BR112018011302B1 - COMPOUNDS, PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF, PHARMACEUTICAL COMPOSITION AND IMMUNOGENIC COMPOSITION COMPRISING SAID COMPOUNDS AND THERAPEUTIC USES THEREOF - Google Patents

Abstract

NEW COMPOUNDS. A compound of Formula (I) wherein Y1, Y2, X1, X2, R1, R2, R3, R4, R5, R6, R8 and R9 are as defined herein; and pharmaceutically acceptable salts and tautomers thereof, compositions, combinations and medicaments containing said compounds and processes for preparing the same. The invention also relates to the use of said compounds, combinations, compositions and medicaments in the treatment of diseases in which modulation of STING (Stimulator of Interferon Genes) is beneficial, for example, in inflammation, allergic and autoimmune diseases, infectious diseases, cancer, precancerous syndromes and as vaccine adjuvants.

Description

FIELD OF INVENTION

[001] The present invention relates to compounds, compositions, combinations and medicines containing said compounds, and processes for preparing them. The invention also relates to the use of said compounds, combinations, compositions and medicaments in the treatment of diseases in which modulation of STING (Stimulator of Interferon Genes) is beneficial, for example, in inflammation, allergic and autoimmune diseases, infectious diseases, human immunodeficiency virus (HIV) infection, AIDS, cancer, precancerous syndromes and as immunogenic composition or vaccine adjuvants.

BASICS OF THE INVENTION

[002] Vertebrates are constantly threatened by the invasion of microorganisms and have developed immune defense mechanisms to eliminate infectious pathogens. In mammals, this immune system comprises two branches: innate immunity and adaptive immunity. The innate immune system is the first line of defense, which is initiated by Pattern Recognition Receptors (PRRs) that detect pathogen ligands as well as molecular patterns associated with damage (Takeuchi O. et al., Cell, 2010 : 140, 805-820). An increasing number of these receptors have been identified, which include Toll-like receptors (TLRs), C-type lectin receptors, retinoic acid-inducible gene-like receptors I (RIG-I), and NOD-like receptors (NLRs), and also double-stranded DNA sensors. Activation of PRRs leads to the upregulation of genes involved in the inflammatory response, including type 1 interferons, pro-inflammatory cytokines and chemokines that suppress pathogen replication and facilitate adaptive immunity.

[003] The adapter protein STING (Stimulator of Inteferon Genes), also known as TMEM 173, MPYS, MITA and ERIS, has been identified as a signaling molecule central to cytosolic nucleic acids in the innate immune response (Ishikawa H and Barber GN, Nature, 2008: 455, 674-678; WO 2013/1666000). Activation of STING results in upregulation of the IRF3 and NFKB pathways, leading to the induction of Interferon-β and other cytokines. STING is critical for responses to cytosolic DNA of pathogenic or host origin and to unusual nucleic acids called Cyclic Dinucleotides (CDNs)

[004] CDNs were identified for the first time as secondary bacterial messengers responsible for controlling numerous responses in the prokaryotic cell. Bacterial CDNs, such as c-di-GMP, are symmetric molecules characterized by two 3'5' phosphodiester bonds.

[005] The direct activation of STING by bacterial CDNs was recently confirmed through X-ray crystallography (Burdette DL and Vance RE, Nature Immunology, 2013: 14, 19-26). Consequently, bacterial CDNs and their analogues have attracted interest as potential vaccine adjuvants (Libanova R. et al., Microbial Biotechnology 2012: 5, 168-176; WO 2007/054279, WO 2005/087238).

[006] More recently, the response to cytosolic DNA was elucidated and showed an involvement in the generation, by an enzyme called cyclic GMP-AMP synthase (cGAS, previously known as C6orf150 or MB21D1), of a novel mammalian CDN signaling molecule identified as cGAMP, which then activates STING. Unlike bacterial CDNs, cGAMP is a non-asymmetric molecule characterized by mixtures of 2', 5' and 3', 5' phosphodiester bonds. (Gao P et al., Cell, 2013: 153, 1-14). The interaction of cGAMP with STING has also been demonstrated by X-ray crystallography (Cai X et al., Molecular Cell, 2014: 54, 289-296).

[007] Interferon was first described as a substance that could protect cells from viral infection (Isaacs & Lindemann, J. Virus Interference. Proc. R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147 , 258-267). In man, type I interferons are a family of related proteins that are encoded by genes on chromosome 9 and encode at least 13 isoforms of interferon alpha (IFNa) and one isoform of interferon beta (IFNβ). Recombinant IFNα was the first approved biological therapy and has become an important therapy in viral infections and cancer. In addition to direct antiviral activity in cells, interferons are known as potent modulators of the immune response, acting on cells of the immune system.

[008] The administration of a small molecular compound, which could stimulate the innate immune response, including the activation of type I interferons and other cytokines, could become an important strategy for the treatment or prevention of human diseases, including viral infections. . This type of immunomodulatory strategy has the potential to identify compounds that may be useful not only in infectious diseases, but also in cancer (Zitvogel, L. et al., Nature Reviews Immunology, 2015 15 (7), p405-414), allergic (Moisan J. et al., Am. J. Physiol. Lung Cell Mol. Physiol., 2006: 290, L987-995), other inflammatory conditions such as irritable bowel disease (Rakoff-Nahoum S., Cell ., 2004, 23, 118 (2): 229-41) and as vaccine adjuvants (Persing et al., Trends Microbiol. 2002: 10 (10 Supplement), S32-7 and Dubensky et al., Therapeutic Advances in Vaccines , published online on September 5, 2013).

[009] Allergic diseases are associated with an immune response to allergens with a Th2 profile. Th2 responses are associated with elevated levels of IgE, which, through its effects on mast cells, promotes hypersensitivity to allergens, resulting in the symptoms seen in, for example, allergic rhinitis and asthma. In healthy individuals, the immune response to allergens is more balanced with a mixed Th2/Th1 and regulatory T cell response. Induction of type I interferons has been shown to result in the reduction of Th2-type cytokines in the local environment and promote Th1/Treg responses. In this context, the induction of type 1 interferons, for example by activation of STING, may offer a benefit in the treatment of allergic diseases such as asthma and allergic rhinitis (Huber JP et al. J Immunol 2010: 185, 813- 817).

[010] In contrast, increased and prolonged production of type I IFN is associated with a variety of chronic infections, including Mycobacteria infections (Collins et al., CHM 2015, Wassermann et al., CHM 2015, Watson et al ., CHM 2015), Franciscella (Storek et Jl 2015; Jin et al., JI 2011), Chlamydia (Prantner et al., JI 2010; Barker et al., Mbio 2013; Zhang et al., JI 2014), Plasmodium (Sharma et al., Immunity, 2011) and HIV (Herzner et al., Nat Immunol 2015; Nissen et al., Clin Exp Immunol 2014; Gao et al., Science 2013; Lahaye et al., Science 2013;)( reviewed in Stifter and Feng, JI 2014). Similarly, excess production of type I interferon is found among patients with complex forms of autoimmune disease. Genetic evidence in humans and support from studies in animal models support the hypothesis that inhibition of STING results in the reduction of type I interferon that triggers autoimmune diseases (Crow YJ et al., Nat. Genet. 2006; 38917- 920, Stetson DB et al., Cell 2008; 134; 587 -598). Therefore, STING inhibitors provide a treatment for patients with chronic production of type I interferon and pro-inflammatory cytokines associated with infections or complex autoimmune diseases. Allergic diseases are associated with an immune response to allergens with a Th2 profile.

[011] Compounds that bind to STING and act as an agonist have been shown to induce type 1 interferons and other cytokines in incubation with human PBMCs. Compounds that induce human interferons may be useful in the treatment of various diseases, for example, in the treatment of allergic diseases and other inflammatory diseases, for example, allergic rhinitis and asthma, in the treatment of infectious diseases, precancerous syndromes and cancer and may also be useful as immunogenic compositions or vaccine adjuvants. Compounds that bind to STING can act as antagonists and may be useful in the treatment of, for example, autoimmune diseases.

[012] It is anticipated that the action of activating or inhibiting agents on STING could be a promising approach for the treatment of diseases in which modulation of the type 1 IFN pathway is beneficial, including inflammatory, allergic and autoimmune diseases, infections, cancer, precancerous syndromes and as immunogenic composition or vaccine adjuvants.

[013] International Patent Applications WO 2014/093936, WO 2014/189805, WO 2013/185052, US2014/0341976, WO 2015/077354, PCT/EP2015/062281 and GB 1501462.4 disclose certain dinucleotides cyclics and their use in inducing an immune response.

[014] An object of the invention is to provide other cyclic dinucleotides suitable for the treatment of cancer.

SUMMARY OF THE INVENTION

[015] The invention is directed to compounds according to Formula (I): wherein Y1, Y2, X1, X2, R1, R2, R3, R4, R5, R6, R8 and R9 are defined as below and pharmaceutically acceptable salts thereof.

[016] In another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[017] In another aspect of the present invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[018] In another aspect of the present invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treating a disease in which modulation of STING is beneficial.

[019] In another aspect of the present invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of inflammation, allergic and autoimmune diseases, infectious diseases, cancer, precancerous syndromes and as immunogenic composition or vaccine adjuvants.

[020] In another aspect of the present invention, there is provided a method of treating a disease in which modulation of STING is beneficial in an individual, which comprises administering a therapeutically effective amount of a compound of Formula (I) or a salt pharmaceutically acceptable thereof.

[021] In another aspect of the present invention, there is provided a method of treating inflammation, allergic and autoimmune diseases, infectious diseases and cancer in an individual, which comprises administering a therapeutically effective amount of a compound of Formula (I) or a salt pharmaceutically acceptable thereof.

[022] In another aspect of the present invention, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament to be used in the treatment of a disease in which the modulation of STING is beneficial.

[023] In another aspect of the present invention, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of inflammatory, allergic and autoimmune diseases, infectious diseases, precancerous syndromes and cancer.

[024] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent.

[025] In another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent and one or more pharmaceutically acceptable excipients.

[026] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent for use in therapy.

[027] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent for use in treating a disease or condition in which modulation of STING is beneficial.

[028] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent for use in the treatment of inflammatory, allergic and autoimmune diseases, infectious diseases, precancerous syndromes and cancer.

[029] In another aspect of the present invention, there is provided a method of treating a disease or condition in which modulation of STING is beneficial in an individual, which comprises administering a therapeutically effective amount of a combination containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent.

[030] In another aspect of the present invention, there is provided a method of treating inflammation, allergic and autoimmune diseases, infectious diseases and cancer in an individual, which comprises administering a therapeutically effective amount of a combination containing a compound of Formula ( I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent.

[031] In another aspect of the present invention, there is provided an immunogenic composition or vaccine adjuvant comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[032] In another aspect of the invention, there is provided a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more immunostimulating agents.

[033] In another aspect of the present invention, there is provided an immunogenic composition comprising an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[034] In another aspect of the present invention, there is provided an immunogenic composition comprising an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of diseases.

[035] In another aspect of the present invention, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of an immunogenic composition comprising an antigen or antigenic composition, for the treatment or prevention of diseases.

[036] In another aspect of the present invention, there is provided a method of treating or preventing diseases which comprises administering an immunogenic composition containing an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human individual who suffers from or is susceptible to disease.

[037] In another aspect of the present invention, there is provided an immunogenic or vaccine composition comprising an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of diseases.

[038] In another aspect of the present invention, there is provided an immunogenic composition comprising an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of diseases.

[039] In another aspect of the present invention, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of an immunogenic or vaccine composition comprising an antigen or antigenic composition for the treatment or prevention of diseases.

[040] In another aspect of the present invention, there is provided a method of treating or preventing diseases which comprises administering a vaccine composition containing an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human individual who suffers from or is susceptible to disease.

[041] In another aspect, there is provided a method of treating an HIV infection in a human who has or is at risk of acquiring the infection, by administering a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof for humans.

[042] In another aspect, there is provided a method of treating an infection in AIDS, in a human having the infection, by administering a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. , to the human.

[043] In another aspect, there is provided a method of treating an HIV infection in a human by administering a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to the human.

BRIEF DESCRIPTION OF THE DRAWINGS

[044] Figure 1 Figure 1 represents the structures of Compounds 1 to 13, where R7 is as defined in Formula (I).

[045] Figure 2 Figure 2 represents the structures of Compounds 14 to 26, where R7 is as defined in Formula (I).

[046] Figure 3 Figure 3 represents the structures of Compounds 27 to 39, where R7 is as defined in Formula (I).

[047] Figure 4 Figure 4 represents the structures of Compounds 40 to 42.

DETAILED DESCRIPTION OF THE INVENTION

[048] This invention relates to new compounds of Formula (I): where: Y1 and Y2 are independently CH2 or O; XI and X2 are independently S or O; R1 is OH and R2 is NH2 or R1 is NH2 and R2 is H; R3 is OH and R4 is NH2 or R3 is NH2 and R4 is H; R5 is selected from: F, OH and OC(O)R7; R6 is selected from: F, OH and OC(O)R7; provided that: when neither R5 nor R6 are F, at least one of Y1 and Y2 is CH2; and R8 and R9 are independently selected from: H, CH2OC(O)R7, CH2OCO2R7, CH2CH2SC(O)R7 and CH2CH2SSCH2R7; provided that: when both X1 and X2 are O, at least one of R8 and R9 is not H; wherein R7 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl, hydroxy and F; and pharmaceutically acceptable salts thereof.

[049] Suitably, in the compounds of Formula (I), at least one of Y1 and Y2 is O. Suitably, in the compounds of Formula (I), both Y1 and Y2 are O.

[050] Suitably, in compounds of Formula (I), at least one of X1 and X2 is S. Suitably, in compounds of Formula (I), X1 is S. Suitably, in compounds of Formula (I), both X1 and X2 are S.

[051] Suitably, in the compounds of Formula (I), R1 is NH2 and R2 is H.

[052] Suitably, in compounds of Formula (I), R5 is OH.

[053] Suitably, in compounds of Formula (I), R6 is F.

[054] Suitably, in compounds of Formula (I), when one of R8 and R9 is not H, it is CH2CH2SC(O)C1-6 alkyl.

[055] Suitably, in compounds of Formula (I), when one of R8 and R9 is not H, it is CH2CH2SSC1-4 alkyl OH.

[056] Suitably, in compounds of Formula (I), when X1 is S, R8 and R9 are H.

[057] Suitably, in compounds of Formula (I), when X2 is S, R8 and R9 are H.

[058] Suitably in the compounds of Formula (I), one of R8 and R9 is H.

[059] Suitably, in the compounds of Formula (I), when X1 and X2 are O, one of R8 and R9 is H.

[060] Suitably, in the compounds of Formula (I), R7 is C12-18 alkyl.

[061] Suitably, in the compounds of Formula (I), R7 is selected from: C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl and F.

[062] Suitably, in the compounds of Formula (I), R7 is C 1-20 alkyl.

[063] Suitably, in the compounds of Formula (I), R7 is tert-butyl.

[064] Suitably, in the compounds of Formula (I), R7 is isopropyl.

[065] Examples of compounds of the present invention include the compounds represented in Figures 1, 2, 3 and 4.

[066] The compounds of Formula (I) can be in the form of a salt.

[067] The compounds of Formula (I) include the compounds of Formula (II): where: Y11 and Y12 are independently CH2 or O; XII is S; X12 is O; R11 is OH and R12 is NH2 or R11 is NH2 and R12 is H; R13 is OH and R14 is NH2 or R13 is NH2 and R14 is H; R15 is selected from: F, OH and OC(O)R17; R16 is selected from: F, OH and OC(O)R17; provided that: when neither R15 nor R16 are F, at least one of Y11 and Y12 is CH2; and R18 and R19 are independently selected from: H, CH2OC(O)R17, CH2OCO2R17, CH2CH2SC(O)R17 and CH2CH2SSCH2R17; wherein R17 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted by one to five substituents independently selected from: aryl, cycloalkyl, hydroxy and F; and pharmaceutically acceptable salts the same.

[068] Suitably, in the compounds of Formula (II), at least one of Y11 and Y12 is O. Suitably, in the compounds of Formula (I), both Y11 and Y12 are O.

[069] Suitably, in compounds of Formula (II), R11 is NH2 and R12 is H.

[070] Suitably, in compounds of Formula (II), R15 is OH.

[071] Suitably, in compounds of Formula (II), R16 is F.

[072] Suitably, in compounds of Formula (II), when one of R18 and R19 is not H, it is CH2CH2SC(O)C1-6 alkyl.

[073] Suitably, in compounds of Formula (II), when one of R18 and R19 is H, that is, CH2CH2SSC1-4 alkyl OH.

[074] Suitably, in the compounds of Formula (II), R18 and R19 are H.

[075] Suitably, in the compounds of Formula (II), one of R18 and R19 is H.

[076] Suitably, in compounds of Formula (II), R17 is C12-18 alkyl.

[077] Suitably, in compounds of Formula (II), R17 is selected from: C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl and F.

[078] Suitably, in compounds of Formula (II), R17 is C 1-20 alkyl.

[079] Suitably, in the compounds of Formula (II), R17 is tert-butyl.

[080] Suitably, in compounds of Formula (II), R17 is isopropyl.

[081] Suitably, the compounds of Formula (II) are in the form of a pharmaceutically acceptable salt.

[082] The compounds of Formula (I) include the compounds of Formula (III): where: Y21 and Y22 are independently CH2 or O; X21 is O; X22 is S; R21 is OH and R22 is NH2 or R21 is NH2 and R22 is H; R23 is OH and R24 is NH2 or R23 is NH2 and R24 is H; R25 is selected from: F, OH and OC(O)R27; R26 is selected from: F, OH and OC(O)R27; provided that: when neither R25 nor R26 are F, at least one of Y21 and Y22 is CH2; and R28 and R29 are independently selected from: H, CH2OC(O)R27, CH2OCO2R27, CH2CH2SC(O)R27 and CH2CH2SSCH2R27; wherein R27 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted by one to five substituents independently selected from: aryl, cycloalkyl, hydroxy and F; and pharmaceutically acceptable salts thereof.

[083] Suitably, in the compounds of Formula (III), at least one of Y21 and Y22 is O. Suitably in the compounds of Formula (III), both Y21 and Y22 are O.

[084] Suitably, in the compounds of Formula (III), R21 is NH2 and R22 is H.

[085] Suitably, in compounds of Formula (III), R25 is OH.

[086] Suitably, in compounds of Formula (III), R26 is F.

[087] Suitably, in compounds of Formula (III), when one of R28 and R29 is not H, it is CH2CH2SC(O)C1-6 alkyl.

