NTPDases), extracellular-5' -nucleotidase (E5NT, also known as CD73), and Alkaline Phosphatase (AP). These enzymes hydrolyze various extracellular nucleotides to nucleosides including adenosine. Extracellular nucleotides are important signaling molecules that trigger cellular responses by acting on their respective receptors (e.g., adenosine activates the P1 receptor, and nucleotides (ADP, ATP) activates the P2 receptor). Adenosine 5' -monophosphate (AMP) is the primary substrate for CD73, and its hydrolysis product is adenosine. Adenosine is ubiquitous in the body and is an important regulator of purinergic cell signaling that is critical to many physiological and pathophysiological processes.

A large body of data indicates that CD73 has enzymatic activity in cancer development and metastasis. CD73 is upregulated in many cancer cell types and tumors, and expression of CD73 has been shown to be associated with tumor neovascularization, invasion, and metastasis. The hydrolytic cascade from extracellular ATP to adenosine is an important immunosuppressive regulatory pathway in the tumor microenvironment. CD73 overexpression impairs adaptive anti-tumor immune responses and promotes tumor growth and metastasis. Extracellular adenosine is also involved in the regulation of the adaptive response to hypoxia. Reduction of e5NT activity with monoclonal antibodies, siRNA and small molecule inhibitors including AMPCP (adenosine [ (α, β) -methylene ] diphosphate) has been shown to attenuate tumor growth and metastasis (see, e.g., Zhou et al, Oncol. Rep.17(2007): 1341-. Tumor growth was also impaired in CD 73-deficient mice, and it has been demonstrated that these effects are primarily due to a reduction in adenosine production in these mice. Thus, the therapeutic potential of CD73 inhibitors against cancer has been actively explored (see, e.g., m.al-Rashida et al, eur.j.med.chem.,115(2016) (484): 484-494, and references therein).

Tumor cells overcome the anti-tumor response, in part by immunosuppressive mechanisms. Several such immune-regulatory mechanisms are known. Among these immune regulatory mechanisms, adenosine is a key factor that can be produced by cancer cells and immune cells in the tumor microenvironment and used to suppress anti-tumor responses. Adenosine Triphosphate (ATP) is catalyzed by two cell surface proteins, CD73 and CD39, to produce adenosine, and this process is enhanced under conditions of metabolic stress, such as tumor hypoxia. Adenosine exerts its immunomodulatory functions through four adenosine receptors (ARs, referred to as a1, A2A, A2B, and A3, respectively) expressed in various immune cells. Overexpression of adenosine producing enzymes (e.g., CD73 and AR) has been associated with tumor development in a variety of cancer types. Since AR signaling increases tumor progression, modulation of this signaling represents a promising therapeutic approach for cancer (m.h. kazemi, et al., j.cell. physiol.,233(2018): 2032-.

As described above, ectonucleotidase is an enzyme located on the surface of cells and used to regulate purinergic (or pyrimidineergic) signaling pathways. The extracellular nucleotidase family exists in four different forms: extracellular nucleotide diphosphodihydrolase (CD39), extracellular nucleotide pyrophosphatase/phosphodiesterase, alkaline phosphatase, and extracellular-5' -nucleotidase (e5NT, also known as CD 73). CD73 is a glycosyl phosphatidylinositol-anchored di-zinc metal phosphatase. CD73 catalyzes the dephosphorylation of extracellular Adenosine Monophosphate (AMP) to produce adenosine. It cooperates with CD39 to form an extracellular enzyme cascade, converting ATP to adenosine. The process of converting AMP to adenosine, catalyzed by CD73, is thought to be a major factor in the elevation of extracellular adenosine levels in the tumor microenvironment (Stagg, j.et al, proc.natl.acad.sci.usa.:107(2010): 1547-. The expression of CD73 is directly upregulated by hypoxia inducible factor-1 α, which explains the increase in extracellular adenosine observed in hypoxic malignancies. CD73 is also expressed by regulatory T cells (Tregs) and promotes Treg-mediated immunosuppression (Stagg J, et al, Cancer Res.71(2011): 2892-2900). In addition, CD73 was induced by transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), Hepatocyte Growth Factor (HGF), interleukin-6 (IL-6), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3(STAT3), interleukin-2 (IL-2), retinoic acid, int/Wingless (WNT), epithelial to mesenchymal transition, and p53 mutations. CD73 is overexpressed in a variety of tumor types and promotes tumor cell invasion, metastasis, and adhesion. CD73 is also associated with immune tolerance and poor prognosis in cancer. Thus, CD73 is a promising target for the development of anticancer drugs. In addition, inhibitors of CD73 have potential in the treatment of other diseases mediated by adenosine and its receptors (y. -p. gong, et al, Expert opin. ther. Pat.,28(2018): 167-charge 171).

The adenosine pathway is also considered to be a major immunosuppressive component of many human tumors (see, e.g., Whiteside, t.l., Expert rev.anticancer ther.,17(2017): 527-535). Adenosine and inosine are key immune checkpoints in cancer. The cooperation of adenosine and the PGE2 pathway in the tumor microenvironment contributes to the suppression of anti-tumor immune effector cells. Thus, targeting the adenosine pathway with pharmacological inhibitors or antibodies is a promising therapeutic strategy in cancer.

In preclinical in vivo studies, activity that blocks extracellular nucleotidase or adenosine receptor signaling has been successful in inhibiting tumor growth and metastasis. The use of adenosine pathway blockade alone or in combination with other immunotherapies (including checkpoint inhibitors) is now in the initial phase I clinical trial in patients with advanced malignancies.

Small molecule inhibitors of CD73 have been reported. For example, Adams et al (international PCT application publication WO2017/098421) describe substituted benzothiadiazine derivatives, which are CD73 inhibitors, pharmaceutical compositions thereof, and their use in treating cancer, precancerous syndrome, and diseases associated with CD73 inhibition.

Debien et al (International PCT application publication WO 2017/120508; U.S. patent application publication US2017/0267710) describe compounds that modulate the conversion of AMP to adenosine by extracellular-5 '-nucleotidase, compositions containing the compounds, methods of synthesizing the compounds, and the use of the compounds and compositions for the treatment and/or prevention of various diseases mediated by extracellular-5' -nucleotidase.

Cacataian et al (International PCT application publication WO 2015/164573) describe purine derivatives and pharmaceutical compositions thereof, which are inhibitors of CD73 and are useful in the treatment of cancer.

Chen et al (International PCT application publication WO 2018/049145) disclose the preparation of nucleotides as extracellular nucleotidase inhibitors and the use of these compounds in the treatment or prevention of cancer.

The contents of all articles and references cited herein are incorporated by reference in their entirety.

Disclosure of Invention

The present invention relates to compounds and compositions that: which comprises a compound that inhibits the activity of extracellular-5' -nucleotidase (also known as e5NT, CD73, NT5E and 5 NT). Inhibition of CD73 enzymatic activity results in the inhibition or modulation of extracellular adenosine levels, thereby modulating the physiological environment of cells and tissues.

The invention also relates to the use of such compounds and compositions in the treatment and/or prevention of diseases, disorders, and conditions mediated in whole or in part by CD 73. CD73 inhibitors have been used in the treatment of a number of diseases, including cancer, fibrosis, neurological and neurodegenerative diseases (e.g., depression and parkinson's disease), ischemic cardiovascular and cerebrovascular diseases, immune related diseases and inflammatory related diseases. In particular embodiments, the CD73 inhibitor compounds and compositions described herein may act to inhibit the immunosuppressive and/or anti-inflammatory activity of CD73, and may be used as therapeutic agents or prophylactic therapies when such inhibition is desired.

In a first broad aspect, the invention provides compounds of formula I, and pharmaceutically acceptable salts or esters thereof:

wherein W is oxygen, sulfur, nitrogen or methylene; x is selected from phosphoryl (-P (═ O) (OR) -), sulfonyl (-S (═ O)₂-) and a carbonyl (-C (═ O) -) fragment, wherein R is hydrogen, an ester-forming group, or a protecting group; y is selected from phosphonate (-PO)₃R₂) Sulfonate (-SO)₃R) and a carboxylate (-CO)₂R), wherein R is hydrogen, an ester-forming group, or a protecting group; r¹Selected from hydroxyl or hydrogen; r²Is chlorine or hydrogen; and R³And R⁴Independently selected from hydrogen, alkyl, alkenyl and alkynyl, wherein R³And R⁴Has 11 to 30 carbon atoms.

In one embodiment, R³And R⁴Independently selected from hydrogen and a ring system containing a bicyclic, tricyclic, spirocyclic, fused or bridged ring carbocyclic (aromatic or non-aromatic) or heterocyclic ring system, said ring system being substituted or unsubstituted, with the proviso that R is³And R⁴Not hydrogen at the same time.

In yet another embodiment, R³Is hydrogen or lower alkyl (e.g. C)_1-6) And R is⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is C_11-30Alkyl radical, C_11-30Alkenyl or C_11-30Alkynyl.

In some embodiments, R³Is hydrogen or lower alkyl, and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is a ring system which is a carbocyclic (aromatic or non-aromatic) or heterocyclic ring system containing bicyclic, tricyclic, spirocyclic, fused, or bridged rings, and which is substituted or unsubstituted.

In one embodiment, the present invention provides compounds of formula II and/or formula III, and pharmaceutically acceptable salts or esters thereof:

wherein, W, X, Y, R¹、R³And R⁴As defined above.

In another embodiment, the present invention provides a compound of formula IV, and pharmaceutically acceptable salts or esters thereof:

wherein, X, Y, R¹、R²、R³And R⁴As defined above.

In another embodiment, the present invention provides a compound of formula V and/or formula VI, and pharmaceutically acceptable salts or esters thereof:

wherein, X, Y, R¹、R³And R⁴As defined above.

In another embodiment, the present invention provides compounds of formula VII and/or formula VIII, and pharmaceutically acceptable salts or esters thereof:

wherein R is hydrogen, an ester-forming group, or a protecting group; and R is¹、R²、R³And R⁴As defined above.

In another embodiment, the invention provides a compound of formula IX and/or XI, and pharmaceutically acceptable salts or esters thereof:

wherein R is hydrogen, an ester-forming group, or a protecting group; and R¹、R²、R³And R⁴As defined above.

In one embodiment, R¹Is a hydroxyl group (i.e., the carbohydrate moiety in the compound is a D-ribose moiety). In yet another embodiment, R¹Is hydrogen (i.e., the carbohydrate moiety in the compound is a 2-deoxy-D-ribose moiety).

In yet another embodiment, R²Is hydrogen. In yet another embodiment, R²Is hydrogen and R¹Is hydroxy (i.e., the compound is an adenosine derivative). In another embodiment, R²Is hydrogen and R¹Is hydrogen (i.e., the compound is a deoxyadenosine derivative). In yet another embodiment, R²Is hydrogen and R³And R⁴None are hydrogen (i.e., the compound is an adenosine derivative or deoxyadenosine derivative having a substituent on the amino group of the adenine moiety). In other embodiments, R²Is chloro and the compound is a 2-chloro-D-adenosine derivative or a 2-chloro-D-deoxyadenosine derivative.

In some embodiments, R³Is hydrogen or lower alkyl (e.g. C)_1-6) And R is⁴Is an alkyl, alkenyl or alkynyl group having 11 to 30 carbon atoms (i.e., C)_11-30Alkyl radical, C_11-30Alkenyl or C_11-30Alkynyl). In some embodiments, R³Is hydrogen or lower alkyl and R⁴Is a group containing adamantyl moieties. R⁴It may be, for example, a substituted or unsubstituted 1-adamantyl group, a substituted or unsubstituted 2-adamantyl group, a substituted or unsubstituted 1-adamantylmethyl group, a substituted or unsubstituted 1-adamantylethyl group, a substituted or unsubstituted 1-adamantylpropyl group, or a substituted or unsubstituted 1-adamantylbutyl group. In some embodiments, R³Is hydrogen or lower alkyl, and R⁴Is a group containing a naphthyl moiety. R⁴Can be, for example, substituted or unsubstituted α -naphthyl, substituted or unsubstituted β -naphthyl, substituted or unsubstituted α -naphthylA methyl group, a substituted or unsubstituted β -naphthylmethyl group, a substituted or unsubstituted naphthylethyl group, a substituted or unsubstituted naphthylpropyl group, or a substituted or unsubstituted naphthylbutyl group.

In another embodiment, R³Is hydrogen or lower alkyl, and R⁴Is a substituent comprising a bicyclic, tricyclic, or polycyclic ring system, wherein the ring system is a fused ring system, a spiro ring system, a bridged ring system, or a parallel (parallel) ring system, and the ring system is a carbocyclic ring, an aliphatic ring, an aromatic ring, a heterocyclic ring, or a combination thereof.

In yet another embodiment, R³Is hydrogen or lower alkyl, and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is an alkyl, alkenyl or alkynyl group having 11 to 30 carbon atoms.