[088] Suitably, in compounds of Formula (III), when one of R28 and R29 is not H, it is CH2CH2SSC1-4 alkyl OH.

[089] Suitably, in the compounds of Formula (III), R28 and R29 are H.

[090] Suitably, in the compounds of Formula (III), one of R28 and R29 is H.

[091] Suitably, in the compounds of Formula (III), R27 is C12-18 alkyl.

[092] Suitably, in compounds of Formula (II I), R27 is selected from: C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl and F.

[093] Suitably, in the compounds of Formula (III), R27 is C1-20 alkyl.

[094] Suitably, in the compounds of Formula (III), R27 is tert-butyl.

[095] Suitably, in the compounds of Formula (III), R27 is isopropyl.

[096] Suitably, the compounds of Formula (III) are in the form of a pharmaceutically acceptable salt.

[097] The compounds of Formula (I) and compounds of Formula (II) include the compounds of Formula (IV): where: X31 is S; X32 is O; R31 is OH and R32 is NH2 or R31 is NH2 and R32 is H; R33 is OH and R34 is NH2 or R33 is NH2 and R34 is H; R35 is selected from: F, OH and OC(O)R37; R36 is selected from: F, OH and OC(O)R37; provided that: at least one of R35 and R36 is F; and R38 and R39 are independently selected from: H, CH2OC(O)R37, CH2OCO2R37, CH2CH2SC(O)R37 and CH2CH2SSCH2R37; wherein R37 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl, hydroxy and F; and pharmaceutically acceptable salts thereof.

[098] Suitably, in compounds of Formula (IV), R31 is NH2 and R32 is H.

[099] Suitably, in compounds of Formula (IV), R35 is OH.

[0100] Suitably, in compounds of Formula (IV), R36 is F.

[0101] Suitably, in compounds of Formula (IV), when one of R38 and R39 is not H, it is CH2CH2SC(O)C1-6 alkyl.

[0102] Suitably, in compounds of Formula (IV), when one of R38 and R39 is not H, it is CH2CH2SSC1-4 alkyl OH.

[0103] Suitably, in the compounds of Formula (IV), R38 and R39 are H.

[0104] Suitably, in the compounds of Formula (IV), one of R38 and R39 is H.

[0105] Suitably, in compounds of Formula (IV), R37 is C12-18 alkyl.

[0106] Suitably, in compounds of Formula (IV), R37 is selected from: C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl and F.

[0107] Suitably, in compounds of Formula (IV), R37 is C1-20 alkyl.

[0108] Suitably, in the compounds of Formula (IV), R37 is tert-butyl.

[0109] Suitably, in compounds of Formula (IV), R37 is isopropyl.

[0110] Suitably, the compounds of Formula (IV) are in the form of a pharmaceutically acceptable salt.

[0111] The compounds of Formula (I) and the compounds of Formula (III) include the compounds of Formula (V): where: X41 is O; X42 is S; R41 is OH and R42 is NH2 or R41 is NH2 and R42 is H; R43 is OH and R44 is NH2 or R43 is NH2 and R44 is H; R45 is selected from: F, OH and OC(O)R47; R46 is selected from: F, OH and OC(O)R47; provided that: at least one of R45 and R46 is F; and R48 and R49 are independently selected from: H, CH2OC(O)R47, CH2OCO2R47, CH2CH2SC(O)R47 and CH2CH2SSCH2R47; wherein R47 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents selected independently of: aryl, cycloalkyl, hydroxy and F; and pharmaceutically acceptable salts thereof

[0112] Suitably, in the compounds of Formula (V), R41 is an NH2 and R42 is H.

[0113] Suitably, in compounds of Formula (V), R45 is OH.

[0114] Suitably, in compounds of Formula (V), R46 is F.

[0115] Suitably, in compounds of Formula (V), when one of R48 and R49 is not H, it is CH2CH2SC(O)C1-6 alkyl.

[0116] Suitably, in compounds of Formula (V), when one of R48 and R49 is not H, it is CH2CH2SSC1-4 alkyl OH.

[0117] Suitably, in the compounds of Formula (V), R48 and R49 are H.

[0118] Suitably in compounds of Formula (V), one of R48 and R49 is H.

[0119] Suitably, in compounds of Formula (V), R47 is C12-18 alkyl.

[0120] Suitably, in compounds of Formula (V), R47 is selected from: C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl and F.

[0121] Suitably, in the compounds of Formula (V), R47 is C1-20 alkyl.

[0122] Suitably, in the compounds of Formula (V), R47 is tert-butyl.

[0123] Suitably, in compounds of Formula (V), R47 is isopropyl.

[0124] Suitably, the compounds of Formula (V) are in the form of a pharmaceutically acceptable salt.

[0125] The compounds of Formula (I) include the compounds of Formula (VI): where: X51 is O; X52 is O; R51 is OH and R52 is NH2 or R51 is NH2 and R52 is H; R53 is OH and R54 is NH2 or R53 is NH2 and R54 is H; R55 is selected from: F, OH and OC(O)R47; R56 is F; R58 and R59 are independently selected from: H, CH2OC(O)R57, CH2OCO2R57, CH2CH2SC(O)R57 and CH2CH2SSCH2R57; wherein R57 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl, hydroxy and F; provided that at least one of R58 and R59 is not H. and pharmaceutically acceptable salts thereof

[0126] Suitably, in compounds of Formula (VI), R51 is NH2 and R52 is H.

[0127] Suitably, in compounds of Formula (VI), R55 is OH.

[0128] Suitably, in the compounds of formula (VI), when one of R58 and R59 is not H, it is CH2CH2SC(O)C1-6 alkyl.

[0129] Suitably, in the compounds of formula (VI), when one of R58 and R59 is not H, it is CH2CH2SSC1-4 alkyl OH.

[0130] Suitably, in the compounds of Formula (VI), one of R58 and R59 is H.

[0131] Suitably, in the compounds of Formula (VI), R57 is C12-18 alkyl.

[0132] Suitably, in the compounds of Formula (VI), R57 is selected from: C1-20 alkyl and C1-20 alkyl substituted by one to 5 substituents independently selected from: aryl, cycloalkyl and F.

[0133] Suitably, in the compounds of Formula (VI), R57 is C1-20 alkyl.

[0134] Suitably, in the compounds of Formula (VI), R57 is tert-butyl.

[0135] Suitably in compounds of Formula (VI), R57 is isopropyl.

[0136] Suitably, the compounds of Formula (VI) are in the form of a pharmaceutically acceptable salt.

[0137] Compounds of Formula (I) include: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)- 9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl -2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione, isomer 1; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl -2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, isomer 2; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl -2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl -2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione, isomer 1; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl -2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, isomer 2; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino -9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10] octadecan-3,12-dione; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino -9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10] octadecan-3,12-dione, isomer 1; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino -9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10] octadecan-3,12-dione, isomer 2; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino -9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10] octadecan-3,12-dione; (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino -9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10] octadecan-3,12-dione, isomer 1; and (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8- (6- amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10 ]octadecan-3,12-dione, isomer 2; and pharmaceutically acceptable salts thereof

[0138] Compound 2 is considered to be a mixture of isomers, as indicated below:

[0139] The isomers of Compound 2 are:

[0140] Compound 28 is considered to be a mixture of isomers, as indicated below:

[0141] The isomers of compound 28 are:

[0142] It is considered that the compounds represented, for example, by the structure: also exist in a protonated form, such as: which represents the same compound.

[0143] It is considered that the compounds represented, for example, by the structure: also exist in a protonated form, such as: which represents the same compound.

[0144] It is considered that the compounds represented, for example, by the structure: also exist in a protonated form, such as: which represents the same compound.

[0145] Typically, the salts of the present invention are pharmaceutically acceptable salts. Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention.

[0146] Salts, including pharmaceutically acceptable salts, are easily prepared by those skilled in the art.

[0147] Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate , camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecylsulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentilate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphate, glycolate, hexylresorcinate, hippurate, hydrabamine (N,N'-di(dehydroabiethyl)-ethylenediamine), hydrobromide, hydrochloride, iodhydrate, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsilate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate (embonate), pantothenate , pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, theoclate (8-chlorotheophyllinate) , thiocyanate, triethiodide, undecanoate, undecylenate and valerate.

[0148] Representative pharmaceutically acceptable base addition salts include, but are not limited to, aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benetamine (N-benzylphenethylamine ), benzathine (N,N'-dibenzylethylenediamine, bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p chlorobenzyl-2-pyrrolyldine-1'-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine , diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t-butylamine and zinc.

[0149] The invention includes all possible stoichiometric and non-stoichiometric forms of compounds of Formula (I) within its scope.

[0150] The compounds of the invention can exist in solid or liquid form. In solid form, the compound of the invention can exist in a continuum of solid states ranging from completely amorphous to completely crystalline. The term “amorphous” refers to a state in which the material does not have long band order at molecular levels and, depending on temperature, can exhibit the physical properties of a solid or a liquid. Typically, these materials do not impart distinctive X-ray diffraction patterns and, in exhibiting the properties of a solid, are more conventionally described as a liquid. After heating, a change from solid to liquid properties occurs, which is characterized by a change of state normally of second order (“glass transition”). The term “crystalline” refers to a solid phase in which the material has a regular internal structure ordered at molecular levels and provides a distinct X-ray diffraction pattern with defined peaks. These materials, when heated sufficiently, will also exhibit the properties of a liquid, but the change from solid to liquid is characterized by a phase change, usually first order (“melting point”).

[0151] The compounds of the invention may have the ability to crystallize in more than one form, a characteristic that is known as polymorphism (“polymorphs”). Polymorphism can generally occur in response to changes in temperature or pressure, or both, and can also result from variations in the crystallization process. Polymorphs can be distinguished by several physical characteristics known in the art, such as X-ray diffraction patterns, solubility and melting point.

[0152] Compounds of Formula (I) can exist in solvated and unsolvated forms. As used herein, the term "solvate" refers to a complex of varying stoichiometry formed by a solute (in this invention, a compound of Formula (I) or a salt) and a solvent. Such solvents, for the purpose of the invention, may not interfere with the biological activity of the solute. The person skilled in the art will understand that pharmaceutically acceptable solvates can be formed for crystalline compounds, in which solvent molecules are incorporated into the crystal lattice during crystallization. The incorporated solvent molecules can be water or non-aqueous molecules, such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine and ethyl acetate molecules. The crystal lattice embedded with water molecules is typically referred to as “hydrates”. Hydrates include stoichiometric hydrates as well as compositions containing varying amounts of water.

[0153] It is also observed that compounds of Formula (I) can form tautomers. “Tautomers” refer to compounds that are alternating forms of a given compound structure, and that vary in terms of the displacement of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted upon treatment with acid or base. It is understood that all tautomers and tautomer mixtures of the compounds of the present invention are included within the scope of the compounds of the present invention. For example, and for absolute clarity, in compounds of Formula (I), when R1 or R3 are OH, the compounds will form the keto tautomer (= O).

[0154] Although the aspects for each variable have generally been listed above, separately for each variable, this invention includes those compounds in which several or each aspect in Formula (I) are selected from each of the aspects listed above. Therefore, this invention intends to include all combinations of aspects for each variable.

[0155] Native CDN molecules may be sensitive to degradation by phosphodiesterases that are present in the blood, on the surface of the host cell or in host cells, for example, in antigen-presenting cells, which absorb vaccine formulations containing said native molecules from CDN. Specific examples are ectonucleotidiases, such as CD39, CD73 and ENPP1, which reside on the cellular plasma membrane, facing the plasma, many of which are known to degrade nucleotides, for example, ATP is converted to AMP by both CD39 and ENPP1. Recently, ENPP1 was identified as a major contributor to the degradation of CDNs that have a 2'-5' phosphodiester linkage (Li, L. et al., 2014, Nature Chemical Biology, 10(12), p10431048). The potency of a CDN that possesses STING agonist activity would be diminished by such degradation, resulting in a lower induced expression level of a signature molecule of innate immunity (e.g., IFN-beta) and therefore exhibits weaker adjuvant potency. . The present invention describes two different and complementary approaches that can be used to improve and maintain the potency of newly described CDNs. As described in detail in the sections below, these approaches are the substitution of sulfur for oxygen at the non-binding positions of the phosphodiester and the use of a prodrug strategy to improve CDN penetration into the cell and protect it from degradation.

[0156] One aspect of the present invention relates to stereochemically defined diastereomers of cyclic purine mono- and dithiodiphosphate dinucleotides that induce STING-dependent TBK 1 activation and their methods of preparation and use.

[0157] The present invention relates to methods of providing a potent STING agonist that is capable of initiating and maintaining a T cell response to tumor antigens, alone or in combination with other immuno-oncological agents, and to methods of providing adjuvant compositions. These compositions are comprised of one or more cyclic purine dinucleotides of Formula (I), wherein the cyclic purine dinucleotides present in the composition are single monothiophosphate diastereomers or substantially pure dithiophosphate diastereomers, methods for their manufacture and methods for the use of them to stimulate an immune response in an animal. The objective of both single agent and vaccine formulation is to provide a combination of antigens and adjuvants capable of generating a sufficient population of memory T cells and/or B cells that react rapidly to a pathogen, tumor cell, etc., containing an antigen of interest.

[0158]Thiophosphates (also referred to as phosphorothioates) are a variant of normal nucleotides in which one of the non-binding oxygens linked to the phosphorus is replaced by a sulfur. A phosphorothioate bond is inherently chiral. The skilled artisan will recognize that each thiophosphate in this structure can exist in the Rou S form. Thus, the Rp and Sp forms are possible on each phosphorus atom. In each case, substantially pure diastereomers of these molecules are preferred. Examples of such CDN thiophosphate molecules are depicted in FIGS. 1 to 4 in this document.

[0159] The term “prodrug”, as used herein, refers to a modification of contemplated compounds, wherein the modified compound is converted within the body (e.g., in a target cell or target organ) back into to the unmodified form through enzymatic or non-enzymatic reactions. In many cases, the prodrug form is inactive or substantially less active than the original non-prodrug form of the contemplated compounds. In certain embodiments, the hydroxyl on a ribose comprises a leaving group of the prodrug (Compounds 13, 26 and 39). In other embodiments, the prodrug form involves the derivatization of one or both phosphates and/or thiophosphates (compounds 3-12, 16-25 and 29-38). Prodrugs can modify the physicochemical, biopharmaceutical and pharmacokinetic properties of medications. The reasons for the development of prodrugs are commonly poor aqueous solubility, chemical instability, low oral bioavailability, lack of penetration of the blood-brain barrier and high first-pass metabolism associated with the original drug. Suitable prodrug moieties are described, for example, “Prodrugs and Targeted Delivery”, J. Rautico, Ed., John Wiley & Sons, 2011. Phosphate prodrugs that are especially relevant to the present invention are found. if described by Wiemer, AJ and Wiemer, DF in “Prodrugs of Phosphonates and Phosphates: Crossing the Membrane Barrier” in Topics of Current Chemistry, (2015) V360, 115-160.

[0160] Preferred cyclic purine dinucleotides of the present invention include the diphosphate CDN prodrugs (such as Compounds 9, 10, 11 and 12 of Figure 1; Compounds 22, 23, 24 and 25 of Figure 2; Compounds 35, 36, 37 and 38 of Figure 3 and Compounds 40, 41 and 42 of Figure 4), the non-prodrugs of monothiophosphates (such as Compounds 2, 15 and 28 of Figures 1-3) and the non-prodrug forms of dithiophosphosphates (such as Compounds 1, 14, and 27 of Figures 1-3) and prodrug forms of both the mono-(such as Compounds 7, 8, 20, 21, 33, and 34 of Figures 1-3) and dithiophosphate (such as Compounds 3-6, 16-19 and 29-32 of Figures 1 to 3).

Definitions

[0161] As used herein, “a compound of the invention” includes all solvates, complexes, polymorphs, radiolabeled derivatives, tautomers, stereoisomers and optical isomers of the compounds of Formula (I) and the salts thereof.

[0162] As used herein, the specific compounds of the invention are designated numerically as indicated in the Figures. For example, Compound 2 is the compound in Figure 1 with “2” below it, and Compound 27 is the compound in Figure 3 with “27” below it. Furthermore, the designations “a” and “b”, etc. correspond to Isomer 1 and Isomer 2, etc., respectively. For example, “Compound 2a” is Isomer 1 of Compound 2, “Compound 27b” is Isomer 2 of Compound 27.

[0163] Unless otherwise defined, the designation “Isomer” or “Diastereomer” is an indication of the order in which a specified compound is eluted from a separation column under the specified conditions. The compound specified with a shorter retention time in the LCMS is designated as “Isomer 1” or “Diastereomer 1”, the compound specified with a longer retention time in the LCMS is designated as “Isomer 2” or “Diastereomer 2”, etc.

[0164] As used herein, the term “effective amount” means the amount of a drug or pharmaceutical agent that will induce the biological or medical response of a tissue, system, animal or human that is sought, for example, by a researcher or clinical. Furthermore, the term “therapeutically effective amount” means any amount that, as compared to a corresponding individual who has not received such amount, results in improved treatment, cure, prevention, or improvement of a disease, disorder, or side effect or decreased rate of advancement of a disease or disorder. The term also includes, within its scope, amounts effective to increase/improve normal physiological function.

[0165] The term “prophylaxis” includes prevention and refers to a measure or procedure that serves to prevent, rather than cure or treat, a disease. Prevention refers to a reduction in the risk of acquiring or developing a disease that causes at least one clinical symptom of the disease to not develop in an individual who may be exposed to a disease-causing agent or in an individual predisposed to the disease before the onset of the disease.

[0166] As used herein, the term “pharmaceutically acceptable” refers to compounds, materials, compositions and dosage forms that are, within the scope of common medical sense, suitable for use in contact with the tissues of humans and animals. without excessive toxicity, irritation or other problem or complication, proportionate to a reasonable risk/benefit ratio.

[0167] As used herein, “pharmaceutically acceptable excipients” includes all diluents, binding carriers, glidants and other components of pharmaceutical formulations with which the compound of the invention is administered.

[0168] “Alkyl” refers to a hydrocarbon chain with the specified number of “member atoms”. For example, C1-C6 alkyl refers to an alkyl group containing 1 to 6 member atoms. For example, C12-C18 alkyl refers to an alkyl group containing 12 to 18 member atoms. For example, C1-C20 alkyl refers to an alkyl group containing 1 to 20 member atoms. Alkyl groups can be saturated, unsaturated, linear or branched. Representative branched alkyl groups have one, two, or three branches. Examples of alkyl include methyl, ethyl, ethylene, propyl (n-propyl and isopropyl), butene, butyl (n-butyl, isobutyl and t-butyl), pentyl and hexyl.

[0169] “Cycloalkyl”, unless otherwise indicated, refers to a saturated or unsaturated non-aromatic hydrocarbon ring system containing three to seven carbon atoms. Cycloalkyl groups are monocyclic or bicyclic ring systems. For example, C3-C7 cycloalkyl refers to a cycloalkyl group containing 3 to 7 member atoms. Examples of cycloalkyl as used herein include: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptyl and spiroheptane.

[0170] “Aryl” refers to an aromatic hydrocarbon ring. Aryl groups are monocyclic, bicyclic and tricyclic ring systems with a total of five to fourteen ring member atoms, wherein at least one ring system is aromatic and wherein each ring in the system contains 3 to 7 member atoms, such as phenyl, naphthalene, tetrahydronaphthalene and biphenyl. Suitably aryl is phenyl.

[0171] “Heteroaryl” refers to a 4 to 8 membered monocyclic aromatic ring containing from 1 to 7 carbon atoms and containing from 1 to 4 heteroatoms, provided that, when the number of carbon atoms is 3, the ring aromatic contains at least two heteroatoms. Heteroaryl groups containing more than one heteroatom may contain different heteroatoms. The described heteroaryl includes: pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furanyl, furazanyl, thienyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, tetrazinyl.

[0172] “Heterocycloalkyl” refers to a saturated or unsaturated non-aromatic ring containing 4 to 12 member atoms, of which 1 to 11 are carbon atoms and 1 to 6 are heteroatoms. Heterocycloalkyl groups containing more than one heteroatom may contain different heteroatoms. Heterocycloalkyl groups are monocyclic ring systems or a monocyclic ring fused to an aryl ring or a heteroaryl ring containing 3 to 6 member atoms. Examples of heterocycloalkyl include: pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothienyl, pyrazolidinyl, oxazolidinyl, oxetanyl, thiazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, morpholinyl, thiamorpholinyl, 1,3-dioxolanyl, 1,3-diox anila, 1 ,4-dioxanyl, 1,3-oxathiolanyl, 1,3-oxathianyl, 1,3-dithyanyl, 1,3oxazolidin-2-one, hexahydro-1H-azepine, 4,5,6,7,tetrahydro-1H-benzimidazole , piperidinyl, 1,2,3,6-tetrahydro-pyridinyl and azetidinyl.

[0173] “Heteroatom”, unless otherwise defined, refers to a nitrogen, sulfur or oxygen atom.

Compositions

[0174] Although it is possible that the compound of the invention can be administered as the raw chemical for use in therapy, it is possible to present the compound of the invention as an active ingredient as a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. Accordingly, the invention further provides pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically acceptable excipients. The excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient. According to another aspect of the invention, there is also provided a process for preparing a pharmaceutical composition including the agent, or pharmaceutically acceptable salts thereof, with one or more pharmaceutically acceptable excipients. The pharmaceutical composition can be used in the treatment and/or prophylaxis of any of the conditions described herein.

[0175] Generally, the compound of the invention is administered in a pharmaceutically effective amount. The amount of compound actually administered will typically be determined by a physician, taking into account relevant circumstances, including the condition being treated, the route of administration chosen, the actual compound administered, the age, weight, response of each patient, the severity of the patient's symptoms and others.

[0176] Pharmaceutical compositions can be presented in single dose forms containing a predetermined amount of active ingredient per single dose. The term "single dosage forms" refers to physically discrete units suitable as single dosages for humans and other mammals, wherein each unit contains a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with an excipient, vehicle or carrier. Typical single dosage forms include ampoules or syringes dosed and prefilled with the liquid compositions, or pills, tablets, capsules or the like in the case of solid compositions.

[0177] Preferred single dosage compositions are those that contain a daily dose or subdose, or an appropriate fraction, of an active ingredient. Such single doses may therefore be administered once or more than once daily. Such pharmaceutical compositions may be prepared by any method well known in the pharmaceutical art.

[0178] The pharmaceutical compositions can be adapted for administration by any appropriate route, for example orally (including buccal or sublingual), rectally, inhaled, intranasal, topical (including buccal, sublingual or transdermal), vaginal or injectable (including subcutaneous , intramuscular, parenteral, intravenous or intradermal). Such compositions can be prepared by any method known in the pharmaceutical art, for example, by associating the active ingredient with the carrier(s) or excipient(s).

[0179] In addition to the routes of administration described above for the treatment of cancer, pharmaceutical compositions can be adapted for administration by intratumoral or peritumoral injection. Intratumoral or peritumoral injection of a compound of the present invention directly into or adjacent to a single solid tumor is expected to induce an immune response that can attack and destroy cancer cells throughout the body, substantially reducing, and in some cases, permanently eliminating, the tumor of the individual affected by the disease. Activating the immune system in this manner to kill tumors at a distant site is commonly known as the abscopal effect and has been demonstrated in animals with multiple therapeutic modalities (van der Jeught, et al., Oncotarget, 2015, 6(3), 1359-1381 ). An additional advantage of local or intratumoral or peritumoral administration is the ability to achieve equivalent efficacy at much lower doses, minimizing or eliminating adverse events that may be seen at much higher systemic doses (Marabelle, A., et al., Clinical Cancer Research, 2014, 20 (7), pp. 1747-1756).

[0180] Pharmaceutical compositions adapted for oral administration can be presented as discrete units, such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or liquid oil-in-water emulsions or liquid water-in-oil emulsions.

[0181] For example, for oral administration in the form of a tablet or capsule, the active drug component can be combined with a pharmaceutically acceptable and non-toxic inert oral excipient, such as ethanol, glycerol, water and the like. Powders are prepared by reducing the compound to a suitable size and mixing with a similarly prepared pharmaceutical excipient, such as an edible carbohydrate, such as starch or mannitol. Flavoring, preservative, dispersant and coloring agents may also be present.

[0182] Capsules are formulated by preparing a powder mixture, as described above, and filling the formed gelatin shells. Excipients including glidants and lubricants, such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol, may be added to the powder mixture prior to the filling operation. A disintegrating or solubilizing agent, such as agar, calcium carbonate or sodium carbonate, may also be added to improve the availability of the medicine when the capsule is ingested.

[0183] Furthermore, when desired or necessary, excipients, including suitable binding agents, glidants, lubricants, sweeteners, flavorings, disintegrants and coloring agents, can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or breaking it, adding a lubricating and disintegrating agent, and pressing it into tablets. A powder mixture is prepared by mixing the compound with a suitable admixture, with a diluent or base, as described above, and, optionally, with a binder, such as carboxymethylcellulose, alliginate, gelatin or polyvinylpyrrolidone, a solution retardant, such as such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder, such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials, and forcing it through a screen. As an alternative to granulation, the powder mixture can be passed through the tableting machine and the result is imperfectly formed pastes broken into granules. Granules can be lubricated to prevent adhesion to tableting matrices by adding stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with a free-flowing inert carrier and compressed into tablets directly without going through the granulation or pulverizing steps. A clear or opaque protective coating consisting of a sealing layer of shellac, a coating of sugar or polymeric material, and a polished wax coating can be provided. Dyes can be added to these coatings to distinguish different single dosages.

[0184] Oral fluids such as solutions, suspensions, syrups and elixirs can be prepared in unit dosage form, so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in an aqueous solution with a suitable flavor, while elixirs are prepared using a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizing and emulsifying agents, such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavoring additives, such as mint oil or natural sweeteners or saccharin or other artificial sweeteners, may also be added.

[0185] When appropriate, single dosage compositions for oral administration can be microencapsulated. The composition may also be prepared to prolong or sustain release, for example, by coating or inserting the particulate material in polymers, wax or the like.

[0186] The compounds of the invention can also be administered in the form of liposome administration systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. The compounds of the invention can also be administered in the form of a nanoparticle administration vehicle, which has multiple compositions and preparation methods. Both polymeric nanoparticles and suitably composed and sized liposomes are particularly advantageous formulations for the treatment of cancer and, in particular, for the delivery of the compounds of the present invention, since they are preferentially targeted to the tumor and lymph nodes. These targeted formulations have several potential advantages, that is: they protect the compounds of the present invention from degradation, increase the amount of active agent for local action, and minimize potential undesirable side effects as a result of excessive systemic exposure (Cai, Shuang et al., 2011. Advanced Drug Delivery Reviews, 2011, V63, pp. 901-908). The potential use of such approaches in formulating a CDN STING agonist has been demonstrated for formulations that act directly on tumors (Nakumura, T. et al., Journal of Controlled Release, 2015, V216, pp. 149-157) and in use as an adjuvant (Hanson, M. et al., Journal of Clinical Investigation, 2015, V125 (6), pp. 2532-2546). Furthermore, there are various modes of administration (intratumoral, subcutaneous, intravenous, intraperitoneal and intramuscular) of nanoparticle and liposome formulations that may be useful especially for the compounds of the present invention. Specifically, similar to native CDN molecules, those of the present invention may be sensitive to degradation by phosphodiesterases that are present in or on host cells, for example, in antigen-presenting cells. The potency of a compound of the present invention may be diminished by such degradation, resulting in a lower level of induced expression of a signature molecule of innate immunity (e.g., IFN-beta). Thus, this degradation may provide weaker potency as measured by the release of IFN-beta by PBMCs or reduced vaccine potency as defined by the magnitude of a measured antigen-specific immune response.

[0187] Pharmaceutical compositions adapted for transdermal administration can be presented as discreet patches intended to remain in intimate contact with the recipient's epidermis for an extended period of time.

[0188] Pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0189] For eye treatments or other external tissues, for example, mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated into an ointment, the active ingredient can be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated into a cream in an oil-in-water cream base or a water-in-oil base.

[0190] Pharmaceutical compositions adapted for topical ocular administration include eye drops, in which the active ingredient is dissolved or suspended in a suitable vehicle, especially an aqueous solvent.

[0191] Pharmaceutical compositions adapted for topical oral administration include pills, lozenges and mouthwashes.

[0192] Pharmaceutical compositions adapted for rectal administration can be presented as suppositories or as enemas.

[0193] Dosage forms for nasal administration or inhalation can be conveniently formulated as aerosols, solutions, suspensions, drops, gels or dry powders.

[0194] Compositions for intranasal administration include aqueous compositions administered into the nose by drops or by pressurized pump. Suitable compositions contain water as a diluent or carrier for this purpose. Compositions for pulmonary or nasal administration may contain one or more excipients, for example, one or more suspending agents, one or more preservatives, one or more surfactants, one or more tonicity-adjusting agents, one or more co-solvents, and may include components to control the pH of the composition, for example, a buffer system. Furthermore, the compositions may contain other excipients, such as antioxidants, for example, sodium metabisulfite, and taste masking agents. The compositions can also be administered into the nose or other regions of the respiratory tract by nebulization.

[0195] Intranasal compositions can allow the compound(s) of Formula (I) or (a) pharmaceutically acceptable salt(s) to be administered to all areas of the nasal cavity (the target tissue) and, in addition, may allow the compound(s) of Formula (I) or (a) pharmaceutically acceptable salt(s) to remain in contact with the target tissue for periods of time larger. A suitable dosage regimen for intranasal compositions would be one in which the patient inhales slowly through the nose after the nasal cavity is clean. During inhalation, the composition would be administered to one nostril while the other would be manually compressed. This procedure would then be repeated for the other nostril. Typically, one or two sprays per nostril will be administered by the above procedure, once, twice or three times a day, ideally once a day. Intranasal compositions suitable for once-daily administration are particularly interesting.

[0196] The suspending agent(s), if included, will typically be present in an amount of 0.1 to 5% (w/w), such as 1. 5% to 2.4% (w/w), based on the total weight of the composition. Examples of pharmaceutically acceptable suspending agents include, but are not limited to, Avicel® (microcrystalline cellulose and sodium carboxymethylcellulose), sodium carboxymethylcellulose, veegum, tragacanth, bentonite, methylcellulose, xanthan gum, carbopol and polyethylene glycols.

[0197] Compositions for pulmonary or nasal administration may contain one or more excipients and may be protected from microbial or fungal contamination and growth by including one or more preservatives. Examples of pharmaceutically acceptable antimicrobial agents or preservatives include, but are not limited to, quaternary ammonium compounds (e.g. benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, lauralkonium chloride and myristyl picolinium chloride), mercurial agents ( e.g. phenylmercuric nitrate, phenylmercuric acetate and thimerosal), alcoholic agents (e.g. chlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterial esters (e.g. parahydroxybenzoic acid esters), chelating agents such as disodium edetate (EDTA ) and other antimicrobial agents, such as chlorhexidine, chlorocresol, sorbic acid and salts thereof (such as potassium sorbate) and polymyxin. Examples of pharmaceutically acceptable antifungal agents or preservatives include, but are not limited to, sodium benzoate, sorbic acid, sodium propionate, methylparaben, ethylparaben, propylparaben and butylparaben. The preservative(s), if included, may be present in an amount of 0.001 to 1% (w/w), such as 0.015% to 0.5% (w/w). /p) based on the total weight of the composition.

[0198] Compositions (for example, in which at least one compound is in suspension) may include one or more surfactants that act as facilitators of the dissolution of drug particles in the aqueous phase of the composition. For example, the amount of surfactants used is an amount that will not lead to foam formation during mixing. Examples of pharmaceutically acceptable surfactants include fatty alcohols, esters and ethers, such as polyoxyethylene (20) sorbitan monooleate (Polysorbate 80), macrogol ethers and poloxamers. The surfactant may be present in an amount of about 0.01 to 10% (w/w), such as 0.01 to 0.75% (w/w), for example, about 0.5% ( w/w), based on the total weight of the composition.

[0199] One or more tonicity-adjusting agent(s) may be included to achieve tonicity with bodily fluids, for example, nasal cavity fluids, resulting in reduced levels of irritation. Examples of pharmaceutically acceptable tonicity-adjusting agents include, but are not limited to, sodium chloride, dextrose, xylitol, calcium chloride, glucose, glycerin and sorbitol. A tonicity adjusting agent, if present, may be included in an amount of 0.1 to 10% (w/w), such as 4.5 to 5.5% (w/w), for example about of 5.0% (w/w), based on the total weight of the composition.

[0200] The compositions of the invention can be buffered by the addition of suitable buffering agents, such as sodium citrate, citric acid, trometamol, phosphates, such as disodium phosphate (for example, the dodecahydrate, heptahydrate, dihydrate and anhydrous forms) or phosphate sodium and mixtures thereof.

[0201] A buffering agent, if present, may be included in an amount of 0.1 to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of the composition.

[0202] Examples of flavor masking agents include sucralose, sucrose, saccharin or a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame, acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhicin, thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, a natural flavoring agent, an artificial flavoring agent and combinations thereof.

[0203] One or more cosolvents may be included to aid the solubility of the drug compound(s) and/or other excipients. Examples of pharmaceutically acceptable cosolvents include, but are not limited to, propylene glycol, dipropylene glycol, ethylene glycol, glycerol, ethanol, polyethylene glycols (e.g., PEG300 or PEG400) and methanol. In one embodiment, the cosolvent is propylene glycol.

[0204] The co-solvent(s), if present, may be included in an amount of 0.05 to 30% (w/w), such as 1 to 25% (w/w). /w), for example, from 1 to 10% (w/w) based on the total weight of the composition.

[0205] Compositions for administration by inhalation include aqueous, organic or aqueous/organic mixtures, dry powder or crystalline compositions administered to the respiratory tract by pressurized pump or inhaler, for example, dry powder reservoir inhalers, dry powder inhalers, single dose, pre-calibrated multidose dry powder inhalers, nasal inhalers or pressurized aerosol inhalers, nebulizers or insufflators. Suitable compositions contain water as a diluent or carrier for this purpose and may be provided with conventional excipients such as buffering agents, tonicity-modifying agents and the like. The aqueous compositions can also be administered to the nose and other regions of the respiratory tract by nebulization. Such compositions may be aqueous solutions or suspensions or aerosols administered from pressurized packaging, such as a metered dose inhaler, with the use of a suitable liquefied propellant.

[0206] Compositions for topical administration to the nose (for example, for the treatment of rhinitis), or to the lung, include pressurized aerosol compositions and aqueous compositions released into the nasal cavities by a pressurized pump. Compositions that are not pressurized and are suitable for topical administrations to the nasal cavity are particularly interesting. Suitable compositions contain water as a diluent or carrier for this purpose. Aqueous compositions for administration to the lung or nose may be provided with conventional excipients, such as buffering agents, tonicity-modifying agents, and the like. Aqueous compositions can also be administered into the nose by nebulization.

[0207] A fluid dispenser can typically be used to deliver a fluid composition to the nasal cavities. The fluid composition may be aqueous or non-aqueous, but typically aqueous. Such a fluid dispenser may have a dispensing nozzle or dispensing orifice through which a metered dose of the fluid composition is dispensed upon application of a force exerted by the user on a pumping mechanism of the fluid dispenser. Such fluid dispensers are generally provided with a reservoir with multiple calibrated doses of the fluid composition, wherein the doses are dispensed upon sequential pump actuations. The dispensing nozzle or orifice may be configured to be inserted into the user's nostrils to dispense the fluid composition by spraying into the nasal cavity. A fluid dispenser of the aforementioned type is described and illustrated in International Patent Application Publication No. WO 2005/044354 (Glaxo Group Limited). The dispenser has a space/housing that houses a fluid dispensing device with a compression pump mounted in a container containing a fluid composition. This space has at least one manually operated side lever that moves inward relative to the space to move the container upward in the space via a cam that causes the pump to compress and pump a calibrated dose of the composition from a rod. of the pump through a space nasal nozzle. In one embodiment, the fluid dispenser is of the general type illustrated in Figures 30-40 of WO 2005/044354.

[0208] An aqueous composition containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, can also be administered by a pump, as disclosed in International Patent Application No. WO 2007/138084 (Glaxo Group Limited), for example, as described with reference to Figures 22-46, or as described in British Patent Application No. GB0723418.0 (Glaxo Group Limited), for example, as described with reference to Figures 7-32 thereof. The pump can be driven by an actuator, as described in Figures 1-6 of document GB0723418.0.

[0209] Dry powder compositions for topical administration to the lung by inhalation may, for example, be presented in capsules and cartridges, for example, of gelatin, or blisters, for example, of laminated aluminum foil, for use in an inhaler or inflator. Powder mixture compositions generally contain a powder mixture for inhalation of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a suitable powder base (carrier substance/diluent/excipient), such as mono-, di-or polysaccharides (e.g. lactose or starch). Dry powder compositions may also include, in addition to the drug and carrier, an additional excipient (e.g., a ternary agent, such as a sugar ester, e.g., cellobiose octaacetate, calcium stearate, or magnesium stearate.