In some embodiments, R³Is hydrogen or lower alkyl, and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is a substituent comprising a bicyclic, tricyclic, or polycyclic ring system, wherein the ring system is a fused ring system, a spiro ring system, a bridged ring system, or a parallel (parallel) ring system, and wherein the ring system is a carbocyclic ring, an aliphatic ring, an aromatic ring, a heterocyclic ring, or a combination thereof.

In one embodiment, R⁴Is a group containing adamantyl moieties. In yet another embodiment, R⁴Is a substituted or unsubstituted 1-adamantyl or 2-adamantyl group. In another embodiment, R⁴Is a substituted or unsubstituted 1-adamantylmethyl group. In some embodiments, R⁴Is 1-adamantylethyl, 1-adamantylpropyl or 1-adamantylbutyl, wherein the adamantyl moiety can be substituted or unsubstituted.

In another embodiment, R⁴Is a group containing a naphthyl moiety. In yet another embodiment, R⁴Is a substituted or unsubstituted alpha-naphthyl or beta-naphthyl. In other embodiments, R⁴Is alpha-naphthylmethyl or beta-naphthylmethyl, with or without additional substituents. In yet another embodiment, R⁴Selected from naphthalenePhenylethyl, naphthylpropyl and naphthylbutyl, wherein the naphthyl moiety can be unsubstituted or substituted.

In yet another embodiment, R³、R⁴Together with the nitrogen to which they are attached form a ternary fused ring system such as, but not limited to, a substituted or unsubstituted carbazolyl moiety.

In some embodiments, compounds of table 1 and pharmaceutically acceptable salts or esters thereof are provided.

Table 1: structures of exemplary Compounds

In some embodiments, the above compounds are provided wherein the C, H, O and N atoms are each independently selected from the group consisting of naturally abundant atoms and isotopically enriched atoms. Isotopically enriched atoms include, but are not limited to, those of carbon¹²C、¹³C and¹⁴c; of hydrogen¹H、²H and³h; of oxygen¹⁶O、¹⁷O and¹⁸o; and of nitrogen¹⁴N and¹⁵N。

in a second broad aspect, the present invention provides a pharmaceutical composition comprising: a compound as defined herein, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a compound of formula I through formula IX, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a compound of formula I through formula IX, or a pharmaceutically acceptable salt or ester thereof, wherein, in the compound, R³And R⁴One of which is not hydrogen or C1-C10 alkyl, alkene or alkynyl. In some embodiments, there is provided a pharmaceutical composition comprising a compound shown in table 1, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises a cream, an emulsion, a gel, a liposome, or a nanoparticle.

In some embodiments, the pharmaceutically acceptable carrier further comprises at least one additional therapeutic agent, such as, but not limited to, a chemotherapeutic agent, an immune and/or inflammation modulator, an anti-hypercholesterolemic agent, or an anti-infective agent. In one embodiment, the at least one additional therapeutic agent is an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors include capraloma, nivolumab, and lanolinzumab (lambrolizumab).

In a third broad aspect, the present invention provides compounds, compositions and methods of inhibiting CD73 activity in a subject in need thereof, comprising administering to the subject an effective amount of a compound and/or pharmaceutical composition described herein.

In particular embodiments, the compounds of the present invention act to inhibit CD73 immunosuppressive and/or anti-inflammatory activity, and are useful as therapeutic or prophylactic treatments when such inhibition is desired. Unless otherwise indicated, when describing the use of the compounds of the present invention herein, it is to be understood that these compounds may be in the form of compositions (i.e., pharmaceutical compositions). As used herein, the terms "CD 73 inhibitor", "CD 73 blocker", "extracellular-5' -nucleotidase inhibitor of adenosine", "NT 5E inhibitor", "5 NT inhibitor" and all other art-accepted terms are used interchangeably to refer to compounds capable of directly or indirectly inhibiting the CD73 receptor in vitro tests, in vivo models, and/or other tests that represent CD73 inhibition and potential therapeutic or prophylactic efficacy. The term also refers to compounds that exhibit at least some therapeutic or prophylactic benefit in a human subject.

Although the compounds of the present invention are believed to act by inhibiting CD73, the practice of the present invention does not require an accurate understanding of the mechanism of action of the compounds. For example, the compounds may also have effects at least in part by modulating (e.g., inhibiting) other components of the purinergic signaling pathway (e.g., CD 39). The purinergic signaling system consists of transporters, enzymes, and receptors for (primarily) synthesis, release, action, and extracellular inactivation of ATP and its extracellular breakdown product adenosine. Because inhibition of CD73 reduces adenosine production, CD73 inhibitors are useful for treating diseases or disorders mediated by adenosine, and which act on adenosine receptors, including a1, A2A, A2B, and A3.

For the purposes of the present invention, the purinergic signaling process described comprises the following components. Purinergic receptors (P1, P2X, and P2Y) are the first components, membrane receptors that mediate various physiological functions (e.g., relaxation of intestinal smooth muscle) in response to ATP or adenosine release; in general, all cells have the ability to release nucleotides into the extracellular environment by regulating exocytosis. The second component is Nucleoside Transporters (NTs), which 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 in the form of a feedback loop linking receptor signaling with transporter function. As previously described, extracellular nucleotidases (CD73 and CD39) hydrolyze nucleotides released into the extracellular environment and contain additional components.

In some embodiments, the present invention provides methods 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 compound or composition described herein. In some embodiments of such methods, at least one CD73 inhibitor compound or composition is administered to the subject in an amount effective to reverse, slow, or prevent the progression of CD 73-mediated immunosuppression. In some embodiments, CD 73-mediated immunosuppression is mediated by Antigen Presenting Cells (APCs).

The type of cancer or tumor that can be treated or prevented using the compounds and compositions of the present invention is not particularly limited. Examples of cancers and tumors that may be treated or prevented using the compounds and compositions described herein include, but are not limited to, prostate, colorectal, pancreatic, cervical, gastric, endometrial, brain, liver, bladder, ovarian, testicular, head, neck, skin (including melanoma and basal carcinoma), mesothelial, white blood cells (including lymphoma and leukemia), esophagus, breast, muscle, connective tissue, lung (including small cell lung cancer and non-small cell lung cancer), adrenal, thyroid, kidney or bone cancers, gliomas, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, skin basal cell carcinoma and testicular seminoma. In some embodiments of the 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.

In some embodiments, the present invention provides methods of treating a subject receiving a bone marrow transplant or peripheral blood stem cell transplant comprising administering a therapeutically effective amount of a CD73 inhibitor compound or composition sufficient to increase delayed-type hypersensitivity to a tumor antigen, delay the time to recurrence of a malignant tumor after transplantation, increase the survival rate after transplantation without recurrence, and/or increase the long-term survival rate after transplantation.

In certain embodiments, the present invention provides 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 compound or composition described herein. In some embodiments, the infectious disorder is a viral infection (e.g., a chronic viral infection), a bacterial infection, a fungal infection, or a parasitic infection. In certain embodiments, the viral infection is human immunodeficiency virus or cytomegalovirus.

In yet other embodiments, the present invention provides methods of treating and/or preventing immune-related diseases, disorders, and conditions, diseases having an inflammatory component, and disorders related thereto, using at least one CD73 inhibitor compound or composition provided herein.

Other diseases, disorders, and conditions that may be treated or prevented, in whole or in part, by inhibiting CD73 activity are also candidate indications for the CD73 inhibitor compounds and compositions provided herein.

In some embodiments, the present invention further provides the use of the CD73 inhibitor compounds and compositions described herein in combination with one or more additional agents. The one or more additional agents may have some CD 73-modulating activity and/or they may act through different mechanisms of action. In some embodiments, such agents comprise radiation (e.g., local or systemic radiotherapy) and/or other forms of treatment of a non-pharmacological nature. When a combination therapy is used, the CD73 inhibitor and one additional agent may be in the form of a single composition or multiple compositions, and the mode of treatment may be administered simultaneously, sequentially or by some other regimen. For example, in some embodiments, embodiments are provided in which a chemotherapy phase is performed after the irradiation phase. The combination therapy may have additive or synergistic effects.

In some embodiments, the present invention provides the use of a CD73 inhibitor compound or composition described herein in combination with bone marrow transplantation, peripheral blood stem cell transplantation, or other types of transplantation therapy.

In a specific embodiment, the present invention provides the use of an inhibitor of CD73 function in combination with an immune checkpoint inhibitor as described herein. Blocking immune checkpoints, which results in the expansion of antigen-specific T cell responses, is shown to be a promising approach in human cancer therapy. Non-limiting examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various types of tumor cells, are candidates for blockade, include PD1 (programmed cell death protein 1), PDL1(PD1 ligand), BTLA (B and T lymphocyte attenuating agents), CTLA4 (cytotoxic T lymphocyte-associated antigen 4), TIM3(T cell membrane protein 3), LAG3 (lymphocyte activator gene 3), A2aR (adenosine A2a receptor A2aR), and killer inhibitory receptors. Non-limiting examples of immune checkpoint inhibitors include capraloman, nivolumab, and lambrolizumab.

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 compound or composition thereof and at least one chemotherapeutic agent, such chemotherapeutic agents including, but not limited to: alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isoflutolamide, dichloromethyldiethylamine, melphalan, and uracil mustard, aziridines such as thiotepa, methanesulfonates such as busulfan, nucleoside analogs (e.g., gemcitabine), nitrosoureas such as carmustine, lomustine, and streptozotocin, topoisomerase 1 inhibitors (e.g., irinotecan), platinum complexes such as cisplatin and carboplatin, bioreductive alkylating agents such as mitomycin, procarbazine, dacarbazine, and hexamethylmelamine); DNA strand breaking agents (e.g., bleomycin); topoisomerase II inhibitors (such as amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, daunorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., Plicamydin); antimetabolites (e.g., folic acid 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 interacting agents (e.g., vincristine, estramustine, vinblastine, docetaxel, epothilone derivatives, and paclitaxel); hormones (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbestrol; chlormadinone; gestrel; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens, such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone); 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-hormonal antigens (e.g., tamoxifen, anti-androgens such as flutamide, and epinephrine such as mitotane and aminoglutethimide). The present invention also provides 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.

In some embodiments directed to methods of treating cancer, a therapeutically effective amount of a CD73 inhibitor is administered in combination with at least one chemotherapeutic agent such that the observed survival rate for cancer is greater than the survival rate for cancer observed with either agent administered alone. In other embodiments directed to methods of treating cancer, the combined administration of a therapeutically effective amount of a CD73 inhibitor and at least one chemotherapeutic agent results in an observed reduction in tumor size or a slower rate of tumor growth than that observed with either agent administered alone.

In another embodiment, the present invention provides a method for treating or preventing cancer in a subject comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor compound or composition, 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).

In other embodiments, the invention provides methods of enhancing rejection of tumor cells in a subject comprising administering a CD73 inhibitor compound or composition in combination with at least one chemotherapeutic agent and/or radiation therapy, wherein the rejection of tumor cells produced is superior to that produced by administering a CD73 inhibitor, chemotherapeutic agent or radiation therapy alone.

In another embodiment, the present invention provides a method for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one immunomodulatory agent that is different from a CD73 inhibitor. It is to be understood that, as used herein, "CD 73 inhibitor" refers to a compound provided herein (e.g., a compound of any one of formula I through formula IX, a compound of table 1), or a pharmaceutically acceptable salt or ester thereof, as well as pharmaceutical compositions thereof.

In some embodiments, the present invention provides a method of treating or preventing a CD 73-associated disease, disorder or condition in a subject in need thereof, comprising administering to the subject an effective amount of at least one CD73 inhibitor or pharmaceutical composition thereof, thereby treating or preventing the CD 73-associated disease, disorder or condition in the subject. In some embodiments, the compound is administered in an amount effective to reverse, slow, or stop the progression of CD 73-mediated immunosuppression in the subject.

In some embodiments, the CD 73-associated disease, disorder, or condition is a cancer, such as, but not limited to, a cancer of the prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin, mesothelial, leukocytes, esophagus, breast, muscle, connective tissue, lung, adrenal gland, thyroid, kidney, or bone. In some embodiments, the cancer is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, or testicular seminoma. In some embodiments, the cancer is melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumor, lymphoma, ovarian cancer, or kaposi's sarcoma.

In some embodiments, the CD 73-associated disease, disorder, or condition is a disease, disorder, or condition selected from the group consisting of rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergy, fibrosis, anemic fibromyalgia, alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, parkinson's disease, infection, crohn's disease, ulcerative colitis, allergic contact dermatitis, eczema, systemic sclerosis, and multiple sclerosis.

In some embodiments, the methods provided herein further comprise administering at least one additional therapeutic agent to the subject. The at least one additional therapeutic agent may be administered simultaneously or sequentially with a compound or composition described herein. In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and/or inflammation modulator, an anti-hypercholesterolemia agent, or an anti-infective agent. In one embodiment, the at least one additional therapeutic agent is an immune checkpoint inhibitor, such as, but not limited to, yipriomama, nivolumab, or lanolizumab (lambrolizumab).

In some embodiments, the present invention provides 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).

In additional embodiments, treatment of an infectious disease is achieved by the combined administration of a vaccine and the administration of a therapeutically effective amount of a CD73 inhibitor provided herein. 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 yet another embodiment, the vaccine is a tumor vaccine (e.g., a vaccine effective against melanoma); the tumor vaccine may comprise genetically modified tumor cells or 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 particular embodiments, the vaccine comprises one or more immunogenic peptides and/or dendritic cells.

In certain embodiments involving treatment of an infection by administration of a CD73 inhibitor and at least one additional therapeutic agent, the symptoms of the infection observed after administration of both are improved compared to the same symptoms of the infection observed with administration of either of the CD73 inhibitor and the additional therapeutic agent alone. In some embodiments, the observed symptom of the infection may be a reduction in viral load, an increase in CD4+ T cell count, a reduction in opportunistic infections, an increase in survival time, eradication of chronic infections, or a combination thereof.

In some embodiments, the present invention provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a compound or composition described herein and an immune checkpoint inhibitor, thereby treating cancer in the subject. The compounds or compositions described herein and the immune checkpoint inhibitor may be administered in combination or sequentially. The compound or composition may be administered after administration of the immune checkpoint inhibitor or before administration of the immune checkpoint inhibitor. In some embodiments, the compound or composition and/or immune checkpoint inhibitor may be administered prior to, concurrently with, or subsequent to other anti-cancer treatments, such as, but not limited to, radiation therapy. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of pleaprima, nivolumab, and lanolinzumab (lambrolizumab).

In a fourth broad aspect, the invention provides a kit comprising a compound or composition described herein. The kit may further comprise a buffer or excipient, and/or instructions for use thereof. In some embodiments, the kit further comprises at least one additional therapeutic agent, such as, but not limited to, a chemotherapeutic agent, an immune and/or inflammation modulator, an anti-hypercholesterolemia agent, an anti-infective agent, or an immune checkpoint inhibitor.

Drawings

For a better understanding of the invention and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which aspects and features according to embodiments of the invention are illustrated, wherein:

fig. 1 is a CD73 inhibition curve (% inhibition vs. log [ Conc. ]/nM) for compound 9; and

figure 2 is a plot of the CD73 inhibition rate of compound 22.

Detailed Description

The number of patients diagnosed with cancer and the number of deaths from cancer continues to increase. Traditional treatment methods, including chemotherapy and radiation therapy, are often intolerable to patients and are also becoming progressively ineffective as cancers (such as tumors) evolve to circumvent such treatments. Recent experimental evidence suggests that CD73 inhibitors may represent an important novel therapeutic modality for the treatment of cancer (e.g., breast cancer).

Promising data also support the role of CD73 function inhibitors in inhibiting the anti-inflammatory activity of CD73 and/or the immunosuppressive activity of CD73, and therefore CD73 inhibitors are useful, for example, in the treatment of immunosuppressive diseases (e.g., HIV and AIDs). Inhibition of CD73 may also be an important therapeutic strategy for subjects with neurological or neuropsychiatric disorders, or depression.

The invention provides, inter alia, small molecule compounds and compositions thereof having CD73 inhibitory activity, and methods of using the compounds and compositions for the treatment and prevention of the diseases, disorders, and conditions described herein. The compounds provided by the present invention are useful as inhibitors of CD73 and, therefore, are useful in the treatment of diseases, disorders, and conditions in which CD73 activity plays a role. In addition, the compounds provided by the present invention are useful as inhibitors of adenosine receptors (e.g., the A2A receptor). Accordingly, the compounds provided herein are useful for the treatment of diseases, disorders, and conditions associated with the activity of one or more adenosine receptors.

In one embodiment, the invention provides a method of treating a subject (e.g., a human) having a cancer or disorder mediated by CD73, comprising administering to the subject a therapeutically effective amount of a CD73 inhibitor provided herein, e.g., a compound provided herein or a pharmaceutically acceptable composition thereof.

It will be understood that the pharmaceutical compositions comprise a compound disclosed herein (or a pharmaceutically acceptable salt or ester thereof) and a pharmaceutically acceptable carrier, additive or vehicle (vehicle). In certain embodiments, the amount of the compound in the composition is such that it is effective as a CD73 inhibitor in a biological sample (e.g., in vitro assay, in vivo model, etc.) or subject. In certain embodiments, the compositions are formulated for administration to a subject in need of such compositions. In some embodiments, the composition is an injectable formulation. In other embodiments, the composition is formulated for oral administration to a subject.

The invention also provides methods of treating patients suffering from adenosine receptors (e.g., A)₂AR) in a subject (e.g., a human) having a cancer or disorder, comprising the step of administering to the subject a therapeutically effective amount of a CD73 inhibitor provided herein, e.g., a compound provided herein or a pharmaceutically acceptable composition thereof. In certain embodiments, the amount of compound in the composition is such that it is effective as an adenosine receptor (e.g., a) in a biological sample (e.g., an in vitro assay, an in vivo model, etc.) or subject₂AR). In certain embodiments, the compositions are formulated for administration to a subject in need of such compositions. In some embodiments, the composition is an injectable formulation. In other embodiments, the composition is formulated for oral administration to a subject. In some embodiments, the composition is in the form of a hard-shelled, soft-shelled, gelatin capsule, cachet, pill, tablet, lozenge, powder, granule, pellet, or dragee. In some embodiments, the combinationThe substance is in the form of a solution, aqueous liquid suspension, non-aqueous liquid suspension, oil-in-water liquid emulsion, water-in-oil liquid emulsion, elixir, or syrup. In some embodiments, the composition has an enteric coating. In some embodiments, the composition is formulated for controlled release.

In yet another embodiment, the present invention provides a method for 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 signaling 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). The invention also provides a method of enhancing rejection of tumor cells in a subject comprising administering a CD73 inhibitor in combination with at least one chemotherapeutic agent and/or radiation therapy, wherein rejection of tumor cells produced is superior to rejection produced by administration of the CD73 inhibitor, chemotherapeutic agent, or radiation therapy alone. In yet another embodiment, the present invention provides a method for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one immunomodulatory agent that is different from a CD73 inhibitor.

In other embodiments, the present invention provides 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).

Definition of

In order to provide a clear and consistent understanding of the terms used in this specification, some definitions are provided below. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" or "an", but it is also consistent with the meaning of "one or more", "at least one" or "one or more". Similarly, the word "another" may mean at least a second or a plurality.

As used in this specification and claims, the word "comprising" (and any form comprising such as "comprises" and "comprises)", "having" (and any form having such as "having", "containing" (and any form containing such as "containing" and "containing") or "containing" (and any form containing such as "containing" and "containing") is inclusive or open-ended and does not exclude additional, unrecited elements or process steps.

The term "about" is used to indicate that the value includes the inherent variation of error with the instruments and methods used in determining the value.

The term "derivative" as used herein is to be understood as meaning a substance which is structurally similar to the compound and differs in some fine structure.

This specification refers to a number of chemical terms and abbreviations used by those skilled in the art. However, for clarity and consistency, definitions of selected terms are provided.

As used herein, the term "alkyl" generally refers to saturated hydrocarbons having 1 to 30 carbon atoms, including straight chain, branched chain and cyclic alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, isopropyl, tert-butyl, sec-butyl, isobutyl, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The term alkyl includes both unsubstituted alkyl and substituted alkyl. The term "C₁-C_nAlkyl "and" C_1-nAlkyl groups "(where n is an integer from 2 to 30) are used interchangeably and represent alkyl groups having from 1 to the indicated" n "carbon atoms. The alkyl residue may be substituted or unsubstituted. In some embodiments, for example, an alkyl group can be substituted with a hydroxyl, amino, carboxyl, carboxylate, amide, carbamate, or aminoalkyl group, among others. In some embodiments, "alkyl" is modified by a range of carbon atom numbers, and thus specifically defines the size of the alkyl. For example, "C₁₁-C₃₀Alkyl "means an alkyl group containing at least 11 carbon atoms and not more than 30 carbon atoms.

As used herein, the term "acyclic" refers to an organic moiety without a ring system. The term "aliphatic group" includes organic moieties characterized as straight or branched chains, typically having from 1 to 15 carbon atoms. Aliphatic groups include acyclic alkyl, alkenyl, and alkynyl groups.

As used herein, the term "alkenyl" refers to a compound having 2 to 30 carbon atomsUnsaturated hydrocarbons, including linear, branched, and cyclic nonaromatic alkenyl groups, and containing from 1 to 6 carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, 1-propen-2-yl, 1-buten-3-yl, 1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl, 1, 3-pentadien-5-yl, cyclopentenyl, cyclohexenyl, ethylcyclopentenyl, ethylcyclohexenyl, and the like. The term alkenyl includes unsubstituted alkenyls and substituted alkenyls. The term "C₂-C_nAlkenyl "and" C_2-nAlkenyl "(where n is an integer of 3 to 30) is used interchangeably to mean alkenyl having 2 to the indicated" n "carbon atoms. In some embodiments, "alkenyl" is modified by a range of numbers of carbon atoms, and thus the size of the alkenyl is specifically defined. For example, "C₁₁-C₃₀Alkenyl "means an alkenyl group containing at least 11 carbon atoms and containing no more than 30 carbon atoms.

As used herein, "alkynyl" refers to unsaturated hydrocarbons having 2 to 30 carbon atoms, including straight, branched, and cyclic non-aromatic alkynyl groups, and containing 1 to 6 carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propyn-3-yl, 1-butyn-4-yl, 2-butyn-4-yl, 1-pentyn-5-yl, 1, 3-pentadiyn-5-yl, and the like. The term alkynyl includes both unsubstituted alkynyl and substituted alkynyl groups. The term "C₂-C_nAlkynyl "and" C_2-nAlkynyl "(where n is an integer from 3 to 30) is used interchangeably to mean alkynyl having 2 to the indicated" n "carbon atoms. In some embodiments, "alkynyl" is modified by a range of carbon atom numbers, and thus specifically defines the size of the alkynyl group. For example, "C₁₁-C₃₀Alkynyl "means an alkynyl group containing at least 11 carbon atoms and containing no more than 30 carbon atoms.

Unless carbon number is limited, "lower" in "lower aliphatic", "lower alkyl", "lower alkenyl" and "lower alkynyl" as used herein means that the moiety has at least one (at least two for alkenyl and alkynyl) and 6 or less carbon atoms.

The terms "cycloalkyl", "alicyclic", "carbocyclic" and equivalents areRefers to a group comprising a saturated or partially unsaturated carbocyclic ring in a monocyclic, spiro (sharing one atom) or fused (sharing at least one bond) carbocyclic ring system, wherein the carbocyclic ring system has from 3 to 15 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopenten-1-yl, cyclopenten-2-yl, cyclopenten-3-yl, cyclohexyl, cyclohexen-1-yl, cyclohexen-2-yl, cyclohexen-3-yl, cycloheptyl, bicyclo [4,3,0]Nonyl, norbornyl, and the like. The term cycloalkyl includes both unsubstituted cycloalkyl and substituted cycloalkyl. The term "C₃-C_nCycloalkyl radicals "and" C_3-nCycloalkyl groups "(where n is an integer from 4 to 15) are used interchangeably and represent cycloalkyl groups having from 3 to the indicated" n "carbon atoms in the ring structure. As used herein, unless otherwise specified for carbon number, a "lower cycloalkyl" group refers to a group having at least 3 and 8 or less carbon atoms in its ring structure.

Cycloalkyl residues may be saturated or contain groups with one or more double bonds in the ring system. In particular, they may be saturated or contain a double bond in the ring system. In unsaturated cycloalkyl residues, the double bond may be present at any suitable position. Monocycloalkyl residues include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl or cyclotetradecyl, which may also be substituted, for example, by C_1-4An alkyl group. Examples of substituted cycloalkyl residues are 4-methylcyclohexyl and 2, 3-dimethylcyclopentyl. Examples of parent structures for bicyclic systems are norbornane, bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane and bicyclo [3.2.1]Octane.

The term "heterocycloalkyl" and equivalents refer to a group containing a saturated or partially unsaturated carbocyclic ring in a monocyclic, spiro (sharing one atom) or fused (sharing at least one bond) carbocyclic ring system having groups of 3 to 15 carbon atoms, including 1 to 6 heteroatoms (e.g., N, O, S, P) or containing heteroatoms (e.g., NH, NRx (Rx is alkyl, acyl, aryl, heteroaryl, or cycloalkyl), PO₂、SO、SO₂Etc.) of. Where possible, the heterocycloalkyl group may be attached to C or to a heteroatom (e.g. via a nitrogen atom). Examples of heterocycloalkyl include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, tetrahydrodithienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thiaxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxiraneyl, thietanyl, oxaazepinyl, diazepinyl, thiazetanyl, 1,2,3, 6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1, 3-dioxolanyl, pyrazolinyl, dithianyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo [3 ], 1,0]Hexyl, 3-azabicyclo [4,1, 0] s]Heptyl, 3H-indolyl, quinolizinyl, and sugars, and the like. The term heterocycloalkyl includes both unsubstituted heterocycloalkyl and substituted heterocycloalkyl. The term "C₃-C_nHeterocycloalkyl "and" C_3-n-heterocycloalkyl "(where n is an integer from 4 to 30) is used interchangeably to mean a heterocycloalkyl group having from 3 to the indicated" n "atoms in the ring structure, including at least one heterogroup or atom as defined above. As used herein, unless otherwise specified for carbon number, "lower heterocycloalkyl" means having at least 3 and equal to or less than 8 carbon atoms in its cyclic structure.