[0210] In one embodiment, a composition suitable for administration by inhalation may be incorporated into a plurality of sealed dose containers, provided in the package(s) containing the medicament, mounted within a suitable inhalation device. The containers may be breakable, peelable or otherwise opened, one at a time, and doses of the dry powder composition are administered by inhalation into a mouthpiece of the inhalation device, as is known in the art. The medicine package can take several different shapes, for example a disc shape or an elongated strip. Representative inhalation devices are the DISKHALER™ and DISKUS™ devices, marketed by GlaxoSmithKline.

[0211] An inhalable dry powder composition may also be provided as a bulk reservoir in an inhalation device, the device being provided with a measuring/calibration mechanism for calibrating a dose of the composition from the reservoir to a channel of inhalation where the calibrated dose can be inhaled by a patient who inhales it into a mouthpiece of the device. Examples of these commercially available devices are TURBUHALER™ (AstraZeneca), TWISTHALER™ (Schering) and CLICKHALER™ (Innovata).

[0212] Another delivery method for an inhalable dry powder composition is for calibrated doses of the composition to be supplied in capsules (one dose per capsule) which are then loaded into an inhalation device, typically by the patient on an as-needed basis. The device has means for rupturing, puncturing or otherwise opening the capsule so that the dose can enter the patient's lungs when the patient inhales into the mouthpiece of the device. Examples of such commercialized devices include ROTAHALER™ (GlaxoSmithKline) and HANDIHALER™ (Boehringer Ingelheim).

[0213] Pressurized aerosol compositions suitable for inhalation may be a suspension or a solution and may contain a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a suitable propellant, such as a hydrogen-containing fluorocarbon or chlorofluorocarbon. or mixtures thereof, particularly hydrofluoroalkanes, especially 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The aerosol composition may optionally contain in the composition additional excipients well known in the art, such as surfactants, for example, oleic acid, lecithin or an oligolactic acid or a derivative thereof, such as, for example, that described in WO 94 /21229 and WO 98/34596 (Minnesota Mining and Manufacturing Company) and cosolvents, for example, ethanol. Pressurized compositions will generally be kept in a can (e.g., an aluminum can) closed with a valve (e.g., a metering valve) and installed in an actuator provided with a nozzle.

[0214] Pharmaceutical compositions adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0215] Pharmaceutical solutions adapted for injectable administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatics and solutes that make the composition isotonic with respect to the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in single-dose or multi-dose containers, e.g., ampoules and sealed vials, and may be stored in a lyophilized condition that requires only the addition of the sterile liquid carrier, e.g., water for injections, immediately prior to administration. use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

[0216] It should be understood that, in addition to the ingredients particularly mentioned above, the compositions may include other agents conventional in the state of the art, taking into account the type of formulation in question, for example, those suitable for oral administration may include agents flavorings.

[0217] Interfering or antisense RNA molecules can be administered to the mammal that needs them. Alternatively, constructs including the same can be administered. Such molecules and constructs can be used to interfere with the expression of the protein of interest, e.g., histone demethylase, and therefore modify histone demethylation. Typically, delivery/administration are the means known in the art.

[0218] Interfering or antisense RNA molecules can be administered in vitro or in vivo to cells, for example, to mammalian tumors. Modes of administration can be used without limitation, including: intravenous, intramuscular, intraperitoneal, intra-arterial, local administration during surgery, endoscopic, subcutaneous and oral. Vectors can be selected for desirable properties for any specific application. Vectors can be viral or plasmid. Adenoviral vectors are useful in this case. Cell, tissue-specific, or otherwise regulatable promoters can be used to control the transcription of inhibitory polynucleotide molecules. Non-viral carriers, such as liposomes or nanospheres, can also be used.

[0219] The compounds of Formula (I), and the pharmaceutically acceptable salts thereof, can also be formulated to produce a composition to be used as an adjuvant to modulate the activity of vaccines. Such compositions may contain antibody(s) or antibody fragment(s) or an antigenic component, including but not limited to proteins, DNA, live or dead bacteria and/or viruses or virus-like particles, together with one or more components with adjuvant activity including, but not limited to, emulsions of aluminum salts, oil and water, heat shock proteins, lipid A preparations and derivatives, glycolipids, other TLR agonists such as CpG DNA or similar agents, cytokines, such as GM-CSF or IL-12 or similar agents.

[0220] A therapeutically effective amount of the agent will depend on a number of factors, including, for example, the age and weight of the individual, the precise condition requiring treatment and its severity, the nature of the formulation and the route of administration, and ultimately at the discretion of the attending physician or veterinarian. In particular, the individual to be treated is a mammal, particularly a human.

[0221] The agent can be administered as a daily dose. This amount can be given in a single dose per day or, generally, in several subdoses (such as two, three, four, five or six) per day, such that the total daily dose is the same.

[0222] Suitably, the amount of the compound of the invention administered, in accordance with the present invention, will be a selected amount of 0.01 mg to 1 g per day (calculated as the free or unsalted compound).

[0223] The compounds of Formula (I), and the pharmaceutically acceptable salts thereof, can be used alone or in combination with other therapeutic agents. The compounds of Formula (I), and the pharmaceutically acceptable salts thereof, and the other pharmaceutically active agents may be administered together or separately and, when administered separately, the administration may occur simultaneously or sequentially, in any order, by any route. convenient separate or combined pharmaceutical compositions.

[0224] The amounts of the compound(s) of Formula (I), or the pharmaceutically acceptable salt(s), and the other pharmaceutically active agent(s)( s), and the relative times of administration will be selected to achieve the desired combined therapeutic effect. The compounds of the present invention and other therapeutic agents can be used in combination by administering simultaneously in a unitary pharmaceutical composition including both compounds. Alternatively, the combination may be administered separately in separate pharmaceutical compositions, each comprising one of the compounds in a sequential manner in which, for example, the compound of the invention is administered first and the other second and vice versa. Such sequential administration may be temporally proximate (e.g., simultaneously) or distant. Furthermore, there is no need for the compounds to be administered in the same dosage form, for example, one compound can be administered topically and the other compound can be administered orally.

[0225] Combinations can be presented as a combined kit. By the term “combination kit” or “component kit”, as used herein, is meant that the pharmaceutical composition or compositions are used to administer the combination according to the invention. When both compounds are administered simultaneously, the combination kit may contain both compounds in a single pharmaceutical composition, such as a tablet, or in separate pharmaceutical compositions. When the compounds are not administered simultaneously, the combination kit will contain each compound in separate pharmaceutical compositions in a single package or in separate pharmaceutical compositions in separate packages.

[0226] The combined kit may also contain instructions, such as dosage and administration instructions. Such dosage and administration instructions may be those that are provided to a physician, for example, by a drug label, or may be those that are provided by a physician, such as instructions to a patient.

[0227] When the combination is administered separately in a sequential manner, in which one is administered first and the other second or vice versa, such sequential administration may be temporally close or distant. For example, administration of the other agent minutes to several minutes after administration of the first agent and administration of the other agent several hours to several days after administration of the first agent, wherein the time lapse is not limited. For example, one agent may be administered once a day and the other agent may be administered 2 or 3 times a day, or one agent may be administered once a week and the other agent may be administered once a day, and so on. against.

[0228] It will be clear to one skilled in the art that, when necessary, the other therapeutic ingredients can be used in the form of salts, for example, as alkali metal or amine salts or as acid addition salts, or prodrugs, or as esters, for example lower alkyl esters, or as solvates, for example hydrates, to optimize the activity and/or stability and/or physical characteristics, such as solubility, of the therapeutic ingredient. It will also be clear that, when necessary, therapeutic ingredients can be used in optically pure form.

[0229] When combined in the same composition, it is understood that the two compounds must be stable and compatible with each other and the other components of the composition and can be formulated for administration. When formulated separately, they may be conveniently provided in any suitable composition in such a manner as is known for such compounds in the art.

[0230] When the compound of Formula (I) is used in combination with a second therapeutic agent active against the same disease, condition or disorder, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily understood by those skilled in the art.

[0231] In one embodiment, the mammal in the methods and uses of the present invention is a human.

[0232] The invention also provides a pharmaceutical composition comprising from 0.5 to 1000 mg of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and from 0.5 to 1000 mg of a pharmaceutically acceptable excipient.

[0233] The compounds of the invention are useful in the treatment of diseases in which STING modulation is beneficial. These include inflammation, allergic and autoimmune diseases, infectious diseases, cancer and precancerous syndromes.

[0234] As modulators of the immune response, compounds of Formula (I) and pharmaceutically acceptable salts thereof may also be useful, independently or in combination, as an adjuvant in the treatment of diseases in which the STING modulation is beneficial.

[0235] In one aspect, the disease or condition is inflammation, allergy and autoimmune disorders. Associated autoimmune diseases include, but are not limited to, systemic lupus erythematosus, psoriasis, insulin-dependent diabetes mellitus (IDDM), dermatomyositis, human immunodeficiency virus (HIV), AIDS, and Sjogren's syndrome (SS). Inflammation represents a group of vascular, cellular, and neurological responses to trauma. Inflammation can be characterized as the movement of inflammatory cells such as monocytes, neutrophils, and granulocytes into tissues. This is often associated with reduced endothelial barrier function and tissue edema. Inflammation can be classified as acute or chronic. Acute inflammation is the body's initial response to harmful stimuli and occurs due to the increased movement of plasma and leukocytes from the blood to injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive change in the type of cells that are present at the site of inflammation and is characterized by the simultaneous destruction and scarring of tissue from the inflammatory process.

[0236] When it occurs as part of an immune response to infection or as an acute response to trauma, inflammation can be beneficial and is usually self-limiting. However, inflammation can be harmful in several conditions. This includes the production of excessive inflammation in response to infectious agents, which can lead to significant organ damage and death (e.g., in the onset of sepsis). Furthermore, chronic inflammation is generally deleterious and is the cause of numerous chronic diseases, causing severe and irreversible tissue damage. In such situations, the immune response is often directed against one's own tissue (autoimmunity), although chronic responses to foreign materials can also lead to bystander damage to one's own tissues.

[0237] The objective of anti-inflammatory therapy is, therefore, to reduce this inflammation, inhibit autoimmunity when present and allow the physiological or tissue healing and repair process to progress.

[0238] The agents can be used to treat inflammation of any tissue and organs in the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, inflammation of the digestive system, ocular inflammation, inflammation of the reproductive system and other inflammations, as exemplified below.

[0239] Musculoskeletal inflammation refers to any inflammatory condition of the musculoskeletal system, particularly conditions that affect the skeletal joints, including the joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knee, ankle and foot and conditions that affect the tissues that connect muscles to bones, such as tendons. Examples of musculoskeletal inflammation that can be treated with compounds of the invention include arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis and osteitis (including, for example, Paget's disease, osteitis pubis and osteitis fibrosa cystica).

[0240] Ocular inflammation refers to inflammation of any structure of the eye, including the eyelids. Examples of ocular inflammation that can be treated with the compounds of the invention include blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis and uveitis.

[0241] Examples of inflammation of the nervous system that can be treated with the compounds of the invention include encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia.

[0242] Examples of inflammation of the vascular or lymphatic system that can be treated with the compounds of the invention include arthrosclerosis, arthritis, phlebitis, vasculitis and lymphangitis.

[0243] Examples of inflammatory conditions of the digestive system that can be treated with the compounds of the invention include cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), ileitis and proctitis.

[0244] Examples of inflammatory conditions of the reproductive system that can be treated with the compounds of the invention include cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis and vulvodynia.

[0245] The agents can be used to treat autoimmune conditions that have an inflammatory component. Such conditions include acute disseminated alopecia, alopecia universalis, Behcet's disease, Chagas disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, Goodpasture syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed tissue disease conjunctiva, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, ORD thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, autoimmune hemolytic anemia type hot, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis and vitiligo.

[0246] The agents can be used to treat T cell-mediated hypersensitivity diseases having an inflammatory component. Such conditions include contact hypersensitivity, contact dermatitis (including that from poison ivy), hives, skin allergies, respiratory allergies (hay fever, allergic rhinitis), and gluten-sensitive enteropathy (celiac disease).

[0247] Other inflammatory conditions that can be treated with the agents include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis , parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, pneumonitis, prostatitis, pyelonephritis and stomatitis, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, intestine and skin allografts, skin homografts and heart valve xenografts, serum sickness and graft-versus-host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary syndrome, congenital adrenal hyperplasia, nonsuppurative thyroiditis , hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity reactions, allergic conjunctivitis, keratitis, herpes ophthalmic zoster, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, chemotherapy for fulminant or disseminated pulmonary tuberculosis, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autoimmune) hemolytic anemia, leukemia and lymphomas in adults, acute leukemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis. Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic lung disease, and inflammation that accompanies infectious conditions (e.g., sepsis ).

[0248] In another aspect of the invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of inflammation, allergy and autoimmune disease.

[0249] In another aspect, there is provided a method of treating inflammation, allergy and autoimmune disease, which comprises administering a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human who needs the same.

[0250] In another aspect, there is provided for the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of inflammation, allergy and autoimmune disease.

[0251] In one aspect, the disease to be treated is asthma.

[0252] The compounds of Formula (I) and the pharmaceutically acceptable salts thereof can be used in combination with one or more other agents that may be useful in the prevention or treatment of allergic diseases, inflammatory diseases, autoimmune diseases, for example; antigenic immunotherapy, antihistamines, steroids, NSAIDs, bronchodilators (e.g., beta 2 agonists, adrenergic agonists, anticholinergic agents, theophylline), methotrexate, leukotriene modulators and similar agents; monoclonal antibody therapy, such as anti-IgE, anti-TNF, anti-IL-5, anti-IL-6, anti-IL-12, anti-IL-1 and similar agents; receptor therapies, e.g., entanercept and similar agents; Non-antigen-specific immunotherapies (e.g., interferon or other cytokines/chemokines, cytokine/chemokine receptor modulators, cytokine agonists or antagonists, TLr agonists, and similar agents).

[0253] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of allergic diseases, inflammation or autoimmune diseases.

[0254] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in treating allergic diseases, inflammation or autoimmune diseases for use in therapy. .

[0255] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of allergic diseases, inflammation or autoimmune diseases, for use in the treatment of allergic diseases, inflammation or autoimmune diseases.

[0256] In another aspect, there is provided for the use of a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of allergic diseases, inflammation or autoimmune diseases. in the manufacture of a medicine for the treatment of allergic diseases, inflammation or autoimmune diseases.

[0257] In another aspect, there is provided a method of treating allergic diseases, inflammation or autoimmune diseases, which comprises administering a therapeutically effective amount of a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in treating allergic diseases, inflammation or autoimmune diseases, to a human in need thereof.

[0258] In another aspect, there is provided a pharmaceutical composition comprising a combination containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of allergic diseases, inflammation or autoimmune diseases. and one or more pharmaceutically acceptable excipients.

[0259] In one aspect, the disease to be treated with this combination is asthma.

[0260] In one aspect, the disease or condition to be treated is cancer.

[0261] Examples of cancerous diseases in which compounds of Formula (I), or pharmaceutically acceptable salts or solvates thereof, may have potentially beneficial antitumor effects include, but are not limited to, cancers of the lung, bone, pancreas, skin, head, neck, uterus, ovaries, stomach, colon, breast, esophagus, small intestine, intestine, endocrine system, parathyroid gland, adrenal gland, urethra, prostate, penis, testicles, ureter, bladder, kidney or liver; rectal cancer; cancer of the anal region; carcinomas of the fallopian tubes, endometrium, cervix, vagina, vulva, renal pelvis, kidney cells; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemagioma; hepatoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphomas; primary CNS lymphoma; CNS neoplasms; spinal cord tumors; squamous cell carcinomas; synovial sarcoma; malignant pleural mesotheliomas; brain stem glioma; pituitary adenoma; bronchial adenoma; chondromatous hamartoma; inesothelioma; Hodgkin's disease or a combination of one or more of the above cancers.

[0262] Suitably, the present invention relates to a method of treating or decreasing the severity of cancers selected from the group consisting of brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden's disease, Lhermitte-Duclos tumor, Wilm's tumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, head and neck cancer, kidney, liver, melanoma, ovarian, pancreatic, adenocarcinoma, ductal adenocarcinoma, adenosquamous carcinoma, acinar cell carcinoma, glucagonoma, insulinoma, prostate, sarcoma, osteosarcoma, giant cell, bone tumor, thyroid, lymphoblastic T-cell leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic neutrophilic leukemia, leukemia acute lymphoblastic T cell, plasmacytoma, large cell immunoblastic leukemia, mantle cell leukemia, multiple myeloma, megakaryotic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, erythroleukemia, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulvar cancer, cervical cancer, endometrial cancer, kidney cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharyngeal cancer, oral cancer, mouth cancer, GIST (gastrointestinal stromal tumor) and testicular cancer.

[0263] Suitably, the present invention relates to a method of treating or lessening the severity of precancerous syndromes in a mammal, including a human, wherein the precancerous syndrome is selected from: cervical intraepithelial neoplasia, monoclonal gammopathy of undetermined significance (MGUS), myelodysplastic syndrome, aplastic anemia, cervical lesions, nevi of the skin (pre-melanoma), prostatic intraepithelial neoplasia (intranslated), ductal carcinoma in situ (DCIS), colon polyps, and severe hepatitis or cirrhosis .

[0264] The compounds of the present invention can also be used as adjuvants to improve the enhanced immune response to any given antigen and/or reduce reactogenicity/toxicity in a patient, particularly a human, in need thereof. To this end, a compound of this invention can be used in combination with vaccine compositions to modify, especially improve, the immune response, for example, increasing the level or duration of protection and/or allowing a reduction in the antigenic dose.

[0265] The compounds of Formula (I) and pharmaceutically acceptable salts thereof can be used in combination with one or more vaccines or immunogenic antigens useful in preventing or treating viral infections. Such vaccines or immunogenic antigens include, without limitation, proteins or particles derived from pathogenic agents, such as attenuated viruses, viral particles and viral proteins normally used as immunogenic substances. Examples of viruses and viral antigens include, without limitation, Poliovirus, Cioronaviridae and Coronavirus, Rhinovirus (all subtypes), Adenovirus (all subtypes), Hepatitis A virus, Hepatitis B, Hepatitis C, Hepatitis D, Human Papillomavirus (including all subtypes), rabies virus, human T-cell lymphotrophic virus (all subtypes), rubella virus, mumps virus, Coxsackie virus A (all subtypes), Cosackie virus B (all subtypes), enterovirus human, herpesviruses, including cytomegalovirus, Epstein-Barr virus, human herpesviruses (all subtypes), herpes simplex virus, varicella zoster virus, human immunodeficiency virus (HIV) (all subtypes), AIDS, Epstein-Barr virus , Reovirus (all subtypes), Filovirus, including Marburg and Ebola viruses (all strains), Arenavirus, including lymphocytic chorioromenitis virus, Lassa virus, Junin virus and Machupo virus, Arbovirus, including West Nile virus, dengue virus (all serotypes), Zika virus, Colorado tick fever virus, Sindbis virus, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae, Poxviruses, including orthopoxviruses (smallpox virus, monkey poxvirus, vaccinia virus, cowpox), the yatapoxviruses (tanapox virus, the Yaba monkey tumor virus), parapoxviruses, molluscum viruses, yellow fever viruses, Hantaviruses, including Hantaan, Seoul, Dobrava, Sin Nombre, Puumala and Saaremaa type Dobrava viruses, human influenza viruses and parainfluenza viruses (all types), H1N1 influenza and swine influenza viruses, respiratory syncytial viruses (all subgroups), rotaviruses, including human rotaviruses A-E, bovine rotavirus, rhesus monkey rotavirus, Polyomaviruses, including simian viruses 40, JC virus, BK virus, Coltivirus, eyach virus, calcivirus and Parvoviridae, including dependovirus, parvovirus and erythrovirus.

[0266] The compounds of the present invention may also be useful in the treatment of one or more diseases affecting mammals that are characterized by cell proliferation in the area of disorders associated with neovascularization and/or vascular permeability, including blood vessel proliferative disorders, including arthritis (rheumatoid arthritis) and restenosis; fibrotic disorders, including liver cirrhosis and atherosclerosis; mesangial cell proliferative disorders, including glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, proliferative retinopathies, organ transplant rejection, and glomerulopathies; and metabolic disorders, including psoriasis, diabetes mellitus, chronic wound healing, inflammation and neurodegenerative diseases.

[0267] In another aspect of the invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treating a disease state selected from the group consisting of: HIV, HBV, HCV, influenza, dermal warts, multiple sclerosis, allergic inflammation and as an adjuvant.Zhijian Chen -Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing - Nature Immunology (2016), 17, 1142-1149. Seng-Ryong Woo -STING-Dependent Cytosolic DNA Sensing Mediates Innate Immune Recognition of Immunogenic Tumors (2014), 41, 830-842. Jenny P.-Y. Ting -NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses - Cell Host and Microbe (2016), 19, 515-528. Zhijian Chen -Pivotal Roles of cGAS-cGAMP Signaling in Antiviral Defense and Immune Adjuvant Effects -Science (2013), 341, 1390-1394. Nuchsupha Sunthamala -E2 Proteins of High Risk Human Papillomaviruses Down- Modulate STING and IFN-k Transcription in Keratinocytes - PLoS (2014), 9, 1-11. Guo, H., et al. (2016). NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses. Cell host & microbe 19, 515-528. Gao, D., et al. (2013). Cyclic GMP-AMP synthesis is an innate immune sensor of HIV and other retroviruses. Science 341, 903-906. Guo, F., et al. (2015). Sting agonists induce an innate antiviral immune response against hepatitis B virus. Antimicrobial Agents and Chemotherapy 59, 1273-1281. Dansako, H., et al. (2016). The cyclic GMP-AMP synthetase-STING signaling pathway is required for both the innate immune response against HBV and the suppression of HBV assembly. FEBS J 283, 144-156. Chang, J., et al. (2015). Treatment of chronic hepatitis B with pattern recognition receptor agonists: Current status and potential for a cure. Antiviral Research 121, 152-159. Li, XD, et al. (2013). Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390-1394. Carroll, E.C., et al. (2016). The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons. Immunity 44, 597-608. Wang, J., et al. (2016). Natural STING Agonist as an “Ideal” Adjuvant for Cutaneous Vaccination. J Invest Dermatol 136, 2183-2191. Holm, C.K., et al. (2016). Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses. Nat Commun 7, 10680. Shirey, K. A., et al. (2011). The anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induces IFN-beta-mediated antiviral activity in vitro and in vivo. J Leukoc Biol 89, 351-357. Nitta, S., et al. (2013). Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type I interferon-dependent innate immunity. Hepatology 57, 46-58. Sunthamala, N., et al. (2014). E2 proteins of high risk human papillomaviruses down-modulate STING and IFN-kappa transcription in keratinocytes. PLoS One 9, e91473. Lau, L., et al. (2015). DNA tumor virus oncogenes antagonize the cGAS-STING DNA-sensing pathway. Science 350, 568-571. Kidd, P. (2003). Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 8, 223-246. Huang, L., et al. (2013). Cutting edge: DNA sensing via the STING adapter in myeloid dendritic cells induces potential tolerogenic responses. J Immunol 191, 3509-3513. Lemos, H., et al. (2014). Activation of the STING adapter attenuates experimental autoimmune encephalitis. J Immunol 192, 5571-5578.

[0268] In another aspect of the invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer and/or precancerous syndromes.

[0269] In another aspect, there is provided a method of treating cancer which comprises administering a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human in need thereof.

[0270] In another aspect, there is provided for the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer and/or precancerous syndromes.

[0271] In one embodiment, the compound of the invention can be used with other therapeutic methods of treating cancer. In particular, in antineoplastic therapy, combination therapy with other chemotherapeutic agents, hormones, antibodies, as well as radiation and/or surgical treatments other than those mentioned above is envisaged.

[0272] In one embodiment, additional anticancer/antineoplastic therapy is surgery and/or radiotherapy.

[0273] In one embodiment, the additional antineoplastic therapy is at least one additional antineoplastic agent.

[0274] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one antineoplastic agent.

[0275] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one antineoplastic agent, for use in therapy.

[0276] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one antineoplastic agent, for use in the treatment of cancer and/or precancerous syndromes.

[0277] In another aspect, there is provided the use of a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one antineoplastic agent, in the manufacture of a medicament for the treatment of cancer and/or or precancerous syndromes.

[0278] In another aspect, there is provided a method of treating cancer comprising administering a therapeutically effective amount of a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one antineoplastic agent to a human who needs the same.

[0279] In another aspect, there is provided a pharmaceutical composition comprising a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent, particularly at least one antineoplastic agent and one or more pharmaceutically acceptable carriers, diluents and excipients.

[0280] Any antineoplastic agent that has activity against a treatable tumor can be used in the combination. Typical useful antineoplastic agents include, but are not limited to, antimicrotubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustard, oxazaphosphorines, alkylsulfonates, nitrosoureas and triazenes; antibiotics, such as anthracyclines, actinomycins and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites, such as purine and pyrimidine analogues and antifolate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; cell cycle signaling inhibitors; immuno-oncological agents and immunostimulatory agents.

Antimicrotubule or antimitotic agents:

[0281] Antimicrotubule or antimitotic agents are phase-specific agents active against tumor cell microtubules during the M phase or mitosis of the cell cycle. Examples of antimicrotubule agents include, but are not limited to, diterpenoids and vinca alkaloids.

[0282] Diterpenoids, which are derived from natural sources, are phase-specific antineoplastic agents that act in the G2/M phases of the cell cycle. Diterpenoids are believed to stabilize the β-tubulin subunit of microtubules by binding to this protein. Disassembly of the protein then appears to be inhibited, which stops mitosis and causes subsequent cell death. Examples of diterpenoids include, but are not limited to, paclitaxel and its analogue, docetaxel.

[0283] Paclitaxel, from 5β, 20-epoxM,2α,4,7β,10β,13α-hexa-hydroxytax-11-en- 9-one 4,10-diacetate 2-benzoate-13-ester with (2R, 3S)-N-benzoyl-3-phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available in the form of an injectable solution -TAXOL®. It is a member of the taxane family of terpenes. Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman et al., Yale Journal of Biology and Medicine, 64:583, 1991; McGuire et al., Ann. Lntem, Med., 111: 273,1989) and for the treatment of breast cancer (Holmes et al., J. Nat. Cancer Inst., 83: 1797, 1991). It is a potential candidate for the treatment of skin neoplasms (Einzig et al., Proc. Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastire et al., Sem. Oncol., 20 :56, 1990). The compound also has potential for the treatment of polycystic kidney disease (Woo et al., Nature, 368: 750, 1994), lung cancer and malaria. Treatment of patients with paclitaxel results in bone marrow suppression (multiple cell lines, Ignoff, RJ et al., Cancer Chemotherapy Pocket Guide, 1998) related to dosing duration above a threshold concentration (50nM)(Kearns, CM et al ., Seminars in Oncolog, 3 (6) p.16-23, 1995).

[0284] Docetaxel, (2R, 3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5β-20-epoxy-1,2α,4,7β,10β,13α-hexa -hydroxytax-11-en-9-one 4-acetate 2-benzoate, trihydrate; It is commercially available in the form of an injectable solution - TAXOTERE®. Docetaxel is indicated for the treatment of breast cancer. Docetaxel is a semi-synthetic derivative of paclitaxel q.v., prepared with a natural precursor, 10-desacetyl-baccatin III, extracted from the needle of the European yew tree.

[0285] Vinca alkaloids are specific antineoplastic phase agents derived from the periwinkle plant. Vinca alkaloids act in the M phase (mitosis) of the cell cycle, specifically binding to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into microtubules. Mitosis is believed to be arrested at metaphase with subsequent cell death. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine and vinorelbine.

[0286] Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBAN® in the form of an injectable solution. Although it has a possible indication as second-line therapy for several solid tumors, it is mainly indicated in the treatment of testicular cancer and several lymphomas, including Hodgkin's disease; and lymphocytic and histiocytic lymphomas. Myelosuppression is the dose-limiting side effect of vinblastine.

[0287] Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commercially available as ONCOVIN® in the form of an injectable solution. Vincristine is indicated for the treatment of acute leukemias and its use in treatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas has also been noted. Alopecia and neurological effects are the most common side effects of vincristine and, to a lesser extent, myelosuppression and the effects of gastrointestinal mucositis occur.

[0288] Vinorelbine, 3',4'-didehydro-4'-deoxy-C'-norvincaleukoblastine [R- (R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)], commercially available in the form of vinorelbine tartrate injectable solution (NAVELBINE®), it is a semi-synthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell prostate cancer, advanced and hormone-refractory prostate cancer. Myelosuppression is the most common dose-limiting side effect of vinorelbine.

Platinum coordination complexes:

[0289] Platinum coordination complexes are non-phase-specific antineoplastic agents that interact with DNA. Platinum complexes enter tumor cells, react with water (aquation) and form intra- and interchain cross-links with DNA, causing adverse biological effects on the tumor. Examples of platinum coordination complexes include, but are not limited to, oxaliplatin, cisplatin and carboplatin.

[0290] Cisplatin, cis-diaminodichloroplatinum, is commercially available as PLATINOL® in the form of an injectable solution. Cisplatin is mainly indicated for the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer.

[0291] Carboplatin, platinum, diamine [1,1-cyclobutane-dicarboxylate (2-)-O,O'], is commercially available as PARAPLATIN® in the form of an injectable solution. Carboplatin is mainly indicated in the first and second line treatment of advanced ovarian carcinoma.

Alkylating agents:

[0292] Alkylating agents are strong electrophilic, non-phase-specific antineoplastic agents. Typically, alkylating agents form covalent bonds, by alkylation, with DNA through nucleophilic portions of the DNA molecule, such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl and imidazole groups. This alkylation disrupts the function of the nucleic acid, which leads to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan and chlorambucil; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine; and triazenes, such as dacarbazine.

[0293] Cyclophosphamide, 2-[bis(2-chloroethyl)amino] tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide monohydrate, commercially available as an injectable solution or tablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of malignant lymphomas, multiple myeloma and leukemias.

[0294]Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available in the form of an injectable solution or tablets such as ALKERAN®. Melphalan is indicated for the palliative treatment of multiple myeloma and unresectable epithelial ovarian carcinoma. Bone marrow suppression is the most common dose-limiting side effect of melphalan.

[0295] Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN® in tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia and malignant lymphomas, such as lymphosarcoma, giant follicular lymphoma and Hodgkin's disease.

[0296] Busulfan, 1,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® in tablets. Busulfan is indicated for the palliative treatment of chronic myeloid leukemia.

[0297] Carmustine, 1,3-[bis(2-chloroethyl)-1-nitrosourea, is commercially available as single vials of lyophilized material such as BiCNU®. Carmustine is indicated for palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas.

[0298] Dacarbazine, 5-(3,3-dimethyl-1-triazene)-imidazol-4-carboxamide, is commercially available as single vials of material such as DTIC-Dome®. Dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second-line treatment of Hodgkin's disease.

Antineoplastic Antibiotics:

[0299] Antineoplastic antibiotics are non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts the common function of nucleic acids, leading to cell death. Examples of antineoplastic antibiotic agents include, but are not limited to, actinomycins, such as dactinomycin, ancocyclines, such as daunorubicin and doxorubicin; and bleomycins.

[0300] Dactinomycin, also known as Actinomycin D, is commercially available in injectable form, as COSMEGEN®. Dactinomycin is indicated for the treatment of Wilm's tumor and rhabdomyosarcoma.

[0301] Daunorubicin, (8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-L-lixohexopyranosyl)oxy]-7,8 hydrochloride, 9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedione is commercially available in injectable liposomal form as DAUNOXOME® or injectable form as CERUBIDINE®. Daunorubicin is indicated for the induction of remission in the treatment of acute non-lymphocytic leukemia and advanced Kaposi's sarcoma associated with HIV.

[0302] Doxorubicin, (8S,10S)-10-[(3-amino-2,3,6-trideoxy-L-junk-hexopyranosyl)oxy]-8-glycolyl hydrochloride, 7,8,9,10 -tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthakenedione, is commercially available in injectable form as RUBEX® or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but it is also a useful component in the treatment of some tumors and solid lymphomas.

[0303] Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is commercially available as BLENOXANE®. Bleomycin is indicated as palliative treatment, as a single agent or in combination with other agents, for squamous cell carcinoma, lymphomas and testicular carcinomas.

Topoisomerase II inhibitors:

[0304] Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.

[0305] Epipodophyllotoxins are phase-specific antineoplastic agents derived from the mandrake plant. Epipodophyllotoxins generally affect cells in the S and G2 phases of the cell cycle, forming a ternary complex with topoisomerase II and DNA, causing breaks in the DNA chain. Chain breaks accumulate, leading to cell death. Examples of epipodophyllotoxins include, but are not limited to, etoposides and teniposides.

[0306] Etoposide, 4'-dimethyl-epipodophyllotoxin 9[4,6-0-(R)-ethylidene-β-D-glucopyranoside], is commercially available in the form of an injectable solution or capsules such as VePESID® and it is commonly known as VP-16. Etoposide is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of non-small cell lung and testicular cancer.

[0307] Teniposide, 4'-dimethyl-epipodophyllotoxin 9[4,6-0-(R)-tenylidene-β-D-glucopyranoside], is commercially available in the form of an injectable solution as VUMON® and is commonly known as VM-26. Teniposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia in children.

Antimetabolite antineoplastic agents:

[0308] Antimetabolite antineoplastic agents are phase-specific antineoplastic agents that act in the S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or inhibiting purine or pyrimidine base synthesis and thus limiting DNA synthesis . Consequently, the S phase does not proceed, which leads to cell death. Examples of antimetabolite antineoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.

[0309] 5-fluorouracil, 5-fluoro-2,4-(1H,3H) pyrimidinedione, is commercially available as fluorouracil. Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into RNA and DNA. The result is usually cell death. 5-fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Other fluoropyrimidine analogs include 5-fluorodeoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate.

[0310] Cytarabine, 4-amino-1-β-D-arabinofuranosyl-2(1H)-pyrimidinone, is commercially available as CYTOSAR-U® and is commonly known as Ara-C. Cytarabine is believed to exhibit cell phase specificity in the S phase, inhibiting DNA chain elongation through incorporation of cytarabine at the terminal end of the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include 5-azacytidine and 2',2'-difluorodeoxycytidine (gemcitabine).

[0311] Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PURINETHOL®. Mercaptopurine exhibits cell phase specificity in the S phase, inhibiting DNA synthesis by an as yet unspecified mechanism. Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. A useful analogue of mercaptopurine is azathioprine.

[0312] Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID®. Thioguanine exhibits cell phase specificity in the S phase, inhibiting DNA synthesis by an as yet unspecified mechanism. Thioguanine is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of acute leukemia. Other purine analogues include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine.

[0313] Gemcitabine, 2'-deoxy-2',2'-difluorocytidine monohydrochloride (β isomer), is commercially available as GEMZAR®. Gemcitabine exhibits cell phase specificity in the S phase and blocks cell progression at the G1/S boundary. Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and, alone, in the treatment of locally advanced pancreatic cancer.

[0314] Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino]benzoyl]-L-glutamic acid, is commercially available as sodium methotrexate. Methotrexate has effects in the cellular phase, specifically in the S phase, inhibiting DNA synthesis, repair and/or replication through the inhibition of dihydrofolic acid reductase, which is necessary for the synthesis of purine- and thymidylate-based nucleotides. Methotrexate is indicated as a single agent or in combination with other chemotherapeutic agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma and carcinomas of the breast, head, neck, ovary and bladder.

Topoisomerase I inhibitors:

[0315] Camptothecins, including camptothecin and camptothecin derivatives, are available, or in development, as inhibitors of topoisomerase I. The cytotoxic activity of camptothecins is believed to be related to their inhibitory activity of topoisomerase I. Examples of camptothecins include, but are not limited to, irinotecan, topotecan and the various optical forms of 7-(4-methylpiperazine-methylene)-10,11-ethylenedioxy-20-camptothecin described below.

[0316] Irinotecan HCl, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino) carbonyloxy]-1H-pyran [3',4',6,7]indolizino [1, 2-b]quinoline-3,14(4H,12H)-dione, is commercially available as the injectable solution CAMPTOSAR®. Irinotecan is a camptothecin derivative that binds, together with its active metabolite SN-38, to the I-DNA topoisomerase complex. Cytotoxicity is believed to occur as a result of irreparable double-strand breaks caused by the interaction of the ternary topoisomerase I:DNA:irintecan or SN-38 complex with replication enzymes. Irinotecan is indicated for the treatment of metastatic cancer of the colon or rectum.