The terms "aryl" and "aryl ring" as used herein refer to an aromatic group having "4 n + 2" (pi) electrons in a conjugated mono-or polycyclic ring system (fused or non-fused) and having 6 to 14 ring atoms, wherein n is an integer from 1 to 7. Polycyclic ring systems include at least one aromatic ring. Aryl groups may be directly linked or through C₁-C₆An alkyl (also known as arylalkyl or aralkyl) linkage. Examples of aryl groups include, but are not limited to, phenyl, benzyl, phenethyl, 1-phenylethyl, tolyl, naphthyl, biphenyl, terphenyl, indenyl, benzocyclooctenyl, benzocycloheptenyl, azulenyl, acenaphthenyl, fluorenyl, phenanthrenyl, anthracenyl, and the like. The term aryl includes both unsubstituted aryl and substituted aryl. Term "C₆-C_nAryl "or" C_6-nAryl "(where n is an integer from 6 to 30) is used interchangeably to mean an aryl group having from 6 to the indicated" n "carbon atoms in the ring structure, including at least one heterocyclic group or atom as defined above.

The terms "heteroaryl" and "heteroaryl ring" refer to an aromatic group having "4 n + 2" (pi) electrons in a conjugated monocyclic or polycyclic ring system (fused or non-fused), and having 5 to 14 ring atoms, wherein n is an integer from 1 to 7, and includes one to six heteroatoms (e.g., N, O, S) or heteroatom-containing (e.g., NH, NRx (Rx is alkyl, acyl, aryl, heteroaryl, or cycloalkyl), SO, etc.) groups. Polycyclic ring systems include at least one heteroaromatic ring. Heteroaryl may be directly linked or through C₁-C₃Alkyl (also known as heteroarylalkyl or heteroaralkyl) linkages. Where possible, the heteroaryl group may be attached to carbon or to a heteroatom (e.g., through a nitrogen atom). Examples of heteroaryl groups include, but are not limited to, pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, tetrazolyl, furanyl, thienyl; isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolidinyl, quinolyl, isoquinolyl, indolyl, isoindolyl, chromenyl, isochromenyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, pyrazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furanyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinolyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, carbazolyl, phenanthridinyl, acridinyl, peryleneyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, dibenzofuranyl, and the like. The term heteroaryl includes unsubstituted heteroaryl and substituted heteroaryl. The term "C₅-C_nHeteroaryl "and" C_5-nHeteroaryl "(wherein n is an integer from 6 to 29) is used interchangeably and denotes heteroaryl having from 5 to the indicated" n "atoms in the ring structure, including at least one heterocyclyl group as defined aboveA group or an atom.

The term "heterocycle" or "heterocyclic" includes heterocycloalkyl and heteroaryl. Examples of heterocycles include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4 α H-carbazolyl, carbolinyl, benzopyranyl (chromanyl), chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5, 2-dithiazinyl, dihydrofuro [2,3-b ] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isobenzopyranyl, isoindolyl, isoindolinyl, Isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolyl, oxadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, nitrophenyl (pyridil), pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, Pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2, 5-thiadiazinyl, 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl, triazinyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, 1,2, 5-triazolyl, 3, 4-triazolyl, xanthenyl, and the like. The term heterocycle includes both unsubstituted heterocyclyl and substituted heterocyclyl groups.

The term "amine" or "amino" as used herein refers to an unsubstituted or substituted group of the general formula-NR_aR_bA fragment of (1), wherein R_aAnd R_bEach independently is hydrogen, alkyl, aryl or heterocyclyl, or R_aAnd R_bTogether with the nitrogen atom to which they are attached form a heterocyclic ring. The term amino refers to a compound or fragment in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom. Thus, as used herein, the terms "alkylamino" and "dialkylamino" refer to a compound having one and at least two C's attached thereto, respectively₁-C₆An amine group of an alkyl group. The terms "arylamino" and "diarylamino" include groups in which a nitrogen atom is bound to at least one or two aryl groups, respectively. The term "amide" or "aminocarbonyl" refers to a compound or fragment containing a nitrogen structure attached to the carbon of a carbonyl or thiocarbonyl group. The term "acylamino" refers to an amino group wherein the amino group is directly attached to an acyl group as defined herein.

The term "bicyclic" or "bicyclic" refers to a two ring system sharing two ring carbon atoms, which may be located anywhere along either ring, and generally refers to bicyclic hydrocarbon radicals, bicyclic aromatic carbon atom ring structural groups, and saturated or partially unsaturated bicyclic carbon atom ring structural groups in which one or more ring carbon atom members have been replaced with a heteroatom such as an O, S, or N atom that is permitted for structural stability. The bicyclic ring system may be a fused ring system, such as bicyclo [4.4.0] decane or naphthalene, or a bridged ring system, such as bicyclo [2.2.2] octane.

The term "tricyclic" or "tricyclic" refers to a ring system having three rings that share three ring carbon atoms, which are located at any position along each ring; the term generally refers to tricyclic hydrocarbon radicals, tricyclic aromatic carbon atom ring structural groups and saturated or partially unsaturated tricyclic carbon atom ring structural groups in which one or more members of the ring carbon atoms have been replaced with a heteroatom such as an O, S or N atom that is permitted for structural stability. The tricyclic system may be three rings in a fused ring arrangement, such as anthracene or tetradecahydroanthracene, or a bridged ring, such as in adamantane or tricyclo [3.3.1.1] decane.

The terms "polycyclic (multicycle)", "polycyclic (multicyclic)" or "multicyclic (multicyclic)" refer to ring systems having more than 3 rings that share more than three ring carbon atoms located anywhere along either ring. The term generally refers to polycyclic hydrocarbon groups, polycyclic aromatic carbon atom ring structure groups, and saturated or partially unsaturated polycyclic carbon atom ring structure groups in which one or more carbon atom ring members have been substituted with heteroatoms such as O, S or N atoms as permitted by structural stability.

The term "fused ring" or "fused" refers to a polycyclic ring system containing fused rings. Typically, the fused ring system contains 2 or 3 rings and/or up to 18 ring atoms. As noted above, the cycloalkyl, aryl, and heterocyclyl groups can form a fused ring system. Thus, the fused ring system may be aromatic, partially aromatic or non-aromatic and may contain heteroatoms. According to this definition, a spiro ring system is not a fused polycyclic, but the fused polycyclic ring systems of the present invention may themselves have a spiro ring attached thereto through a single ring atom of the system. Examples of fused ring systems include, but are not limited to, naphthyl (e.g., 2-naphthyl), indenyl, phenanthryl, anthryl, pyrenyl, benzimidazole, benzothiazole, and the like.

The term "spiro" or "spiro" refers to an organic compound that exhibits a distorted structure of two or more rings (ring systems) in which 2 or 3 rings are joined together by a common atom. Spiro compounds may be fully carbocyclic (all carbon), such as but not limited to spiro [5.5] undecane, or heterocyclic (having one or more non-carbon atoms), including but not limited to carbocyclic spiro compounds, heterocyclic spiro compounds, and polyspiro compounds.

The term "bridged ring" or "bridged" refers to a carbocyclic or heterocyclic moiety in which two or more atoms are shared in two or more ring structures, wherein the shared atom is C, N, S or other heteroatoms arranged in a chemically sound substitution pattern. Alternatively, a "bridged ring" compound also refers to a carbocyclic or heterocyclic structure: one atom at any position of the main ring is bonded to a second atom on the main ring, which does not comprise part of the main ring structure, by a chemical bond or an atom other than a bond. The first and second atoms may or may not be adjacent to each other in the main ring. Illustrated below are specific non-limiting examples of bridged ring structures contemplated herein. Other carbocyclic or heterocyclic bridged ring structures are also contemplated, including bridged rings in which the bridging atoms are C or heteroatoms arranged in a chemically sound substitution pattern, as is known in the art.

The term "nitro" means-NO₂(ii) a The terms "halo" and "halogen" refer to a bromo, chloro, fluoro, or iodo substituent; the term "thiol", "thio" or "mercapto" refers to-SH; and the term "hydroxyl" or "hydroxy" refers to-OH. The term "alkylthio" is an alkyl group having a mercapto group attached thereto. Suitable alkylthio groups include groups having from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term "alkylcarboxyl" as used herein refers to an alkyl group having a carboxyl group attached thereto.

The term "alkoxy" or "lower alkoxy" as used herein refers to an alkyl group having an oxygen atom attached thereto. Representative alkoxy groups include groups having from 1 to about 6 carbon atoms such as methoxy, ethoxy, propoxy, tert-butoxy and the like. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, propoxy, butoxy, pentyloxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy and the like. The term "alkoxy" includes unsubstituted or substituted alkoxy, as well as perhaloalkoxy and the like.

The term "carbonyl" or "carboxyl" refers to compounds and moieties containing a carbon attached to an oxygen atom by a double bond. Examples of carbonyl containing moieties include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, and the like.

The term "acyl" is a carbonyl group having a carbon atom attached to a hydrogen atom (i.e., formyl), an aliphatic radical (C)₁-C₂₉Alkyl radical, C₁-C₂₉Alkenyl radical, C₁-C₂₉Carbonyl on alkynyl radicals, e.g. acetyl), cycloalkyl (C)₃-C₈Cycloalkyl), heterocyclyl (C)₃-C₈HeterocycloalkanesRadical and C₅-C₆Heteroaryl) and aryl (C)₆Aryl, such as benzoyl). The acyl group may be an unsubstituted or substituted acyl group (e.g., salicyloyl).

It is to be understood that the term "substituted" or "substituted" includes the implicit proviso that such substitution results in a stable compound (e.g., the compound is not capable of undergoing spontaneous rearrangement, cyclization, elimination, etc.) as a function of the valency of the substituent atom and the substituent group. As used herein, the term "substituted" is meant to encompass all permissible substituents of organic compounds. Broadly, permissible substituents include acyclic and cyclic, branched and unbranched branches of organic compounds, carbocyclic and heterocyclic, aromatic and nonaromatic substituents. The permissible substituents may be one or more. The term "substituted" means that when the above groups are substituted at one or more positions, the substituents are, for example, acyl, amino (including simple amino, monoalkylamino and dialkylamino, monoaryl and diarylamino, and alkylarylamino), acylamino (including carbamoyl and ureido), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, alkoxycarbonyl, carboxy (carboxylate), carboxylate, aminocarbonyl, monoalkylaminocarbonyl and dialkylaminocarbonyl, cyano, azido, halogen, hydroxy, nitro, trifluoromethyl, thio, alkylthio, arylthio, alkylthiocarbonyl, thiocarboxylate, lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, lower alkoxy, aryloxy, aryloxycarbonyloxy, benzyloxy, benzyl, sulfinyl, ureido, cyano, azido, halogen, hydroxy, nitro, trifluoromethyl, thio, alkylthio, arylthio, thiocarboxylate, lower alkyl, lower alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl, lower alkoxy, aryloxy, aryloxyca, Alkylsulfinyl, sulfonyl, sulfate, sulfonate, sulfonamide, phosphate, phosphonate, phosphoryl, oxo, guanidine, imino, formyl, and the like. Any of the above substituents may be further substituted, if permitted, for example with alkyl, aryl or other groups.

The term "solvate" refers to a physical association of a compound with one or more solvent molecules (whether organic or inorganic). The physical association includes hydrogen bonding. In some cases, the solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystal. "solvate" encompasses both solution phases and solvates which can be separated. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, hemi-ethanolates, and the like.

"pharmaceutically acceptable salts" of a compound refers to salts of a pharmaceutically acceptable compound. Salts of the desired compounds may retain or improve the biological effectiveness and properties of the free acids and bases of the parent compound as defined herein, or may contribute to the inherent basic, acidic or charged functionality of the molecule, which is otherwise biologically undesirable. Examples of pharmaceutically acceptable Salts may be mentioned by Berge et al in "Pharmaceutical Salts", J.pharm.Sci.66,1-19 (1977). Non-limiting examples of such salts include:

(1) forming an acid addition salt on a basic or positively charged functional group by adding an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, carbonate formers, and the like; organic acids such as acetic acid, propionic acid, lactic acid, oxalic acid, glycolic acid, pivalic acid, tert-butylacetic acid, beta-hydroxybutyric acid, valeric acid, caproic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, succinic acid, malic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, cyclohexylsulfamic acid, benzenesulfonic acid, sulfanilic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3-phenylpropionic acid, laurylsulfonic acid, laurylsulfuric acid, oleic acid, palmitic acid, stearic acid, lauric acid, pamoic acid (pamoic acid), palmitic acid, pantothenic acid, lactobionic acid, alginic acid, galactaric acid, galacturonic acid, Gluconic acid, glucoheptonic acid, glutamic acid, naphthoic acid, hydroxynaphthoic acid, salicylic acid, ascorbic acid, stearic acid, muconic acid, and the like.

(2) Base addition salts formed when an acidic proton present in the parent compound is replaced with a metal ion, including alkali metal ions (e.g., lithium, sodium, potassium), alkaline earth metal ions (e.g., magnesium, calcium, barium) or other metal ions such as aluminum, zinc, iron, and the like, or is coordinated with an organic base; wherein the organic base is selected from ammonia, ethylamine, diethylamine, ethylenediamine, N' -dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocaine, procaine, choline, lysine, etc.

Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Typically, such salts are prepared by reacting the free acid or base form of the compound with an equal stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two. Salts may be prepared in situ during the final isolation or purification of the compound or by reacting the compound in its free acid or base form with the corresponding base or acid as required and then isolating the salt thus formed. The term "pharmaceutically acceptable salts" also includes zwitterionic compounds that contain a cationic group covalently bonded to an anionic group, which are referred to as "inner salts". It is to be understood that all acid, salt, base and other ionic and non-ionic forms encompassed by the compounds of the present invention are within the scope of the present invention. For example, if the compound of the present invention is an acid, the salt form of the compound is also encompassed within the scope of the present invention. Likewise, if a compound of the present invention is a salt, the acid and/or base form of the compound is also encompassed within the scope of the present invention.

The compounds provided herein may include atomic isotopes at one or more of the atoms constituting such compounds in non-natural proportions thereof. Unnatural proportions of isotopes can be defined from amounts found in nature to amounts consisting of 100% of the atoms in question. For example, the compounds may incorporate a radioisotope, such as tritium (A), (B), (C), (D), (³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) Or a non-radioactive isotope such as deuterium (²H) Or carbon-13 (¹³C) In that respect Such isotopic variations may provide additional applications to those described elsewhere in this application. For example, isotopic variations of the compounds of the present invention can find additional utility, including but not limited to, as diagnostic and/or imaging agents,or as cytotoxic/radiotoxic therapeutic agents. In addition, isotopic variants can have altered pharmacokinetic and pharmacodynamic profiles, which can help to enhance safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds provided herein, whether radioactive or not, are encompassed by the present invention.

Isotopic enrichment is the form of an element in which one particular isotope is enriched (i.e., increased) and the other isotopic form is reduced or depleted by altering the relative abundance of the isotopes of a given element. As used herein, an "isotopically enriched" compound or derivative means that one or more specific isotopic forms of one or more specific isotopes in the compound are increased, i.e., one or more elements are enriched. Typically, in isotopically enriched compounds or derivatives, the specific isotopic form of a particular position of the compound is increased. It is understood, however, that the isotopic form of two or more elements in the compound can be increased. Further, isotopically enriched compounds can be in the form of mixtures of isotopically enriched forms enriched in more than one isotope, enriched in more than one element, or both. As used herein, an "isotopically enriched" compound or derivative has a level of isotopic form that is higher than the natural abundance of the form. The level of isotopic enrichment will vary depending on the natural abundance of the particular isotopic form. In some embodiments, the isotopically enriched level of the compound or the isotopically enriched level of the element in the compound can be from about 2 to about 100 mole percent (%), for example, about 2%, about 5%, about 17%, about 30%, about 51%, about 83%, about 90%, about 95%, about 96%, about 97%, about 98%, and greater than about 98%, about 99%, or 100%.

As used herein, "naturally abundant element" or "naturally abundant atom" refers to an element or atom, respectively, of atomic mass that is most abundant in nature. For example, the natural abundance of hydrogen is¹H, nitrogen in natural abundance is¹⁴N, oxygen in natural abundance is¹⁶O, carbon in natural abundance of¹²C and the like. By "non-isotopically enriched" compounds is meant all of the compounds in the groupCompounds in which the atoms or elements are all naturally abundant isotopes, i.e. all atoms or elements have the atomic mass most abundant in nature.

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

The terms "administration," "applicator," or the like, when applied to, for example, a subject, cell, tissue, organ, or biological fluid, refer to contacting, for example, a CD73 inhibitor, a pharmaceutical composition or diagnostic agent comprising the CD73 inhibitor, with the subject, cell, tissue, organ, or biological fluid. In the case of cells, administration includes contacting the agent with the cell (e.g., in vitro or ex vivo), and contacting the agent with a fluid, wherein the fluid is in contact with the cell.

The terms "treat", "treating", "treatment", and the like refer to an action taken (e.g., administration of a CD73 inhibitor or a pharmaceutical composition comprising the same) after a disease, disorder, or condition or symptom thereof has been diagnosed, observed, so as to temporarily or permanently eliminate, alleviate, inhibit, slow down, or ameliorate at least one underlying cause of the disease, disorder, or condition afflicting the subject, or at least one symptom associated with the disease, disorder, or condition afflicting the subject. Thus, treatment includes inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms associated therewith) the active disease.

As used herein, the term "in need of treatment" refers to a judgment made by a physician or other caregiver that a subject is in need of treatment or will benefit from treatment. This determination is made based on a variety of factors within the expertise of the physician or caregiver.

The terms "prevent", "preventing", "prevention", and the like, refer to measures taken (e.g., administration of a CD73 inhibitor or a pharmaceutical composition comprising the same) to temporarily or permanently prevent, inhibit, suppress, or reduce the risk of a subject suffering from a disease, disorder, or condition, etc. (as determined, for example, by lack of clinical symptoms) or delay the onset thereof, typically in the context of a subject susceptible to the particular disease, disorder, or condition (e.g., prior to onset of the disease, disorder, condition, or symptoms thereof). In certain instances, the term also refers to slowing the progression of a disease, disorder or condition or inhibiting its development into a harmful or other undesirable state.

The term "in need of prevention" as used herein refers to the judgment made by a physician or other caregiver that a subject is in need of or will benefit from preventive care. Such a determination is made based on a variety of factors within the expertise of the physician or caregiver.

The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and refer to an amount of an agent that, when administered to a subject, is administered to the subject, either alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses, in an amount capable of producing any detectable positive effect on any symptom, aspect or feature of a disease, disorder or condition. The therapeutically effective amount can be determined by measuring the relevant physiological effects, and can be adjusted according to the dosing regimen and diagnostic analysis of the subject's condition, among others. For example, measuring the serum level of the CD73 inhibitor (or e.g., a metabolite thereof) at a particular time after administration can indicate whether a therapeutically effective amount has been used. In some embodiments, the terms "therapeutically effective amount" and "effective amount" refer to an amount or dose of a therapeutic agent, e.g., a compound, that provides a desired therapeutic, diagnostic, or prognostic effect in a subject following administration to the subject in a single or multiple doses. The effective amount can be readily determined by the attending physician or diagnostician by known techniques and by observing the results obtained under analogous circumstances. In determining the effective amount or dose of the compound to be administered, a number of factors are considered, including but not limited to: the size, age, and general health of the subject; the specific diseases involved; the degree of involvement or severity of the disease or disorder to be treated; responses of the subject individuals; the particular compound administered; a mode of administration; the bioavailability characteristics of the administered formulation; selecting a dosage regimen; the use of concomitant medication; and other related considerations.

The term "substantially pure" is used herein to indicate that the component comprises greater than about 50% of the total composition, and typically greater than about 60% of the total composition. More typically, "substantially pure" means that the composition has a target component that is at least 75%, at least 85%, at least 90% or more of the total composition. In some cases, the component of interest will comprise greater than about 90%, or greater than about 95% of the total content of the composition.

As used herein, the terms "CD 73-associated disease, disorder or condition" and "CD 73-mediated disease, disorder or condition" are used interchangeably to refer to any disease, disorder or condition that may benefit from treatment with a CD73 inhibitor. In general, diseases, disorders, and conditions related to or mediated by CD73 are those in which CD73 activity plays a biological, mechanistic, or pathological role. Such diseases, disorders and conditions may also be associated with the activity of one or more adenosine receptors. Non-limiting examples of CD 73-associated diseases, disorders, and conditions include tumor-associated disorders (cancer, tumors, etc.), immune-related disorders, inflammatory component conditions, microbial-associated conditions, CNS-related conditions and neurological conditions, and other diseases (such as, but not limited to, cardiovascular diseases, gastrointestinal diseases, metabolic diseases, liver diseases, lung diseases, ophthalmic diseases, and kidney diseases).

For example, inhibitors of CD73 may be useful in the prevention or treatment of proliferative disorders, cancer or tumors; increasing or enhancing an immune response; improving vaccination, including increasing vaccine titer; and increase inflammation. The CD73 inhibitor disclosed by the invention can be used for treating immunodeficiency diseases related to immunodeficiency, immunosuppressive drug treatment, acute and/or chronic infection and aging. CD73 inhibitors may also be used to stimulate the immune system of patients with iatrogenically induced immunosuppression, including those who have received bone marrow transplantation, chemotherapy, or radiation therapy. In other embodiments, the CD73 inhibitor may be used to treat or prevent any viral, bacterial, fungal, parasitic or other infectious disease, disorder or condition, including but not limited to HIV and AIDS.

In some embodiments, the CD73 inhibitor may be used to prevent or treat an immune-related disease, disorder or condition selected from the group consisting of: rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergy, fibrosis, anemic fibromyalgia, alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, parkinson's disease, infections, crohn's disease, ulcerative colitis, allergic contact dermatitis, eczema, systemic sclerosis, and multiple sclerosis.

The pharmaceutical compositions provided herein 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 may be used in combination with other therapeutically active agents or compounds described herein to treat or prevent the CD 73-related diseases, disorders, and conditions discussed herein.

Pharmaceutical compositions containing the active ingredient (e.g., CD73 inhibitor) may be in a form suitable for oral use, for example, 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 may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents such as sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically acceptable preparations. Tablets, capsules and the like typically contain the active ingredient in admixture with non-toxic pharmaceutically acceptable carriers or excipients which are suitable for the manufacture of tablets. These carriers or excipients may be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may be uncoated or coated using known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action. For example, a time delay material such as glycerol monostearate or glycerol distearate may be employed. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Other agents include biodegradable or biocompatible particulate or polymeric materials such as polyesters, poly-amine acids, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate or lactide/glycolide copolymers, polylactide/glycolide copolymers or ethylene vinyl acetate copolymers to control delivery of the administered composition. For example, oral formulations may be embedded in microcapsules prepared using hydroxymethylcellulose by coacervation techniques or by interfacial polymerization, or gelatin microcapsules or poly (methylmethacylate) microcapsules, or in colloidal drug delivery systems. 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.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (for example, calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose); or in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture thereof. Such excipients may be suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as naturally occurring phosphatides (for example lecithin), or condensation products of an alkylene oxide with fatty acids (for example polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (for example heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (for example polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (for example polyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Such as those described above, such as sweetening agents, may be added, and flavoring agents may also be added to provide a palatable oral preparation.

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 known in the art.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil (for example, olive oil or arachis oil) or a mineral oil (for example, liquid paraffin), or mixtures thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia and gum tragacanth; naturally occurring phospholipids, such as soy, lecithin and esters or partial esters derived from fatty acids; hexitols, such as sorbitan monooleate; condensation products of partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.

The pharmaceutical compositions generally comprise a therapeutically effective amount of a CD84 inhibitor compound provided herein and one or more pharmaceutically and physiologically acceptable formulating agents. Suitable pharmaceutically or physiologically acceptable diluents, carriers or excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium disulfide), preservatives (e.g., benzyl alcohol, methyl paraben, ethyl or n-propyl paraben), emulsifiers, suspending agents, dispersants, solvents, fillers, detergents, buffers, vehicles, diluents and/or adjuvants. For example, a suitable vehicle may be a physiological saline solution or citrate buffered saline, possibly supplemented with other substances common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. One of skill in the art will readily know of various buffers that may be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. For example, the buffer component may 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-morpholine) ethanesulfonic acid (MES), 2- (N-morpholine) ethanesulfonic acid sodium salt (MES), 3- (N-morpholine) propanesulfonic acid (MOPS), and Ntris [ hydroxymethyl ] methyl-3-aminopropanesulfonic acid (TAPS). After the pharmaceutical composition is formulated, it may be stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form, a lyophilized form requiring reconstitution prior to use, a liquid form requiring dilution prior to use, or other acceptable forms.

In some embodiments, the pharmaceutical composition is contained in a single-use container (e.g., a single-use vial, ampoule, syringe, or auto-injector), while in other embodiments, is contained in a multiple-use container (e.g., a multiple-use vial).

The formulation may also include a carrier to protect the composition from rapid degradation or disappearance in vivo, such as a controlled release formulation, including liposomes, hydrogels, 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 syringe pumps and devices, all of which are well known to those skilled in the art.

The pharmaceutical compositions may also be in the form of sterile injectable aqueous or oleaginous suspensions. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which are 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 as a solution in 1, 3-butanediol. Acceptable diluents, solvents and dispersion media which can be employed include water, ringer's solution, isotonic sodium chloride solution, Cremophor ELTM (BASF, Parsippany, N.J.) or Phosphate Buffered Saline (PBS), ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols) and suitable mixtures thereof. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Furthermore, fatty acids (such as oleic acid) may be used in the preparation of injectables. Prolonged absorption of a particular injectable formulation can be brought about by the inclusion of agents that delay absorption (e.g., aluminum monostearate or gelatin).