[0317] Topotecan HCl, (S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1H-pyran[3',4',6,7]indolizino monohydrochloride [1, 2-b]quinoline-3,14-(4H,12H)-dione, is commercially available in the form of HYCAMTIN® injectable solution. Topotecan is a camptothecin derivative that binds to the topoisomerase I:DNA complex and prevents the rebinding of single-strand breaks caused by Topoisomerase I in response to torsional deformation of the DNA molecule. Topotecan is indicated for the second-line treatment of metastatic carcinoma of ovarian cancer and small cell cancer.

Hormones and hormonal analogues:

[0318] Hormones and hormone analogues are compounds useful for treating cancer in which there is a relationship between the hormone(s) and the growth and/or lack of growth of cancer. Examples of hormones and hormone analogues useful in the treatment of cancer include, but are not limited to, adrenocorticosteroids, such as prednisone and prednisolone, which are useful in the treatment of malignant lymphoma and acute leukemia in children; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazol, vorazol and exemestane useful in the treatment of adrenocortical carcinoma and hormone-dependent breast carcinoma containing estrogen receptors; progestrins such as megestrol acetate useful in the treatment of hormone-dependent breast cancer and endometrial carcinoma; estrogens, estrogens and antiestrogens, such as fulvestrant, flutamide, nilutamide, bicalutamide, cyproterone acetate and 5a-reductases, such as finasteride and dutasteride, useful in the treatment of prostatic carcinoma and benign prostatic hypertrophy; antiestrogens, such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene, as well as selective estrogen receptor modulators (SERMS), such as those described in United States Patent Nos. 5,681,835, 5,877,219 and 6,207,716, useful in the treatment of hormone-dependent breast carcinoma and other susceptible cancers; and gonadotropin-releasing hormone (GnRH) and its analogues that stimulate the release of leutinizing hormone (LH) and/or follicle-stimulating hormone (FSH) for the treatment of prostate carcinoma, for example, LHRH agonists and antagonists, such as LHRH acetate goserelin and luprolide.

Signal transduction pathway inhibitors:

[0319] Signal transduction pathway inhibitors are those inhibitors that block or inhibit a chemical process that induces an intracellular change. As used herein, this change is cellular proliferation or differentiation. Signal transduction inhibitors useful in the present invention include inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3 domain blockers, serine/threonine kinases, phosphodylinositol-3 kinases, myoinositol signaling and Ras oncogenes.

[0320] Various protein tyrosine kinases catalyze the phosphorylation of specific tyrosyl residues in various proteins involved in regulating cell growth. Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.

[0321] Receptor tyrosine kinases are transmembrane proteins with an extracellular ligand-binding domain, a transmembrane domain and a tyrosine kinase domain. Receptor tyrosine kinases are involved in the regulation of cell growth and are generally referred to as growth factor receptors. Inappropriate or uncontrolled activation of many of these kinases, i.e. aberrant growth factor receptor kinase activity, for example by overexpression or mutation, has been shown to result in uncontrolled cell growth. Consequently, aberrant activity of such kinases has been associated with the growth of malignant tissue. Consequently, inhibitors of these kinases could provide cancer treatment methods. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), erbB2, erbB4, ret, vascular endothelial growth factor receptor ( VEGFr), the tyrosine kinase with homology domains to immunoglobulin-like epidermal growth factor (TIE-2), insulin receptor growth factor-I (IGFI), macrophage colony-stimulating factor (CFMS), BTK, ckit, cmet, fibroblast growth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin receptors (eph), and the RET protooncogene. Several growth receptor inhibitors are in development and include ligand antagonists, antibodies, tyrosine kinase inhibitors, and antisense oligonucleotides. Growth factor receptors and agents that inhibit growth factor receptor function are described, for example, in Kath, John C., Exp. Opin. The R. Patents (2000) 10 (6): 803-818; Shawver et al DDT Vol 2, No. 2 February 1997; and Lofts, F.J. et al., "Growth factor receptors as targets," New Molecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr, David, CRC press 1994, London.

[0322] Tyrosine kinases, which are not growth factor receptor kinases, are called non-receptor tyrosine kinases. Non-receptor tyrosine kinases useful in the present invention, which are targets or potential targets of antineoplastic drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (focal adhesion kinase), Brutons tyrosine kinase and Bcr-Abl . Such non-receptor kinases and agents that inhibit non-receptor tyrosine kinase function are described in Sinh, S. and Corey, S.J., (1999) Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80; and Bolen, JB, Brugge, JS, (1997), Annual review of Immunology. 15: 371-404.

[0323] SH2/SH3 domain blockers are agents that disrupt the binding of the SH2 or SH3 domain on a variety of enzymes or adapter proteins, including the PI3-K p85 subunit, Src family kinases, adapter molecules (Shc, Crk, Nck, Grb2) and Ras-GAP. The SH2/SH3 domains as targets for antineoplastic drugs are discussed in Smithgall, TE (1995), Journal of Pharmacological and Toxicological Methods. 34 (3) 125-32.

[0324] Serine/Threonine kinase inhibitors include blockers of the MAP kinase cascade, which include blockers of Raf kinases (rafk), Extracellular or Mitogen Signal Regulated Kinases (MEKs) and Extracellular Signal Regulated Kinases (ERKs); and blockers of members of the protein kinase C family, including PKC blockers (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta). IkB family kinases (IKKa, IKKb), PKB family kinases, akt kinase family members, and TGF beta receptor kinases. Such serine/threonine kinases and their inhibitors are described in Yamamoto, T., Taya, S., Kaibuchi, K. (1999), Journal of Biochemistry. 126 (5) 799-803; Brodt, P, Samani, A. and Navab, R. (2000), Biochemical Pharmacology, 60. 1101-1107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys. 27: 41-64; Philip, PA and Harris, AL (1995), Cancer Treatment and Research. 78:3-27, Lackey, K. et al. Bioorganic and Medicinal Chemistry Letters, (10), 2000, 223-226; US Patent No. 6,268,391; and Martinez-Iacaci, L., et al., Int. J. Cancer (2000), 88 (1), 44-52.

[0325] Inhibitors of members of the phosphotidyl inositol-3 kinase family, including PI3-kinase, ATM, DNA-PK and Ku blockers are also useful in the present invention. Such kinases are discussed in Abraham, RT (1996), Current Opinion in Immunology. 8 (3) 412-8; Canman, CE, Lim, DS (1998), Oncogene 17 (25) 3301-3308; Jackson, SP (1997), International Journal of Biochemistry and Cell Biology. 29 (7): 935-8; and Zhong, H. et al, Cancer Res, (2000) 60 (6), 1541-1545.

[0326] Also useful in the present invention are Myo-inositol signaling inhibitors, such as phospholipase C blockers and myo-inositol analogues. Such signal inhibitors are described in Powis, G. and Kozikowski A., (1994) New Molecular Targets for Cancer Chemotherapy ed., Paul Workman and David Kerr, CRC press 1994, London.

[0327] Another group of signal transduction pathway inhibitors are Ras oncogene inhibitors. Such inhibitors include inhibitors of the proteases farnesyltransferase, geranylgeranyl transferase and CAAX, as well as antisense oligonucleotides, ribozymes and immunotherapy. Such inhibitors have been shown to block ras activation in cells containing mutant and wild-type ras, consequently acting as antiproliferation agents. Inhibition of the Ras oncogene is discussed in Scharovsky, OG, Rozados, VR, Gervasoni, SI Matar, P. (2000), Journal of Biomedical Science. 7 (4) 292-8; Ashby, MN (1998), Current Opinion in Lipidology. 9(2) 99-102; and BioChem. Biophys. Acta, (19899) 1423 (3): 19-30.

[0328] As mentioned above, antibody antagonists to receptor kinase receptor binding can also serve as signal transduction inhibitors. This group of signal transduction pathway inhibitors includes the use of humanized antibodies to the extracellular ligand-binding domain of the receptor tyrosine kinase. For example, the C225 EGFR-specific antibody Imclone (see Green, MC et al., Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev., (2000), 26 (4), 269-286); Herceptin® and antibody to erbB2 (see Tyrosine Kinase Signaling in Breast cancer: erbB Family Receptor Tyrosine Kinases, Breast cancer Res, 2000, 2 (3), 176-183); and the specific antibody for 2CB VEGFR2 (see Brekken, RA et al., Selective Inhibition of VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice, Cancer Res. (2000) 60, 5117-5124).

Antiangiogenic agents:

[0329] Antiangiogenic agents, including non-receptor MEK angiogenesis inhibitors may be useful. Antiangiogenic agents, such as those that inhibit the effects of vascular edothelial growth factor (e.g., the anti-vascular endothelial cell growth factor antibody -bevacizumab [Avastin™] and compounds that work by other mechanisms (e.g., linomide, inhibitors of the function of integrin αvβ3, endostatin and angiostatin);

Immunotherapeutic agents:

[0330] Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of Formula (I). Immunotherapy approaches, including, for example, ex vivo and in vivo approaches to increase the immunogenicity of the patient's tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4, or granulocyte-macrophage colony-stimulating factor , addresses decreasing T cell anergy, approaches using transfected immune cells such as dendritic cells transfected with cytokines, approaches using tumor cell lines transfected with cytokines, and approaches using anti-idiotypic antibodies

Pro-aptotic agents:

[0331] Agents used in pro-apoptotic regimes (for example, bcl-2 antisense oligonucleotides) can also be used in the combination of the present invention.

Cell cycle signaling inhibitors

[0332] Cell cycle signaling inhibitors inhibit molecules involved in cell cycle control. A family of protein kinases called cyclin-dependent kinases (CDKs) and their interaction with a family of proteins called cyclins controls progression through the eukaryotic cell cycle. Coordinated activation and inactivation of different cyclin/CDK complexes are necessary for normal progression through the cell cycle. Several inhibitors of cell cycle signaling are in development. For example, examples of cyclin-dependent kinases, including CDK2, CDK4 and CDK6, and inhibitors therefor, are described, for example, in Rosania et al., Exp. Opin. The R. Patents (2000) 10 (2): 215-230.

[0333] In one embodiment, the combination of the present invention comprises a compound of Formula I, or a salt or solvate thereof, and at least one antineoplastic agent selected from antimicrotubule agents, platinum coordination complexes, alkylating agents , antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-MEK receptor tyrosine angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents and signaling inhibitors cell cycle.

[0334] In one embodiment, the combination of the present invention includes a compound of Formula I, or a salt or solvate thereof, and at least one antineoplastic agent, which is an antimicrotubule agent selected from diterpenoids and vinca alkaloids. .

[0335] In another embodiment, at least one antineoplastic agent is a diterpenoid.

[0336] In another embodiment, at least one antineoplastic agent is a vinca alkaloid.

[0337] In one embodiment, the combination of the present invention includes a compound of Formula I, or a salt or solvate thereof, and at least one antineoplastic agent, which is a platinum coordination complex.

[0338] In another embodiment, at least one antineoplastic agent is paclitaxel, carboplatin or vinorelbine.

[0339] In another embodiment, at least one antineoplastic agent is carboplatin.

[0340] In another embodiment, at least one antineoplastic agent is vinorelbine.

[0341] In another embodiment, at least one antineoplastic agent is paclitaxel.

[0342] In one embodiment, the combination of the present invention includes a compound of Formula I, or salts or solvates thereof, and at least one antineoplastic agent, which is an inhibitor of the signal transduction pathway.

[0343] In another embodiment, the signal transduction pathway inhibitor is an inhibitor of a growth factor receptor kinase VEGFR2, TIE 2, PDGFR, BTK, erbB2, EGFr, IGFR-1, TrkA, TrkB, TrkC or c-fms.

[0344] In another embodiment, the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase rafk, akt or PKC-zeta.

[0345] In another embodiment, the signal transduction pathway inhibitor is an inhibitor of a non-receptor tyrosine kinase selected from the src family of kinases.

[0346] In another embodiment, the signal transduction pathway inhibitor is a c-src inhibitor.

[0347] In another embodiment, the signal transduction pathway inhibitor is a Ras oncogene inhibitor selected from farnesyl transferase and geranyl transferase inhibitors.

[0348] In another embodiment, the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase selected from the group consisting of PI3K.

[0349] In another embodiment, the signal transduction pathway inhibitor is a dual EGFr/ erbB2 inhibitor, for example, N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6 -[5-({[2-(methanesulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (structure below):

[0350] In one embodiment, the combination of the present invention includes a compound of Formula I, or a salt or solvate thereof, and at least one antineoplastic agent, which is an inhibitor of cell cycle signaling.

[0351] In another embodiment, the cell cycle signaling inhibitor is an inhibitor of CDK2, CDK4 or CDK6.

Immunostimulating agents:

[0352] As used herein, "immunostimulating agent" refers to any agent that can stimulate the immune system. As used herein, immunostimulatory agents include, but are not limited to, vaccine adjuvants such as Toll-like receptor agonists, T cell checkpoint blockers such as mAbs to PD-1 and CTL4, and T cell checkpoint agonists. T cells, such as agonist mAbs to OX-40 and ICOS.

[0353] Additional examples of another active ingredient or ingredients (antineoplastic agent) for use in combination or co-administered with the compound of Formula (I), invented and described here, are the anti-PD-L1 agents.

[0354] Anti-PD-L1 antibodies and methods for producing them are known in the art.

[0355] Such antibodies to PD-L1 may be polyclonal or monoclonal and/or recombinant and/or humanized.

[0356] Examples of PD-L1 antibodies are disclosed in: US Patent No. 8,217,149; 12/633,339; US Patent No. 8,383,796; 13/091,936; US Patent No. 8,552,154; 13/120,406; US Patent Publication No. 20110280877; 13/068337; US Patent Publication No. 20130309250; 13/892671; WO 2013019906; WO 2013079174; US Application No. 13/511,538 (filed August 7, 2012), which is the US National Phase of International Application No. PCT/US10/58007 (filed 2010); and US Application No. 13/478,511 (filed May 23, 2012).

[0357] Examples of additional antibodies to PD-L1 (also referred to as CD274 or B7-H1) and methods for use are described in US Patent No. US 7,943,743; US 20130034559, WO 2014055897, US Patent No. 8,168,179; and US Patent No. 7,595,048. PD-L1 antibodies are in development as immunomodulatory agents for the treatment of cancer.

[0358] In one embodiment, the antibody to PD-L1 is an antibody described in US Patent No. 8,217,149. In another embodiment, the anti-PD-L1 antibody comprises the CDRs of an antibody described in US Patent No. 8,217,149.

[0359] In another embodiment, the antibody to PD-L1 is an antibody described in US Application No. 13/511,538. In another embodiment, the anti-PD-L1 antibody comprises the CDRs of an antibody described in US Application No. 13/511,538.

[0360] In another embodiment, the antibody to PD-L1 is an antibody disclosed in Application No. 13/478,511. In another embodiment, the anti-PD-L1 antibody comprises the CDRs of an antibody disclosed in US Application No. 13/478,511.

[0361] In one embodiment, the anti-PD-L1 antibody is BMS-936559 (MDX-1105). In another embodiment, the anti-PD-L1 antibody is MPDL3280A (RG7446). In another embodiment, the anti-PD-L1 antibody is MEDI4736.

[0362] Additional examples of another active ingredient or ingredients (antineoplastic antagonist agent) for use in combination or co-administered with the compound of Formula (I), invented and described herein, are PD-1 antagonists

[0363] "PD-1 antagonist" means any chemical compound or biological molecule that blocks the binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and, Preferably, it also blocks the binding of PD-L2 expressed on a cancer cell to PD-1 expressed on the immune cell. Alternative or synonymous names for PD-1 and its ligands include: PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any embodiment of the aspects or embodiments of the present invention in which a human subject is to be treated, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of human PD-L1 and PD-L2 to human PD-1. The amino acid sequences of human PD-1 can be found in NCBI Locus No.: NP_005009. The amino acid sequences of human PD-L1 and PD-L2 can be found in NCBI Locus No.: NP_054862 and NP_079515, respectively.

[0364] PD-1 antagonists useful in any aspect of the present invention include a monoclonal antibody (mAb) or antigen-binding fragment thereof, which specifically binds to PD-1 or PD-L1, and, therefore, Preferably, it specifically binds to human PD-1 or PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments.

[0365] Examples of mAbs that bind to human PD-1 and are useful in various aspects and embodiments of the present invention are described in documents US 7488802, US 7521051, US 8008449, US 8354509, US 8168757, WO 2004/ 004771, WO 2004/072286, WO 2004/056875 and US 2011/0271358.

[0366] Specific anti-human PD-1 MAbs useful as PD-1 antagonists in any of the aspects and embodiments of the present invention include: MK-3475, a humanized IgG4 MAb with the structure described in WHO Drug Information, vol. 27, No. 2, pages 161-162 (2013) and comprising the heavy and light chain amino acid sequences shown in Figure 6; nivolumab, a human IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013) and comprising the heavy and light chain amino acid sequences shown in Figure 7; the humanized antibodies h409A11, h409A16 and h409A17, described in documents WO 2008/156712 and AMP-514, are being developed by Medimmune.

[0367] Other PD-1 antagonists, which are useful in any of the aspects and embodiments of the present invention, include an immunoadhesin that specifically binds to PD-1, and, preferably, specifically binds to human PD-1 , for example, a fusion protein that contains the PD-1 or extracellular binding portion of PD-L1 or PD-L2 fused to a constant region, such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in WO 2010/027827 and WO 2011/066342. Specific fusion proteins useful as a PD-1 antagonist in the method of treatment, medicaments and uses of the present invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and if binds to human PD-1.

[0368] Other examples of mAbs that bind to human PD-L1 and useful in the method of treatment, medicines and uses of the present invention are described in documents WO 2013/019906, W02010/077634 A1 and US 8383796. The PD-L1 mAbs Specific anti-human L1 useful as a PD-1 antagonist in the treatment method, medicaments and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C.

[0369] KEYTRUDA/pembrolizumab is an anti-PD-1 antibody marketed by Merck for the treatment of lung cancer. The amino acid sequence of pembrolizumab and methods of use are described in US Patent No. 8,168,757.

[0370] Opdivo/nivolumab is a fully human monoclonal antibody marketed by Bristol Myers Squibb directed against the surface PD-1 receptor of human negative immunoregulatory cells (programmed death-1 or programmed cell death-1/PCD-1) with immunopotentiating. Nivolumab binds and blocks the activation of PD-1, a transmembrane protein of the Ig superfamily, by its ligands PD-L1 and PD-L2, resulting in the activation of T cells and cell-mediated immune responses against tumor cells or pathogens. Activated PD-1 negatively regulates T cell activation and effector function through suppression of P13k/Akt pathway activation. Other names for nivolumab include: BMS-936558, MDX-1106, and ONO-4538. The amino acid sequence for nivolumab and methods of use and manufacture are disclosed in US Patent No. US 8,008,449.