The CD73 inhibitor compounds and compositions provided herein can be administered to a subject in any suitable manner known in the art. Suitable routes of administration include, but are not limited to, oral, parenteral (e.g., intramuscular), intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal and intracerebroventricular), nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal, and inhalation. Depot injections, typically administered subcutaneously or intramuscularly, may also be used to release the CD73 inhibitors disclosed herein over a defined period of time. In certain embodiments, the CD73 inhibitor compounds and compositions are administered orally to a subject in need thereof.

The CD73 inhibitor compounds and compositions provided herein can be administered to a subject in such amounts: the amount depends on, for example, the administration target (e.g., desired resolution); the age, weight, sex, health and physical condition of the subject to which the formulation is administered; the route of administration; and the condition of a disease, disorder, condition, or symptom thereof. The dosing regimen also takes into account the presence, nature and extent of any adverse effects associated with the administered agent. Effective dosages and dosage regimens can be readily determined, for example, by safety and dose escalation assays in vivo studies (e.g., animal models) and other methods known to those of skill in the art. Generally, the dosage parameters dictate that the dosage be less than the amount that is likely to have irreversible toxicity 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, for example, by pharmacokinetic and pharmacodynamic parameters associated with ADME, taking into account route of administration and other factors.

In some embodiments, the CD73 inhibitor may be administered once daily, or multiple times daily, at a dosage level of about 0.01mg/kg to about 50mg/kg, or about 1mg/kg to about 25mg/kg, of the subject's body weight to achieve the desired therapeutic effect. For use in oral formulations, the compositions may be provided in the form of tablets or capsules containing from 1.0 mg to 1000 mg of the active ingredient, particularly 1mg, 3mg, 5mg, 10mg, 15mg, 20 mg, 25mg, 50mg, 75 mg, 100mg, 150mg, 200mg, 250mg, 300 mg, 400 mg, 500 mg, 600 mg, 750 mg, 800 mg, 900 mg or 1000 mg of the active ingredient.

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

The invention also provides kits comprising CD73 inhibitor compounds or compositions. Kits are generally in the form of physical structures containing various components and can be used, for example, to perform the methods provided herein. For example, a kit may include one or more CD73 inhibitors disclosed herein (e.g., provided in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The CD73 inhibitor may be provided in a ready-to-use form (e.g., a tablet or capsule) or in a form that requires reconstitution or dilution, e.g., prior to administration (e.g., a powder). When the CD73 inhibitor is in a form that requires reconstitution or dilution by the user, the kit may further include diluents (e.g., sterile water), buffers, pharmaceutically acceptable excipients, and the like, packaged together with or separately from the CD73 inhibitor. When combination therapy is employed, the kit may contain several therapeutic agents separately, or they may already be combined in the kit. The components of the kit may be enclosed in separate containers, and all of the various containers may be in a single package. The kits of the invention can be designed to suitably maintain the conditions required for the components contained therein (e.g., refrigeration or freezing).

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

Examples

The invention will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not to be construed as limiting the scope of the invention in any way.

Unless defined otherwise or clear from context to be otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Synthesis of Compounds

The compounds provided by the present invention may be prepared by conventional methods and as described in the examples below.

Amine compound RNH₂Are available from commercial sources or are prepared by methods described in the literature.

Triethylammonium bicarbonate buffer (TEAC) was prepared. A 1M solution of TEAC was prepared by slowly adding dry ice to a 1M solution of triethylamine in water over several hours until the pH of the solution reached about 7.4-7.6 (measured using a pH meter).

2-chloropurine nucleoside derivative S-x (1.0mmol, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The solution was cooled by an ice bath. To this cold solution was added a solution of methylene bis phosphorus chloride (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was stirred at 0 ℃ for 2-4h and the reaction was checked by Thin Layer Chromatography (TLC). The reaction was quenched by the addition of TEAC solution and the pH of the reaction was adjusted to 7 to 8. The mixture was extracted with Dichloromethane (DCM), the aqueous phase was separated and concentrated. The residual material was purified by reverse phase C18-column chromatography to give the product as a colorless solid.

Example 1: synthesis of Compound 1

DIEA (diisopropylethylamine, 7.5mmol, 969mg, 1.5eq.) was added to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2236mg, 1.0eq.) and benzylamine (5.0mmol, 536mg, 1.0eq.) in dioxane (25 mL). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The crude product was purified by column chromatography. Dissolve the intermediate in 50mL NH₃/CH₃OH solution and stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and the crude product was purified by column chromatography to give 2-chloropurine nucleoside derivative S-1(1818 mg).

S-1(1.0mmol, 392mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and then cooled under ice bath conditions. To the cold solution was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The reaction mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched by TEAC solution and the pH of the reaction was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 1(369mg) as an off-white solid.

¹H NMR(500MHz,CD₃OD-d₄)δppm 2.46(t，2H)，4.23-4.74(m，7H)，6.01(d，1H)，7.19-7.38(m，5H)，8.59(s，1H)；¹³C NMR(125MHz,CD₃Cl-d₃)δppm 40.12，43.84，63.92，69.87，74.64，83.71，88.50，115.22，126.92，127.47，128.13，149.27，154.08，154.96，160.55；³¹P NMR(200MHz,CD₃Cl-d₃)δppm 12.94，18.11；m/z(ESI⁺)550.1。

Example 2: synthesis of Compound 6

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.2g, 1.0eq.) and 1-naphthylmethylamine (5.0mmol, 786mg, 1.0eq.) in dioxane (25 mL). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The solvent was removed (using rotary evaporation) and the residue was purified by column chromatography. This intermediate was dissolved in 50mL NH₃/CH₃OH solution and stirred at 35 ℃ overnight. The solvent was evaporated in vacuo and the residue was purified by column chromatography to give S-6(1.3 g).

S-6(1.0mmol, 442mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and then cooled under ice bath conditions. To the cold solution was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The reaction mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM. The aqueous phase was separated and concentrated, and the residue was purified by reverse phase column chromatography (C18 column) to give compound 6(110mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 0.88-0.91(m,3H),1.34-1.43(m,4H),1.67-1.68(m,2H),2.15-2.26(m,2H),4.16-4.22(m,2H),4.25-4.31(m,2H),4.38-4.41(m,1H),4.53-4.57(m,1H),4.74-4.76(m,H),6.13-1.15(m,1H),8.70-8.75(m,1H)；¹³C NMR(125MHz,D₂O)δppm 13.20,21.60,27.24,27.55,63.46,67.12,70.15,74.34,84.08,84.14,87.38,120.98,142.83,150.24,152.49,153.22；³¹P NMR(200MHz,D₂O)δ16.15,18.97；m/z(ES^-)571.8。

Example 3: synthesis of Compound 7

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.2g, 1.0eq.) and 2-naphthylmethylamine (5.0mmol, 786mg, 1.0eq.) in dioxane (25 mL). The reaction was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The crude product was purified by column chromatography. Dissolve the intermediate in 50mL NH₃/CH₃OH solution and stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-7(1.15 g).

S-7(1.0mmol, 442mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and then cooled under ice bath conditions. To the cold solution was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The reaction mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched by a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 7(105mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 2.11(t,J＝19.7Hz,2H),4.10(s,2H),4.29(s,1H),4.42(s,1H),4.55(s,1H),4.88(s,2H),5.79(s,1H),7.36-7.44(m,4H),7.71(d,J＝7.9Hz,1H),7.76(d,J＝7.2Hz,1H),7.87(d,J＝7.8Hz,1H),8.13(s,1H)；¹³C NMR(125MHz,D₂O)δppm 27.57,42.23,63.39,70.05,74.16,83.59,86.92,117.78,122.74,125.48,125.88,126.05,126.26,128.24,128.43,130.48,132.19,133.08,139.02,148.62,153.86,154.44.³¹P NMR(202MHz,D₂O)δppm15.17,19.58；m/z(ES^-)598.2。

Example 4: synthesis of Compound 8

2-chloropurine nucleoside derivative S-8(1.0mmol, 415mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL), and the solution was cooled under ice bath conditions. To the cold solution was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give the product as an off-white solid (58 mg).

¹H NMR(500MHz,D₂O)δppm 2.12(t,J＝19.7Hz,2H),4.08(s,2H),4.27(s,1H),4.40(s,1H),4.53(s,1H),4.65(s,2H),5.75(s,1H),7.36(s,3H),7.65(s,2H),7.69(d,J＝8.0Hz,2H),8.28(s,1H)；¹³C NMR(125MHz,D₂O)δppm 44.04,63.41,70.06,74.19,83.73,86.90,125.50,125.60,126.00,126.34,127.38,128.15,132.13,132.71,135.18,139.21,153.96,154.73；³¹P NMR(202MHz,D₂O)δppm 15.86,19.01；m/z(ES^-)598.4。

Example 5: synthesis of Compound 9

DIEA (12.5mmol, 1.6g, 2.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.2g, 1.0eq.) and memantine hydrochloride (5.0mmol, 1.0g, 1.0eq.) in 25mL dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL), washed with water (2 × 30mL) and concentrated. The residue was purified by column chromatography to give an intermediate. Dissolve the intermediate in 50mL NH₃/CH₃OH solution and the mixture was stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-9(1.1 g).

S-9(1.0mmol, 463mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and the mixture was cooled under ice bath conditions. To the cold mixture was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. After completion of the reaction, the reaction was quenched with a TEAC solution. The pH of the mixture was adjusted to 7 to 8. The mixture was extracted with DCM and the aqueous phase was separated. The aqueous solution was concentrated, and the residue was purified by reverse phase column chromatography (C18 column) to give compound 9(200mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 0.76(s,6H),0.98-1.12(m,2H),1.22(s,2H),1.31(d,J＝11.3Hz,2H),1.70(dd,J＝29.4,11.9Hz,4H),1.90(s,2H),2.11(t,J＝19.9Hz,3H),4.07(s,2H),4.28(s,1H),4.44(dd,J＝6.5,2.4Hz,1H),4.67-4.63(m,1H),5.91(d,J＝5.7Hz,1H),8.33(s,1H)；¹³C NMR(125MHz,D₂O)δppm 25.96,26.95,29.97,32.08,39.23,42.32,50.32,55.08,63.73,70.04,74.33,83.78,87.45,116.10,138.34,148.62,153.57,154.16；³¹P NMR(200MHz,D₂O)δppm 18.12；m/z(ES^-)620.2。

Example 6: synthesis of Compound 10

2-chloropurine nucleoside derivative S-10(1.0mmol, 451mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL), and the solution was cooled under ice bath conditions. To the cold solution was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 10(60mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 2.27(t,J＝19.4Hz,2H),4.17(s,2H),4.35(s,1H),4.46(s,1H),6.02(s,1H),7.27(s,4H),7.54(s,2H),7.97(s,2H),8.33(s,1H)；¹³C NMR(125MHz,D₂O)δppm 16.73,25.47,26.48,27.50,57.39,63.69,70.09,74.31,83.84,87.70,113.16,120.00,122.84,124.66,126.42,138.03,143.08,148.86,152.70,154.12；³¹P NMR(200MHz,D₂O)δppm 17.43,19.35-19.97；m/z(ES^-)608.0。

Example 7: synthesis of Compound 11

2-chloropurine nucleoside derivative S-11(1.0mmol, 564mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The solution was cooled under ice bath conditions and a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5mL) was added to the cold solution. The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched by a TEAC solution and then the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 11(100mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 0.77(d,J＝6.5Hz,3H),1.22(s,14H),1.55(s,2H),1.92(s,4H),2.12(t,J＝19.8Hz,2H),2.67(d,J＝38.2Hz,4H),4.03(s,2H),4.19(s,1H),4.40(s,1H),4.60(s,1H),5.20(d,J＝5.6Hz,4H),5.88(d,J＝4.4Hz,1H),8.51(s,1H)；¹³C NMR(125MHz,D₂O)δppm 13.90,22.48,24.71,25.52,27.10,29.26,29.64,31.41,36.86,37.49,63.89,70.44,74.37,84.15,86.83,119.95,127.82,129.70,149.34,152.45,152.86,164.88,174.66；³¹P NMR(200MHz,D₂O)δppm 16.04,18.65；m/z(ES^-)720.4。

Example 8: synthesis of Compound 12

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and 2-naphthylamine (5.0mmol, 715mg, 1.0eq.) in 25mL dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The DCM solution was concentrated to dryness and the residue was purified by column chromatography to give an intermediate. This intermediate was dissolved in 50mL NH₃/CH₃OH solution and the mixture was stirred at 35 ℃ overnight. The solvent was evaporated in vacuo and the residue was purified by column chromatography to give compound S-12(670 mg).

S-12(1.0mmol, 427mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and the solution was cooled under ice bath conditions. To the cold solution was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched by a TEAC solution and the pH of the quenched reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 12(100mg) as an off-white solid.

¹H NMR(D₂O,500MHz)δppm 2.21-2.29(m,2H),4.20-4.22(m,2H),4.40-4.44(m,1H),4.56-4.60(m,1H),4.78-4.80(m,1H),6.09-6.10(m,1H),7.58-7.64(m,3H),7.71-7.73(m,1H),7.97-7.99(m,1H)8.02-8.04(m,2H),8.55(s,1H)；¹³C NMR(D₂O,125MHz)δppm 26.38,27.37,28.36,63.61,70.33,84.01,86.76,118.06,121.57,122.25,125.52,126.23,126.41,139.82,149.74,153.39,153.47；³¹P NMR(D₂O,200MHz)δppm 16.14,18.96；m/z(ES^-)583.9。

Example 9: synthesis of Compound 15

2-chloropurine nucleoside derivative S-15(1.0mmol, 513mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture was cooled under ice bath conditions, followed by the addition of a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched by a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 15(30mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 0.70(t,J＝6.3Hz,3H),1.06(s,16H),1.27(s,2H),1.58(s,2H),2.10(t,J＝19.7Hz,2H),4.12(d,J＝32.1Hz,4H),4.27(s,1H),4.46(s,1H),4.66(s,1H),6.00(d,J＝4.7Hz,1H),8.60(s,1H)；¹³C NMR(125MHz,D₂O)δppm 13.73,22.40,25.51,27.55,28.40,29.07,29.36,31.66,63.64,66.70,70.27,74.36,83.95,87.18,120.57,142.65,150.05,152.38,153.2；³¹P NMR(200MHz,D₂O)δppm 15.64,18.89；m/z(ES^-)670.1。

Example 10: synthesis of Compound 16

2-chloropurine nucleoside derivative S-16(1.0mmol, 478mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and the mixture was cooled under ice bath conditions, followed by addition of a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched by a TEAC solution and the pH of the mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 16(250mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 1.64(s,11H),1.91(s,4H),2.16(t,J＝19.8Hz,2H),2.29(s,2H),4.14(dddd,J＝6.8,5.9,4.1,1.6Hz,3H),4.30-4.37(m,1H),4.56-4.46(m,1H),6.11(d,J＝5.1Hz,1H),8.72(s,1H)；¹³C NMR(125MHz,D₂O)δppm 16.75,27.23,28.38,33.41,36.07,42.03,51.44,63.47,70.17,74.37,84.19,87.49,115.91,121.90,143.36,149.28,153.15,173.66；³¹P NMR(200MHz,D₂O)δppm 16.37,18.85；m/z(ES^-)634.1。

Example 11: synthesis of Compound 17

DIEA (12.5mmol, 1.6g, 2.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and 2-amantadine hydrochloride (5.0mmol, 0.94g, 1.0eq.) in 25mL dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The organic layer was concentrated and the residue was purified by column chromatography to give an intermediate. Dissolve the intermediate in 50mL NH₃/CH₃OH solution and stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and then the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-17(880 mg).

Compound S-17(1.0mmol, 435mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL), and the mixture was cooled under ice bath conditions. To the cold mixture was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 17(30mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 1.60(d,J＝12.7Hz,2H),1.71(s,2H),1.82(d,J＝20.8Hz,7H),1.96(d,J＝19.6Hz,4H),2.14(t,J＝19.8Hz,2H),4.10(s,2H),4.20(s,1H),4.31(s,1H),4.47(t,J＝4.2Hz,1H),5.96(d,J＝5.6Hz,1H),8.41(s,1H)；¹³C NMR(125MHz,D₂O)δppm16.70,26.77,30.81,31.44,36.48,36.86,57.36,63.51,70.23,74.20,83.97,86.73,139.05,154.39；³¹P NMR(200MHz,D₂O)δppm 15.85,19.04；m/z(ES^-)592.0。

Example 12: synthesis of Compound 18

2-chloropurine nucleoside derivative S-18(1.0mmol, 512mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture was cooled under ice bath conditions, and then a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5mL) was added. The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by thin layer chromatography. The reaction was quenched with a TEAC solution and the pH of the mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 18(70mg) as an off-white solid.

¹H NMR(500MHz,CD₃OD-d₄)δppm 0.93(t,J＝6.8Hz,3H),1.52(s,18H),1.60-1.74(m,2H),2.33(t,J＝19.8Hz,2H),3.43(t,J＝6.6Hz,2H),4.27(d,J＝21.4Hz,3H),4.47-4.61(m,1H),4.71(t,J＝5.3Hz,1H),6.13(d,J＝5.4Hz,1H),8.76(s,1H)；¹³C NMR(125MHz,CD₃OD-d₄)δppm 13.01,22.30,26.62,28.98,29.28,31.63,39.56,63.95,70.57,74.91,84.44,87.86,118.78,142.32,150.79,151.78,151.97,154.17；³¹P NMR(200MHz,CD₃OD-d₄)δppm16.03,20.25；m/z(ES^-):669.2。

Example 13: synthesis of Compound 19

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and di-n-dodecylamine (5.0mmol, 1.8g, 1.0eq.) in 25mL dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The organic layer was concentrated to dryness and the residue was purified by column chromatography to give an intermediate compound. This intermediate was dissolved in 50mL NH₃/CH₃OH solution and the mixture was stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-19(1.3 g).

S-19(1.0mmol, 637mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture was cooled in an ice bath, followed by the addition of a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue is passed through a reverse phase¹⁸Purification by column chromatography gave compound 19(150mg) as an off-white solid.

¹H NMR(500MHz,CD₃OD-d₄)δppm 0.93(t,J＝6.6Hz,6H),1.32-1.46(m,36H),1.72(s,4H),2.36(t,J＝20.0Hz,2H),3.70(s,2H),4.11-4.32(m,5H),4.47(s,1H),4.66(t,J＝5.2Hz,1H),6.04(d,J＝5.4Hz,1H),8.37(s,1H)；¹³C NMR(125MHz,CD₃OD-d₄)δppm 13.08,22.34,26.44,29.08,29.34,31.68,64.24,70.51,74.51,83.84,87.46,118.27,137.84,151.64,153.46,154.25；³¹P NMR(200MHz,CD₃OD-d₄)δppm 16.20,19.99；m/z(ES^-):794.6。

Example 14: synthesis of Compound 20

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to the mixture of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl-. beta. -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and 2-aminoanthracene (5.0mmol, 1.0g, 1.0eq.) in 25mL of dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The organic layer was concentrated to dryness and the residue was purified by column chromatography to give an intermediate compound. This intermediate was dissolved in 50mL NH₃/CH₃OH solution and stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and then the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-20(770 mg).

S-20(1.0mmol, 477mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture was then cooled in an ice bath, followed by the addition of a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 20(80mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 2.25(t,J＝19.4Hz,2H),4.28(d,J＝58.1Hz,3H),4.46(s,2H),5.56(s,1H),7.19(s,3H),7.57(d,J＝73.7Hz,4H),7.85(s,2H),8.21(s,1H)；³¹P NMR(200MHz,D₂O)δppm 18.42,19.15；m/z(ES^-):633.9。

Example 15: synthesis of Compound 22

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and 1-adamantanamine (5.0mmol, 756mg, 1.0eq.) in 25mL dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). After removal of the solvent, the residue was purified by column chromatography to give an intermediate compound. This intermediate was dissolved in 50mL NH₃/CH₃In the OH solution, adding a solvent into the solution,and the mixture was stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and then the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-22(770 mg).

S-22(1.0mmol, 435mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture was then cooled in an ice bath, followed by the addition of a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 22(100mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 1.64(s,6H),2.06(d,J＝33.5Hz,9H),2.17(d,J＝19.9Hz,2H),4.09(s,2H),4.30(d,J＝0.9Hz,1H),4.45(ddd,J＝5.9,3.0,2.0Hz,1H),4.68-4.65(m,1H),5.94(d,J＝5.2Hz,1H),8.38(s,1H)；¹³C NMR(125MHz,D₂O)δppm 26.22,27.22,29.28,35.72,40.80,53.50,63.57,70.19,74.20,83.95,86.81,138.60,148.74,153.76,154.43；³¹P NMR(200MHz,D₂O)δppm 16.38,18.81；m/z(ES-)592.2。

Example 16: synthesis of Compound 23

2-chloropurine nucleoside derivative S-23(1.0mmol, 481mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL), and the mixture was cooled in an ice bath. To the cold mixture was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 23(141mg) as an off-white solid.

¹H NMR(D₂O,500MHz)δppm 0.90-0.97(m,9H),1.18-1.20(m,1H),1.36-1.40(m,1H),1.75-1.80(m,1H),1.90-1.97(m,1H),2.00-2.30(m,2H),2.40-2.45(m,1H),4.20-4.25(m,2H),4.40-4.44(m,1H),4.44-4.57(m,1H),4.72-4.73(m,1H),4.91-5.05(m,1H),6.43-6.45(m,1H),9.02(m,1H)；¹³C NMR(D₂O,125MHz)δ12.74,18.03,18.90,35.91,44.47,47.41,48.45,63.50,70.16,74.34,83.22,84.06,84.13,87.37,120.84,142.72,150.23,152.41,153.24,153.45ppm；³¹P NMR(D₂O,200MHz)δ16.49,18.79ppm；m/z(ES^-)638.0。

Example 17: synthesis of Compound 31

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl-. beta. -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and 3-azaspiro [ 4.5.5 ] ring]Decane (5.0mmol, 696mg, 1.0eq.) in 25mL of dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The organic layer was concentrated to dryness and the residue was purified by column chromatography to give an intermediate compound. This intermediate was dissolved in 50mL NH₃/CH₃OH solution and stirred at 35 ℃ overnight. The solvent was evaporated under vacuum, and then the residue was purified by column chromatography to give the corresponding 2-chloropurine nucleoside derivative S-31(1.1 g).

S-31(1.0mmol, 423mg, 1.0eq.) was dissolved in trimethyl phosphate (10mL) and the mixture was cooled in an ice bath strip. To the cold mixture was added a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the mixture was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was collected and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 31(140mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 1.43(t,J＝22.8Hz,10H),1.83(d,J＝52.0Hz,2H),2.15(t,J＝19.7Hz,2H),3.38(s,1H),3.58(s,1H),3.79(s,1H),40.1(s,1H),4.12(s,2H),4.32(s,1H),4.49(t,J＝4.4Hz,1H),4.67-4.70(m,1H),5.97-5.98(d,J＝5.4Hz,1H),8.36-8.37(d,J＝6.8Hz,1H)；¹³C NMR(125MHz,D₂O)δppm 7.67,8.70,9.72,22.92,27.41,34.49,45.49,47.78,69.61,73.58,74.79,86.08,87.43,118.15,139.22,150.08,153.06,153.84；³¹P NMR(200MHz,D₂O)δppm 15.71,19.11；m/z(ES^-):580.0。

Example 18: synthesis of Compound 51

DIEA (7.5mmol, 969mg, 1.5eq.) was added dropwise to a solution of 2, 6-dichloro-9- (2',3',5' -tri-O-acetyl- β -D-ribofuranosyl) purine (5.0mmol, 2.23g, 1.0eq.) and nortropine (5.0mmol, 636g, 1.0eq.) in 25mL dioxane. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in DCM (100mL) and washed with water (2X 30 mL). The residue was purified by column chromatography to give an intermediate compound. This intermediate was dissolved in 50mL NH₃/CH₃OH solution and the mixture was stirred at 35 ℃ overnight. After removal of the solvent (under vacuum), the residue was purified by column chromatography to give 2-chloropurine nucleoside derivative S-51(1.2 g).

Compound S-51(1.0mmol, 411mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture was cooled in an ice bath, followed by the addition of a solution of bis (dichlorophosphoryl) methane (4.0eq.) in trimethyl phosphate solution (5 mL). The resulting mixture was then stirred at 0 ℃ for 2-4h and the reaction was checked by TLC. The reaction was quenched with a TEAC solution and the pH of the reaction was adjusted to 7 to 8. The mixture was extracted with DCM, the aqueous phase was separated and concentrated. The residue was purified by reverse phase column chromatography (C18 column) to give compound 51(160mg) as an off-white solid.

¹H NMR(500MHz,D₂O)δppm 1.78-2.42(m,11H),4.06(s,1H),4.12(s,2H),4.28-4.37(m,1H),4.45-4.55(m,1H),4.82-4.88(m,1H),5.38(dd,J＝3.4,2.4Hz,1H),5.99(d,J＝5.6Hz,1H),8.40(s,1H)；¹³C NMR(125MHz,D₂O)δppm 26.05,27.10,27.68,37.08,38.20,53.79,54.40,64.38,70.20,74.09,83.99,86.71,117.78,138.54,151.12,154.22；³¹P NMR(200MHz,D₂O)δppm 16.90,18.63；m/z(ES^-):568.0。

Biological assay

Screening assays for CD73 inhibitors

The inhibitory effect of compounds on CD73 was evaluated using the Malachite Green phosphate assay kit (R & D, Cat # DY 996). Briefly, compounds were dissolved and diluted to the desired concentration with phosphate-free buffer (Tris-HCl, pH 7.3). mu.L of the compound solution was added to an equal volume of CD73 protein solution (2 Xconcentration, 0.5. mu.g/mL, Novoprotein, Cat # C446) followed by incubation at room temperature for 5 min. To each well 10 μ L of Malachite Green reagent a was added, mixed well and incubated at room temperature for 10 minutes. Then, 10 μ L of Malachite Green reagent B was added to each well, mixed well and incubated at room temperature for 20 minutes. Finally, the optical density per well was determined using a microplate reader at 620 nm. The inhibitory activity of selected compounds is shown in table 2.