[0371] Additional examples of another active ingredient or ingredients (antineoplastic agent) for use in combination or co-administered with the compound of Formula (I), invented and described here, are immunomodulators.

[0372] As used herein, "immunomodulators" refer to any substance that includes monoclonal antibodies that affect the immune system. The ICOS binding proteins of the present invention can be considered immunomodulators. Immunomodulators can be used as antineoplastic agents for the treatment of cancer. For example, modulators include, but are not limited to, anti-CTLA-4 antibodies, such as ipilimumab (YERVOY) and anti-PD-1 antibodies (Opdivo/nivolumab and Keytruda/pembrolizumab). Other immunomodulators include, but are not limited to, antibodies against OX-40, antibodies against PD-L1, antibodies against LAG3, antibodies against TIM-3, antibodies against 41BB and antibodies against GITR.

[0373] Yervoy (ipilimumab) is a fully human CTLA-4 antibody marketed by Bristol Myers Squibb. The protein structure of ipilimumab and methods of use are described in US Patent Nos. 6,984,720 and 7,605,238.

[0374]CD134, also known as OX40, is a member of the TNFR superfamily of receptors that are not constitutively expressed on resting naïve T cells, unlike CD28. OX40 is a secondary costimulatory molecule, expressed 24 to 72 hours after activation; its ligand, OX40L, is also not expressed in resting antigen-presenting cells, but is expressed after their activation. OX40 expression depends on complete T cell activation; without CD28, OX40 expression is delayed and fourfold lower. Antibodies against OX-40, OX-40 fusion proteins and methods of using the same are disclosed in United States Patent Nos.: US 7,504,101; US 7,758,852; US 7,858,765; US 7,550,140; US 7,960,515; WO 2012027328; WO2013028231.

[0375] The term "Toll-like receptor" (or "TLR"), as used herein, refers to a member of the Toll-like receptor family of proteins, or a fragment thereof, that detects a microbial product and/or or initiates an adaptive immune response. In one embodiment, a TLR activates a dendritic cell (DC). Toll-like receptors (TLRs) are a family of pattern recognition receptors that were initially identified as innate immune system sensors that recognize microbial pathogens. TLRs recognize distinct structures in microbes, often referred to as "PAMPs" (pathogen-associated molecular patterns). Ligand binding to TLRs triggers a cascade of intracellular signaling pathways that induce the production of factors involved in inflammation and immunity. In humans, ten TLRs have been identified. TLRs that are expressed on the surface of cells include TLR-1, -2, -4, -5 and -6, while TLR-3, -7/8 and -9 are expressed with the ER compartment. Subsets of human DCs can be identified based on distinct TLR expression patterns. By way of example, the myeloid or "conventional" subset of DC (mDC) expresses TLRs 1-8 when stimulated and a cascade of activation markers (e.g., CD80, CD86, MHC class I and II, CCR7), pro- -inflammatory and chemokines are produced. One result of this stimulation and resulting expression is the priming/primary activation of antigen-specific CD4+ and CD8+ T cells. These DCs acquire an enhanced capacity to absorb/uptake antigens and present them appropriately to T cells. In contrast, the plasmacytoid subset of DCs (pDC) expresses only TLR7 and TLR9 upon activation, with a resulting activation of NK cells as well. As dying tumor cells can adversely affect DC function, it has been suggested that activation of DC with TLR agonists may be beneficial for the primary activation of antitumor immunity in an immunotherapy approach to the treatment of cancer. It has also been suggested that successful treatment of breast cancer using radiation and chemotherapy requires activation of TLR4.

[0376] TLR agonists known in the art and useful in the present invention include, but are not limited to, the following: Pam3Cys, a TLR1/2 agonist; CFA, a TLR2 agonist; MALP2, a TLR2 agonist; Pam2Cys, a TLR2 agonist; FSL-1, a TLR-2 agonist; Hib-OMPC, a TLR-2 agonist; polyribosinic acid: polyribocidic acid (Poly I:C), a TLR3 agonist; polyadenosine-polyureidic acid (polyAU), a TLR3 agonist; polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Hiltonol), a TLR3 agonist; bacterial flagellin, a TLR5 agonist; imiquimod, a TLR7 agonist; resiquimod, a TLR7/8 agonist; ioxoribine, a TLR7/8 agonist; and unmethylated CpG dinucleotide (CpG-ODN), a TLR9 agonist.

[0377] Additional TLR agonists known in the art and that have utility in the present invention further include, but are not limited to, aminoalkyl glucosaminide phosphates (AGPs) that bind to the TLR4 receptor are known to be useful as adjuvants of vaccine and immunostimulatory agents to stimulate the production of cytokines, activating macrophages, promoting the innate immune response and increasing the production of antibodies in immunized animals. An example of a naturally occurring TLR4 agonist is bacterial LPS. An example of a semisynthetic TLR4 agonist is monophosphoryl lipid A (MPL). AGPs and their immunomodulatory effects through TLR4 are disclosed in Patent Publications such as WO 2006/016997, WO 2001/090129, and/or US Patent No. 6,113,918 and have been reported in the literature. Additional AGP derivatives are disclosed in US Patent No. 7,129,219, US Patent No. 6,525,028 and US Patent No. 6,911,434. Some AGPs act as TLR4 agonists, while others are recognized as TLR4 antagonists.

[0378] In addition to the immunostimulating agents described above, the compositions of the present invention may also comprise one or more additional substances that, due to their adjuvant nature, can stimulate the immune system to respond to cancer antigens present in inactivated tumor cells. Such adjuvants include, but are not limited to, lipids, liposomes, inactivated bacteria that induce innate immunity (e.g., inactivated or attenuated Listeria monocytogenes), compositions that mediate innate immune activation through NOD-like receptors (NLRs), retinoic acid-inducible gene-like receptors I (RIG-I)(RLRs) and/or C-type lectin receptors (CLRs). Examples of PAMPs include lipoproteins, lipopolypeptides, peptidoglycans, zymosan, lipopolysaccharides, Neisseria porins, flagellin, profilin, galactoceramid, muramyl dipeptide. Peptidoglycans, lipoproteins and lipoteichoic acid are cell wall components of gram-positive bacteria. Lipopolysaccharides are expressed by most bacteria, MPL being an example. Flagellin refers to the structural component of bacterial flagella that is secreted by pathogenic and commensal bacteria. rt-galactosylceramide (rt-GalCer) is an activator of natural killer T (NKT) cells. Muramyl dipeptide is a bioactive peptidoglycan motif common to all bacteria.

[0379] Due to their adjuvant qualities, TLR agonists are preferably used in combination with other vaccines, adjuvants and/or immunomodulators, and can be combined in various combinations. Thus, in certain embodiments, compounds of Formula (I) described herein that bind to STING and induce STING-dependent TBKI activation and an inactivated tumor cell that expresses and secretes one or more cytokines that stimulate the induction, recruitment and/or maturation of DCs, as described herein, can be administered in conjunction with one or more TLR agonists for therapeutic purposes.

[0380] Additional examples of another active ingredient or ingredients (antineoplastic agent) for use in combination or co-administered with the compound of Formula (I), invented and described here, are antibodies against ICOS.

[0381] The CDRs for murine antibodies against human ICOS with agonist activity are shown in document PCT/EP2012/055735 (WO 2012/131004). Antibodies to ICOS are also disclosed in WO 2008/137915, WO 2010/056804, EP 1374902, EP 1374901 and EP 1125585.

[0382] Indoleamine 2,3-dioxygenase 1 (IDO1) is a key immunosuppressive enzyme that modulates the antitumor immune response by promoting the generation of regulatory T cells and blocking the activation of effector T cells, thereby facilitating growth tumor by allowing cancer cells to escape the control of the immune system. (Lemos H, et al., Cancer Res. 2016 Apr 15;76(8):2076-81), (Munn DH, et at., Trends Immunol. 2016 Mar;37(3):193-207). Other active ingredients (antineoplastic agents) for use in combination or co-administered with the compounds of Formula (I), invented and described herein, are IDO inhibitors. Epacadostat, ((Z)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-[2-(sulfamoylamino)ethylamino]-1,2,5-oxadiazol-3-carboxamidine), is a Selective and highly potent oral inhibitor of the IDO1 enzyme that reverses tumor-associated immune suppression and restores effective antitumor immune response. Epacadostat is disclosed in US Patent No. 8,034,953.

[0383] Additional examples of another active ingredient or ingredients (antineoplastic agent) for use in combination or co-administered with the compound of Formula (I), invented and described here, are CD73 inhibitors and adenosine A2a and A2b antagonists.

[0384] In one aspect, the disease to be treated is an infectious disease caused, for example, by bacteria or viruses.

[0385] In another aspect of the invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of infectious diseases.

[0386] In another aspect, there is provided a method of treating infectious diseases which comprises administering a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human in need thereof.

[0387] In another aspect, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of infectious diseases.

[0388] In one embodiment, the compound of the invention can be used with other therapeutic methods of treating infectious diseases. In particular, antiviral and antibacterial agents are anticipated.

[0389] The compounds of Formula (I) and their pharmaceutically acceptable salts thereof can be used in combination with one or more agents useful in the prevention or treatment of bacterial and viral infections. Examples of such agents include, without limitation; polymerase inhibitors, such as those described in WO 2004/037818-A1, as well as those described in WO 2004/037818 and WO 2006/045613; JTK-003, JTK-019, NM-283, HCV-796, R-803, R1728, R1626, as well as those described in documents WO 2006/018725, WO 2004/074270, WO 2003/095441, US 2005/0176701, WO 2006/020082, WO 2005/080388, WO 2004/064925, WO 2004/065367, WO 2003/007945, WO 02/04425, WO 2005/014543, WO 2003/000254, EP 106 5213, WO 01/47883, WO 2002 /057287, WO 2002/057245 and similar agents; replication inhibitors such as acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine and similar agents; protease inhibitors, such as HIV protease inhibitors, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir, brecanavir, atazanavir, tipranavir, palinavir, lasinavir and HCV protease inhibitors, BILN2061, VX-950, SCH503034; and similar agents; nucleoside and nucleotide reverse transcriptase inhibitors such as zidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavidine, adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine, alovudine, amdoxovir, elvucitabine and similar agents; non-nucleoside reverse transcriptase inhibitors (including an agent with antioxidant activity such as immunocal, oltipraz etc.) such as nevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz, capravirine, TMC-278, TMC-125, etravirine and other agents similar; entry inhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140, TNX-355, BMS-806, 5-Helix and similar agents; integrase inhibitors, such as L-870,180 and similar agents; budding inhibitors, such as PA-344 and PA-457, and similar agents; chemokine receptor inhibitors such as vicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427857), TAK449, as well as those described in WO 02/74769, WO 2004/054974, WO 2004/ 055012, WO 2004/055010, WO 2004/055016, WO 2004/055011 and WO 2004/054581, and similar agents; neuraminidase inhibitors such as CS-8958, zanamivir, oseltamivir, peramivir and similar agents; ion channel blockers such as amantadine or rimantadine and similar agents; and RNA interference and antisense oligonucleotides, such as ISIS-14803 and similar agents; antiviral agents with an undetermined mechanism of action, for example those described in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin and similar agents. The compounds of Formula (I) and the pharmaceutically acceptable salts thereof may also be used in combination with one or more other agents which may be useful in the prevention or treatment of viral infections, for example, in immunological therapies (e.g. interferon or other cytokines/chemokines, cytokine/chemokine receptor modulators, cytokine agonists or antagonists and similar agents); and therapeutic vaccines, antifibrotic agents, anti-inflammatory agents such as corticosteroids or NSAIDs (non-steroidal anti-inflammatory agents) and similar agents.

[0390] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of infectious diseases.

[0391] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of infectious diseases for use in therapy.

[0392] In another aspect, there is provided a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in the treatment of infectious diseases, for use in the treatment of infectious diseases.

[0393] In another aspect, there is provided the use of a combination comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one additional therapeutic agent useful in treating infectious diseases in the manufacture of a medicament for the treatment of infectious diseases.

[0394] In another aspect, there is provided a method of treating infectious disease comprising administering a therapeutically effective amount of a combination containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent. useful in the treatment of infectious diseases, to a human who needs it.

[0395] In another aspect, there is provided a pharmaceutical composition comprising a combination containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent useful in treating infectious diseases, and one or more pharmaceutically acceptable excipients.

[0396] In another aspect, there is provided a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and one or more immunostimulating agents.

[0397] Furthermore, an immunogenic composition or vaccine adjuvant comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is provided.

[0398] Furthermore, an immunogenic composition is provided comprising an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0399] Furthermore, there is provided a vaccine composition comprising an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0400] Furthermore, there is provided a method of treating or preventing disease which comprises administering an immunogenic composition containing an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human subject who suffers from or is susceptible to the disease.

[0401] Furthermore, there is provided a method of treating or preventing disease which comprises administering a vaccine composition containing an antigen or antigenic composition and a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a human subject who suffers from or is susceptible to the disease.

[0402] Furthermore, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of an immunogenic composition containing an antigen or antigenic composition, for the treatment or prevention of diseases.

[0403] Furthermore, there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of a vaccine composition containing an antigen or antigenic composition, for the treatment or prevention of diseases.

[0404] It is considered that the compounds represented in the application can be drawn using different conventions. For example, the following two compounds are considered equivalent in chemical and stereochemical structure. Preparation and examples of compounds

[0405] Compounds of Formula (I), in which Y1, Y2, X1, X2, R1, R2, R3, R4, R5, R6, R8 and R9 are as defined above, can be prepared by methods known in the art of organic synthesis, as established in the schemes and examples below. In all methods, it is well understood that protecting groups for sensitive or reactive groups can be employed when necessary in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (PGM Wuts and TW Green (2007) Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons). These groups are removed in a suitable synthesis phase of the compound using methods that are readily apparent to those skilled in the art. The selection of processes, as well as the reaction conditions and the order of their execution, must be consistent with the preparation of the compounds of Formula (I).

[0406] The compounds of Formula (I) and their salts can be prepared by the methodology described below, constituting other aspects of this invention.

[0407] Therefore, there is provided a process for preparing a compound of Formula (I), wherein R5 is OC(O)R7 and R6 is F, and both Y1 and Y2 are O, both X1 and X2 are S-, as illustrated in formula (IIa), wherein R1, R2, R3 and R4 are as defined above for a compound of Formula (I). The process comprises the acylation of a compound of Formula (IIIa): wherein R1, R2, R3, R4 and R7 are as defined above for a compound of formula (IIa) and thereafter, if necessary, preparing a salt of the compound so formed.

[0408] Example 1: A compound of formula (IIIa) and any myristic anhydride in a suitable solvent, for example, dimethylformamide (DMF), in the presence of a base such as pyridine, is stirred at room temperature or heated to a temperature suitable, for example at 60°C, for a suitable period of time, for example 2 to 48 hours. The product, of formula (IIa), is isolated by removing volatile compounds and purifying, if necessary.

[0409] Furthermore, there is provided a process for preparing a compound of Formula (I), wherein R5 is OH and R6 is F, and both Y1 and Y2 are O, both X1 and X2 are S, and both R8 and R9 are CH2OC(O)tBu, illustrated as formula (IVa), wherein R1, R2, R3 and R4 are as defined above for a compound of Formula (I). The process comprises adding a carbonyloxymethyl group to a compound of formula (IIIa):

[0410] Example 2: A compound of formula (IIIa) and chloromethyl pivalate (POM-Cl) in a suitable solvent, for example, dimethylformamide (DMF), in the presence of a base, such as Et3N, is stirred at room temperature for a suitable period of time, for example 48 hours. The product, formula (IVa), is isolated by removal of volatiles and purification, if necessary.

[0411] A compound of formula (IIIa) can be prepared by deprotecting a compound of formula (Va): wherein, R1, R2, R3 and R4 are as defined above for a compound of formula (IIIa) and P1 is a suitable protecting group such as tert-butyldimethylsilyloxy (TBDMS) and then, if necessary, by preparing a salt of the compound then formed.

[0412] Example 3: A compound of formula (Va), in a suitable solvent, for example, pyridine, is heated to a suitable temperature, for example, 50 ° C, then treated with a mixture of triethylamine trihydrofluoride and triethylamine for a suitable period of time, for example 2 to 3 hours. The product, of formula (IIIa), is isolated by precipitation by addition of a solvent, for example, acetone, or by removal of volatiles and purification, if necessary.

[0413] A compound of formula (Va) can be prepared by deprotecting a compound of formula (VIa): wherein P1 is a protecting group as defined for the compound of formula (Va) and R12, R13, R14 and R15 are defined as R12 is OH and R13 is NHCOiPr, or R12 is NHBz and R13 is H; R14 is OH and R15 is NHCOiPr or R14 is NHBz and R15 is H;

[0414] Example 4: A compound of formula (VIa) is dissolved in a suitable mixture, for example, methylamine in methanol or aqueous ammonia in methanol, and heated to a suitable temperature, for example, 50 to 55 ° C, for a suitable period of time, for example, 2 to 72 hours. The product, of formula (Va), is isolated by removal of the solvent and purification if necessary.

[0415] A compound of formula (VIa) can be prepared by reacting a compound of formula (VIa): wherein P1, R12, R13, R14 and R15 are as defined above for a compound of formula (VIa).

[0416] Example 5: A compound of formula (VIIa) is dissolved in a suitable solvent, for example, pyridine, treated with a suitable coupling reagent, for example, 2-chloro-5,5-dimethyl-1,3, 2-dioxaphosphorinane 2-oxide and stirred at a suitable temperature, for example 20°C, for a suitable period of time, for example 0.5 to 2 hours. The reaction was inhibited by adding a suitable solvent, e.g. water, then a sulfating agent, e.g. 3H-benzo[c][1,2]dithiol-3-one, was added and stirred at a suitable temperature. , for example at 20 °C, for a suitable period of time, for example 5 to 10 minutes. The reaction was inhibited by adding a suitable solvent, for example, aqueous NaHCO3 solution. The product, of formula (VIa), was extracted with a suitable organic solvent, such as EtOAc. The product, of formula (VIa), is isolated by removal of the solvent and purification if necessary.

[0417] A compound of formula (VIIa) can be prepared by reacting a compound of formula (VIIIa) with a compound of formula (IXa): wherein, P1, R12, R13, R14 and R15 are as defined above for a compound of formula (VIIa) and DMTr is a 4,4-dimethoxytrityl protecting group.