TABLE 2 inhibitory Activity of Compounds in CN73 enzyme assay

Compound numbering	Efficacy	¹
			1	+++
2	++
		6	+++
7	+++
		8	++
9	+++
		10	++
11	++
		12	++
15	+++
		16	++
17	+++
		18	++
19	+
		20	++
22	+++
		23	++
31	+++
		51	+++

¹"+" denotes IC₅₀>100 nM; "+ +" denotes IC₅₀10-100 nM; "+ + + +" denotes IC₅₀<10nM。

Cellular level analysis of CD73 Activity

The 5 × compound solution was prepared by dissolving and diluting the compound to the desired concentration in serum-free RMPI-1640 medium with 1 μ M AMP. A375 cells were collected and washed twice with PBS, then allowed to stand at 1.125X 10⁵The density of/mL was suspended in serum-free RMPI-1640 medium. Each aliquot of 80. mu.L of the cell suspension was placed in a 96-well plate, followed by addition of 20. mu.L of a 5X compound solution, gentle mixing, and 5% CO at 37 ℃₂The cells were incubated for 16 hours. After incubation, 50 μ Ι _ of supernatant was transferred from each well to a new 96-well plate. Subsequently, 2. mu.L of 2.5. mu.M ATP and 50. mu.L of Celltiter Glo reagent were added to each well in sequence. Fluorescence was measured using pherastar (bmg).

3. Method for evaluating pharmacokinetics of compound

After a single intravenous (1mg/kg) or intragastric (3mg/kg) administration of the compound to fasted SD male rats, blood samples were collected at 0.08h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h after administration. Plasma was separated by centrifugation (8000rpm) and frozen (-20 ℃) until sample analysis was performed. The concentration of the compound in rat plasma was determined by HPLC-MS/MS. Plasma was dispensed into appropriate tubes containing internal standard and methanol or acetonitrile. The tubes were mixed vigorously for 3 minutes to achieve deproteinization, followed by centrifugation at 8000rmp for 5 minutes. The supernatant was transferred to an autosampler vial and injected into the chromatography system. Pharmacokinetic parameters, including AUC, were calculated using WinMonlin 6.3 software_0-t，Cmax，tmax，t_1/2MRT, C1 and Vd. The absolute bioavailability was calculated as follows: f ═ AUC (i.g.) x dose (i.v.)]/[ AUC (i.v.) x dose (i.g.)]×100％。

While the invention has been described in detail with reference to the embodiments thereof, the embodiments are provided for the purpose of illustration and not for the purpose of limitation. Other embodiments that can be derived from the principles of the invention are intended to be within the scope of the invention as defined by the claims.

Claims

1. A compound of formula I, or a pharmaceutically acceptable ester or salt thereof

Wherein:

w is oxygen, sulfur, nitrogen or methylene;

x is selected from phosphoryl (-P (═ O) (OR) -), sulfonyl (-S (═ O)₂-) and a carbonyl (-C (═ O) -) moiety, wherein R is hydrogen, an ester-forming group, or a protecting group;

y is selected from phosphonate (-PO)₃R₂) Sulfonate (-SO)₃R) and a carboxylate (-CO)₂R), wherein R is hydrogen, an ester-forming group, or a protecting group;

R¹is hydroxy or hydrogen;

R²is chlorine or hydrogen; and

R³and R⁴Independently selected from hydrogen, alkyl, alkenyl and alkynyl, wherein R³And R⁴Has 11 to 30 carbon atoms; or

R³And R⁴Independently selected from hydrogen and a ring system containing a bicyclic, tricyclic, spirocyclic, fused or bridged ring carbocyclic or heterocyclic ring system, said carbocyclic system being aromatic or non-aromatic, with the proviso that R is³And R⁴Not hydrogen at the same time.

2. The compound of claim 1, wherein R³Is hydrogen or lower alkyl; and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is C₁₁To C₃₀Alkyl, alkenyl or alkynyl.

3. The compound of claim 1, wherein R³Is hydrogen or lower alkyl; and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is a ring system having a carbocyclic or heterocyclic ring system containing bicyclic, tricyclic, spirocyclic, fused, or bridged rings, the carbocyclic ring system being aromatic or non-aromatic, and the heterocyclic ring system being substituted or unsubstituted.

4. The compound of any one of claims 1 to 3, wherein the compound is a compound of formula II or formula III, or a pharmaceutically acceptable salt or ester thereof:

5. a compound according to any one of claims 1 to 3, wherein the compound is of formula IV:

6. a compound according to any one of claims 1 to 3, wherein the compound is of formula V or formula VI:

7. a compound according to any one of claims 1 to 3, wherein the compound is of formula VII or VIII:

wherein:

r is hydrogen, an ester-forming group, or a protecting group.

8. A compound according to any one of claims 1 to 3, wherein the compound is of formula IX, or a pharmaceutically acceptable salt or ester thereof:

wherein:

r is hydrogen, an ester-forming group, or a protecting group;

R¹is hydroxy or hydrogen;

R²is hydrogen or chlorine; and

R³is hydrogen or lower alkyl, and R⁴Is alkyl, alkenyl or alkynyl having 11 to 30 carbon atoms; or

R³Is hydrogen or lower alkyl, and R⁴Is a substituent comprising a bicyclic, tricyclic, or polycyclic ring system, wherein the ring system is a fused ring system, a spiro ring system, a bridged ring system, or a parallel ring system, and the ring system is a carbocyclic ring, an aliphatic ring, an aromatic ring, a heterocyclic ring, or a combination thereof; or

R³Is hydrogen or lower alkyl, and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is alkyl, alkenyl or alkynyl having 11 to 30 carbon atoms; or

R³Is hydrogen or lower alkyl, and R⁴is-C (═ O) R⁵OR-C (═ O) OR⁵Wherein R is⁵Is a substituent containing a carbocyclic or heterocyclic ring system containing a bicyclic, tricyclic, spirocyclic, fused or bridged ring, said carbocyclic ring system being aromatic or non-aromatic, said heterocyclic ring systemIs substituted or unsubstituted, wherein the ring is carbocyclic, aliphatic, aromatic, heterocyclic, or a combination thereof; or

R³Is hydrogen or lower alkyl, and R⁴Is unsubstituted or substituted 1-adamantyl, alpha-naphthylmethyl or beta-naphthylmethyl; or

R³、R⁴And the nitrogen atoms to which they are attached form a fused three-membered ring structure.

9. The compound of claim 8, wherein R, R¹And R²As defined in claim 8; r³Is hydrogen or lower alkyl; and R is⁴Is a group containing adamantyl moieties.

10. The compound of claim 9, wherein R⁴Is a substituted or unsubstituted 1-adamantyl group or a substituted or unsubstituted 2-adamantyl group.

11. The compound of claim 9, wherein R⁴Is a substituted or unsubstituted 1-adamantylmethyl group.

12. The compound of claim 9, wherein R⁴Is a substituted or unsubstituted 1-adamantylethyl group, a substituted or unsubstituted 1-adamantylpropyl group, or a substituted or unsubstituted 1-adamantylbutyl group.

13. The compound of claim 8, wherein R, R¹And R²As defined in claim 8; r³Is hydrogen or lower alkyl; and R is⁴Is a group containing a naphthyl moiety.

14. The compound of claim 13, wherein R⁴Is a substituted or unsubstituted alpha-naphthyl group, or a substituted or unsubstituted beta-naphthyl group.

15. Root of herbaceous plantThe compound of claim 13, wherein R⁴Is a substituted or unsubstituted alpha-naphthylmethyl group, or a substituted or unsubstituted beta-naphthylmethyl group.

16. The compound of claim 13, wherein R⁴Selected from the group consisting of substituted or unsubstituted naphthylethyl, substituted or unsubstituted naphthylpropyl, and substituted or unsubstituted naphthylbutyl.

17. The compound of claim 8, wherein the fused three-membered ring structure is a substituted or unsubstituted carbazolyl moiety.

18. The compound of any one of claims 1 to 17, wherein the compound is a compound listed in table 1, or a pharmaceutically acceptable salt or ester thereof.

19. The compound of any one of claims 1 to 18, wherein C, H, O and the N atom in the compound are each independently selected from a naturally abundant atom and an isotopically enriched atom.

20. The compound of claim 19, wherein said isotopically enriched atoms are selected from those of carbon¹²C、¹³C and¹⁴c; selected from hydrogen¹H、²H and³h; selected from oxygen¹⁶O、¹⁷O and¹⁸o; and is selected from nitrogen¹⁴N and¹⁵N。

21. a pharmaceutical composition comprising a compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutically acceptable carrier comprises a cream, an emulsion, a gel, a liposome, or a nanoparticle.

23. The pharmaceutical composition of claim 21 or 22, wherein the composition is suitable for oral administration.

24. The pharmaceutical composition of claim 23, wherein the composition is in the form of a hard-shelled, soft-shelled, gelatin capsule, cachet, pill, tablet, lozenge, powder, granule, pellet, or dragee.

25. The pharmaceutical composition of claim 23, wherein the composition is in the form of a solution, an aqueous liquid suspension, a non-aqueous liquid suspension, an oil-in-water liquid emulsion, a water-in-oil liquid emulsion, an elixir, or a syrup.

26. The pharmaceutical composition of any one of claims 23 to 25, wherein the composition has an enteric coating.

27. The pharmaceutical composition of any one of claims 23 to 26, wherein the composition is formulated for controlled release.

28. The pharmaceutical composition of claim 20 or 21, wherein the composition is injectable.

29. A method of treating or preventing a CD 73-related disease, disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-20 or the pharmaceutical composition of any one of claims 21-28, thereby treating or preventing a CD 73-related disease, disorder or condition in the subject.

30. The method of claim 29, wherein the compound is administered in an amount effective to reverse, slow or stop the progression of CD 73-mediated immunosuppression in the subject.

31. The method of claim 29 or 30, wherein the CD 73-associated disease, disorder or condition is cancer.

32. The method of claim 31, wherein the cancer is a cancer of the prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin, mesothelial intima, white blood cells, esophagus, breast, muscle, connective tissue, lung, adrenal gland, thyroid, kidney, or bone.

33. The method of claim 31, wherein the cancer is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, or testicular seminoma.

34. The method of claim 31, wherein the cancer is selected from the group consisting of melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumors, lymphoma, ovarian cancer, and kaposi's sarcoma.

35. The method of claim 29 or 30, wherein the CD 73-associated disease, disorder or condition is a disease, disorder or condition selected from the group consisting of rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergy, fibrosis, anemic fibromyalgia, alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, parkinson's disease, infection, crohn's disease, ulcerative colitis, allergic contact dermatitis, eczema, systemic sclerosis and multiple sclerosis.

36. The pharmaceutical composition of any one of claims 21 to 28, further comprising at least one additional therapeutic agent.

37. The pharmaceutical composition of claim 36, wherein the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and/or inflammation modulator, an anti-hypercholesterolemic agent, or an anti-infective agent.

38. The pharmaceutical composition of claim 36, wherein the at least one additional therapeutic agent is an immune checkpoint inhibitor.

39. The method of any of claims 29 to 35, further comprising: administering at least one additional therapeutic agent to the subject.

40. The method of claim 39, wherein the at least one additional therapeutic agent is administered simultaneously or sequentially with the compound or composition.

41. The method of claim 39 or 40, wherein the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and/or inflammation modulator, an anti-hypercholesterolemic agent, or an anti-infective agent.

42. A kit comprising a compound or pharmaceutically acceptable salt or ester according to any one of claims 1 to 20, and at least one additional therapeutic agent.

43. The kit of claim 42, wherein said at least one additional therapeutic agent is a chemotherapeutic agent, an immune and/or inflammation modulator, an anti-hypercholesterolemic agent, an anti-infective agent, or an immune checkpoint inhibitor.

44. The kit of claim 42 or 43, further comprising a buffer or excipient, and/or instructions for use thereof.

45. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt or ester thereof, and an immune checkpoint inhibitor, thereby treating cancer in the subject.

46. The method of claim 42, wherein said administering is prior to, concurrent with, or subsequent to radiation therapy.

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

48. The method of claim 45, wherein the compound and the immune checkpoint inhibitor are administered sequentially.

49. The method of claim 48, wherein the compound is administered after the immune checkpoint inhibitor.

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

51. The pharmaceutical composition of claim 38, the kit of claim 43, or the method of any one of claims 45 and 47-50, wherein the immune checkpoint inhibitor is selected from the group consisting of Yiprioman, Natuzumab, and Lanrolizumab.