[0418] Example 6: A compound of formula (IXa), in a suitable solvent, e.g. acetonitrile in the presence of molecular sieves, is treated with a solution of a compound of formula (VIIIa) dissolved in a suitable solvent, e.g. , acetonitrile, and stirred at a suitable temperature, for example 20°C, for a suitable period of time, for example 0.5 to 2 hours. A solution of a suitable sulfating agent, for example N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (DDTT), is added and the mixture is stirred. at a suitable temperature, for example 20°C, for a suitable period of time, for example 0.5 to 1 hour. After evaporation of the solvent, the residue is dissolved in a suitable solvent, for example a mixture of dichloromethane and water, and treated with a suitable reagent, for example dichloroacetic acid, and stirred at a suitable temperature, for example at 20°C. °C for a suitable period of time, for example 15 minutes. A solution containing the product of formula (VIIa) is obtained by adding a suitable solvent, for example, pyridine, and concentrating by evaporation.

[0419] A compound of formula (VIIIa) can be prepared by reacting a compound of formula (Xa). wherein, R12 and R13 are as defined above for a compound of formula (VIIIa) and DMTr is a 4,4-dimethoxytryl protecting group.

[0420] Example 7: A compound of formula (Xa) is dissolved in a suitable mixture, for example acetonitrile containing water, treated with pyridinium trifluoroacetate and stirred at a suitable temperature, for example at 20 ° C, for a appropriate period of time, for example 1 to 5 minutes. Then, tert-butylamine is added and the mixture is stirred at a suitable temperature, for example 20°C, for a suitable period of time, for example 10 minutes. The product is isolated by evaporation of the solvent and then dissolved in a suitable solvent, e.g. dichloromethane containing water, and treated with dichloroacetic acid and stirred at a suitable temperature, e.g. 20°C, for a suitable period of time. for example, 15 minutes. A concentrated solution of the product, of formula (VIIIa), in acetonitrile is obtained by adding pyridine followed by azeotropic breakdown of the mixture with anhydrous acetonitrile.

[0421] Phosphoramidites of formula (IXa) and (Xa) are known in the literature or are commercially available from suppliers, such as Sigma, Chemgenes and CarboSynth or can be prepared by known methods.

[0422] Example 8 - Compound 1b (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18 -hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0423] Intermediate 1: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl) methoxy) methyl hydrogen phosphonate )-4-fluorotetrahydrofuran-3-yl

[0424] To a solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) -4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2190 mg, 2.5 mmol) in acetonitrile (15 mL) and water (0.090 mL, 5.00 mmol) at room temperature was added 2.2 Pyridine ,2-trifluoroacetate (579 mg, 3.00 mmol). The mixture was stirred for 1 minute, after which LCMS indicated complete conversion to the first intermediate, m/z (M+H) = 793.3. Then, 2-methylpropan-2-amine (13.14 mL, 125 mmol) was added and the mixture was stirred for 10 minutes, after which LCMS indicated consumption of the first intermediate formed.

[0425] The mixture was concentrated in vacuum to generate a white foam. The foam was then dissolved in acetonitrile (20 mL) and concentrated. This process was repeated once more. The crude material was dissolved in dichloromethane (10 mL) and purified by two-batch chromatography (silica gel, gradient elution of 0-30% methanol in dichloromethane). The desired fractions were combined and evaporated to provide two separate white solids which were then dissolved in dichloromethane, combined and evaporated to obtain the title compound (780 mg, 1.055 mmol, 42.2% yield) as a white solid. LCMS m/z 740.4 (M+H).

[0426] Intermediate 2: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl) tetrahydrofuran-3-yl hydrogen phosphonate

[0427] Intermediate 2 was produced, in general, according to the procedure below. Minor modifications may be used, such as, for example, those described for Intermediate 2 in other Examples.

[0428] A hydrogen phosphonate solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-fluorotetrahydrofuran-3-yl (775 mg, 1.048 mmol) in dichloromethane (20 mL) and water (0.188 mL, 10.48 mmol) at room temperature was added 2,2-dichloroacetic acid (0.655 mL, 8.38 mmol). The mixture was stirred at room temperature for 30 minutes.

[0429] The reaction was then inhibited by the addition of pyridine (1.356 mL, 16.76 mmol) and concentrated in vacuum to obtain a colorless oil. The material was stored under nitrogen at 4 °C. After storage at 4°C, the material solidified to give the impure title compound as a waxy white solid, which was used without further purification. LCMS m/z 438.3 (M+H).

[0430] Intermediate 3: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2R, 3R, 4R, 5R)-2 -(6-benzamido-9H-purin-9-yl)-4- ((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl) -4-fluorotetrahydrofuran-3-yl

[0431] Intermediate 3 was produced, in general, according to the procedure below. Minor modifications may be used, such as, for example, those described for Intermediate 3 in other Examples.

[0432] (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (458 mg, 1.047 mmol) solid and impure obtained above was subjected to azeotropic breakdown with anhydrous acetonitrile (3 x 20 mL). After the last concentration, 10 mL of acetonitrile was kept in the flask. At room temperature, (2R, 3R, 4R, 5R)-2-(6-benzamido-9H-purin-9-yl)- 5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) was added -4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (1345 mg, 1.361 mmol) was subjected to azeotropic drying with anhydrous acetonitrile (3 x 20 mL). After the last concentration, 5 mL of acetonitrile was kept in the flask and 3Â molecular sieves (~40 spheres) were added. The solution was left standing on the molecular sieves at room temperature for 1 hour.

[0433] To the dry suspension of hydrogen phosphonate of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl ( 458 mg, 1.047 mmol) in acetonitrile (10 mL) at room temperature under a nitrogen atmosphere, pre-dried solution of (2R, 3R, 4R, 5R)-2-(6-benzamido-9H-purin) was added. -9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (1345 mg, 1.361 mmol) in acetonitrile (5 mL) per syringe. The color of the solution changed from orange to light yellow. The mixture was stirred under a nitrogen atmosphere at room temperature for 1 hour, after which LCMS indicated minimal starting material remaining. Then, (E)-N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (237 mg, 1.152 mmol) was added to the reaction mixture and the mixture was stirred at room temperature for 30 minutes. The solvent was evaporated in vacuo and the residue was taken up in dichloromethane (40 mL) and water (0.188 mL, 10.47 mmol), followed by the addition of 2,2-dichloroacetic acid (1.037 mL, 12.57 mmol). The mixture was stirred at room temperature for 10 minutes, after which LCMS indicated the formation of the desired product. The reaction was inhibited with pyridine (10 mL, 124 mmol), then the mixture was concentrated in vacuo to give the crude title compound as an orange oil. LCMS: m/z 1054 (M+H). The product was stored under nitrogen at 4 °C and used in the next step without further purification.

[0434] Intermediate 4: N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H-purin-9-yl)-18-[( tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanilidene-2,4,7,11,13,16-hexaoxa-3X5,12À5-diphosphatricycle [13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide

[0435] A hydrogen phosphonate solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2R, 3R, 4R, 5R)-2 -(6-benzamido-9H-purin-9-yl)-4- ((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl) Crude -4-fluorotetrahydrofuran-3-yl (1104 mg, 1.047 mmol) in pyridine (~20 mL) was evaporated and then redissolved in pyridine (20 mL) and concentrated to approximately 10 mL. To this solution under nitrogen, 2-chloro-5,5-dimethyl-1,3,2-dioxaphophinan 2-oxide (677 mg, 3.67 mmol) was added in one portion. The reaction mixture was stirred at room temperature under nitrogen for 30 minutes, after which LCMS indicated consumption of the starting material, then the reaction was inhibited by the addition of water (0.660 mL, 36.7 mmol). 3H-Benzo[c][1,2]dithiol-3-one (264 mg, 1.571 mmol) was then added and the mixture was stirred at room temperature for 5 minutes before pouring into a water solution. (160 mL) containing sodium bicarbonate (4400 mg, 52.4 mmol). This mixture was stirred for 5 minutes, then EtOAc (150 mL) was added and the mixture was stirred for a further 10 minutes. The solution was transferred to a separatory funnel, and the aqueous layer was separated from the organic layer. The aqueous layer was further extracted with EtOAc (150 mL), then the combined organic layers were dried (Na2SO4), filtered and evaporated in vacuo until an orange oil was obtained. The material was stored under nitrogen at 4 °C overnight.

[0436] After warming to room temperature, the crude material was diluted with toluene (20 mL) and evaporated to remove excess pyridine. The crude product was purified by chromatography (silica gel, ISCO Teledyne Gold, 80 g). Gradient elution was performed from 0-10% methanol in dichloromethane over 15 minutes, followed by a 5 minute isocratic hold at 10% methanol in dichloromethane. Then the gradient increased from 10-20% methanol in dichloromethane over 15 minutes followed by a 10 minute isocratic hold at 20% methanol in dichloromethane. The desired fractions were combined and evaporated in vacuo to obtain the title compound (570 mg, 0.342 mmol, 32.6% yield) as a yellow solid.

[0437] Four isomers were observed by LCMS [m/z 1068.5 (M+H)] in an approximate ratio of 8:4:2:1 with retention times of 1.13, 1.23, 1.18 and 1.08 minutes, respectively. The product was stored under nitrogen at 4 °C and used in the next step without further purification.

[0438] Intermediate 5: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl )oxy]-9-fluoro-3,12-disulfanyl-2.4.7.11.13.16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0439] A solution of N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H-purin-9-yl)-18-[( tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanilidene-2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide obtained above (570 mg, 0.534 mmol) in methylamine (33% by weight in ethanol)(25 mL, 201 mmol) he was stirred at room temperature for 50 minutes, after which the LCMS indicated consumption of the starting materials. The reaction mixture was concentrated in vacuo to obtain an orange residue. The material was stored under nitrogen at 4 °C overnight.

[0440] The material was left to warm to room temperature and dissolved in methanol/DMSO (4 mL in total). A portion was purified using reverse phase HPLC (10-90% acetonitrile:water (with 0.1% NH4OH modifier), Gemini C18 50x30 mm column, 47 mL/min, 8 minute gradient, UV collection = 214 nm).

[0441] Another portion was purified using reverse phase HPLC (10-50% acetonitrile: water (with 0.1% NH4OH modifier), Gemini C18 50x30 mm column, 47 mL/min, 8 minute gradient, collection of UV = 214 nm).

[0442] The fractions from the two purifications were combined and concentrated to obtain three isomeric products: • Isomer 1 of the title compound as a bisammonium salt, with the exact stereochemistry at the two phosphorus centers being undetermined (6 mg, purity by LCMS = 70%, 4.99 μmol, 0.936% yield) as an off-white gum, LCMS m/z 807.2 (M+H), tRET = 0.68 min. • Isomer 2 of the title compound as a bisammonium salt, the exact stereochemistry at the two phosphorus centers being undetermined (64 mg, LCMS purity = 22%, 0.017 mmol, 3.14% yield) as a colorless gum, LCMS m/z 807.2 (M+H), tRET = 0.80 min. • Isomer 3 of the title compound as a bisammonium salt, the exact stereochemistry at the two phosphorus centers being undetermined (26 mg, LCMS purity = 50%, 0.015 mmol, 2.90% yield) as a white solid, LCMS m/z 807.2 (M+H), tRET = 0.92 min.

[0443] The last elution isomer, isomer 3 of the title compound, was the main product, as determined by the area under the chromatography peak (UV @ 214 nm) and was used in the next deprotection step.

[0444] Example 8: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy -3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12X5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0445] Isomer 3 of (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert- butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione (25 mg, 0.031 mmol) obtained from the previous step was suspended in pyridine (0.5 mL) and triethylamine (0.5 mL). The mixture was stirred and heated to 50°C, then triethylamine trihydrofluoride (0.5 mL, 3.07 mmol) was added and the mixture was stirred at 50°C for a further 2 hours, at which time LCMS indicated the total consumption of the starting material and conversion to the desired product.

[0446] The mixture was left to cool to room temperature, then acetone (~ 10 mL) was added and the solvent was evaporated and the material was stored under nitrogen at 4 °C overnight.

[0447] The crude residue was purified using reverse phase HPLC (0-20% acetonitrile in water (0.1% NH4OH), Gemini 50x30 mm column, 47 mL/min, 8 minute gradient, UV @ detection 214nm). The desired fractions were combined and evaporated to obtain the title compound as a bisammonium salt (2.3 mg) as a single diastereomer, the exact stereochemistry at the two phosphorus centers being undetermined. The product was a white solid. LCMS m/z 693.1 (M+H). 1H NMR (600 MHz, DMSO-d6 with a drop of D2O): δ ppm 8.69 (s, 1H), 8.33 (s, 1H), 8.14-8.17 (m, 1H), 8 .12 (br s, 1H), 6.24 (br dd, J = 14.5, 3.2 Hz, 1H), 6.08 (br d, J = 8.3 Hz, 1H), 5.71 -5.86 (m, 1H), 5.22 (br t, J = 8.7 Hz, 2H), 4.49-4.54 (m, 1H), 4.32 (br s, 1H), 4.16 (br s, 1H), 4.08-4.15 (m, 2H), 4.03-4.06 (m, 1H), 4.00-4.03 (m, 1H), 3 .73 (br s, 1H).13C NMR (150 MHz, DMSO-d6 with a drop of D2O): δ ppm 156.1, 155.9, 153.2, 152.9, 150.4, 149.1 , 119.1, 118.4, 90.6, 85.3, 83.6, 83.0, 80.6, 77.7, 71.6, 71.1, 67.2, 63.3.31P NMR (162 MHz, DMSO-d6 with a drop of D2O) δ ppm 53.84 and 49.04.

[0448] It is noted that Example 8 produced Compound 1b - Isomer 2. Due to the small scale of the reaction, no Compound 1a - Isomer 1 was isolated. Both Isomer 1 and Isomer 2 of Compound 1 are prepared in Examples 8a and 8b below.

[0449] Examples 8a and 8b - Compounds 1a and 1b (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9 - fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0450] Intermediate 1: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl hydrogen phosphonate )-4- fluorotetrahydrofuran-3-yl

[0451] To a solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) -4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (10 g, 11.42 mmol) in acetonitrile (65 mL) and water (0.411 mL, 22.83 mmol) at room temperature was added 2.2 Pyridine,2-trifluoroacetate (2.65 g, 13.70 mmol). The mixture was stirred for 1 minute, after which LCMS indicated complete conversion to the first intermediate m/z 793.7 (M+H). Then, 2-methylpropan-2-amine (60.0 mL, 571 mmol) was added and the mixture was stirred for 15 minutes, after which LCMS indicated consumption of the first intermediate formed.

[0452] The mixture was concentrated in vacuum to obtain a white foam. The foam was then dissolved in acetonitrile and concentrated (50 mL). This process was repeated once more. The crude material was dissolved in dichloromethane and purified by chromatography (silica gel, ISCO RediSep, 120 g silica) and eluted with a gradient of 0-30% methanol in dichloromethane. The desired fractions were combined and evaporated in vacuo to obtain the title compound (4.8 g, 6.49 mmol, 56.8% yield) as a white solid. LCMS m/z 740.3 (M+H).

[0453] Intermediate 2: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl) tetrahydrofuran-3-yl hydrogen phosphonate

[0454] Intermediate 2 was produced, in general, according to the procedure below. Minor modifications may be used, such as, for example, those described for Intermediate 2 in other Examples.

[0455] A hydrogen phosphonate solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-4-fluorotetrahydrofuran-3-yl (4.8 g, 6.49 mmol) in dichloromethane (100 mL) and water (1.169 mL, 64.9 mmol) at room temperature, added acid 2,2- dichloroacetic acid (4.06 mL, 51.9 mmol). The mixture was stirred at room temperature for 30 minutes. The reaction was then quenched with pyridine (8.40 mL, 104 mmol) and concentrated in vacuo to obtain the crude title compound as a colorless oil. The material was immediately used as is in the next step. A final mass was not determined. LCMS m/z 438.3 (M+H).

[0456] Intermediate 3: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2R, 3R, 4R, 5R)-2 -(6-benzamido-9H-purin-9-yl)-4- ((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl) -4-fluorotetrahydrofuran-3-yl

[0457] Intermediate 3 was produced, in general, according to the procedure below. Minor modifications may be used, such as, for example, those described for Intermediate 2 in other Examples.

[0458] Solid and impure solid (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate ( 2.84 g, 6.49 mmol) obtained above was subjected to azeotropic cracking with anhydrous acetonitrile (3 x 60 mL). After the last concentration, 20 mL of acetonitil was kept in the flask. At room temperature, (2R, 3R, 4R, 5R)-2-(6-benzamido-9H-purin-9-yl)- 5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- was added 4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (8.00 g, 8.10 mmol) was subjected to azeotropic drying with anhydrous acetonitrile (3 x 60 mL). After the last concentration, 30 mL of acetonitrile was kept in the flask and 3Å molecular sieves (~40 spheres) were added. The solution was left standing on the molecular sieves at room temperature for 1 hour.

[0459] To the dry suspension of hydrogen phosphonate of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl ( 2.84 g, 6.49 mmol) in acetonitrile (40 mL) at room temperature under a nitrogen atmosphere, pre-dried solution of (2R, 3R, 4R, 5R)-2-(6-benzamido -9H- purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite ( 8.00 g, 8.10 mmol) in acetonitrile (30 mL) per syringe. The mixture was stirred under a nitrogen atmosphere at room temperature for 1 hour, after which LCMS indicated minimal starting material remaining. Then, (E)-N,N-dimethyl-N'-(3-thio-oxo-3H-1,2,4-dithiazol-5-yl) formimidamide (1.467 g, 7.5 g) was added to the reaction mixture. 14 mmol) and the mixture was stirred at room temperature for 45 minutes. The solvent was evaporated in vacuo and the residue was taken into dichloromethane (100 mL) and water (1.170 mL, 64.9 mmol), followed by the addition of 2,2-dichloroacetic acid (6.43 mL, 78 mmol). The mixture was stirred at room temperature for 10 minutes, after which LCMS indicated the formation of the desired product. The reaction was inhibited with pyridine (12.61 mL, 156 mmol), then the mixture was concentrated in vacuo to obtain the crude title compound as an orange oil. LCMS indicated the formation of two isomers with two overlapping peaks between 0.98-1.04 min. The material was immediately used as is in the next step. A final mass of the sample was not determined. LCMS m/z 1054.6 (M+H).

[0460] Intermediate 4: N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H-purin-9-yl)-18-[( tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanilidene-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricycle [13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide

[0461] A hydrogen phosphonate solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2R, 3R, 4R, 5R)-2 -(6-benzamido-9H-purin-9-yl)-4- ((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl) Crude -4-fluorotetrahydrofuran-3-yl (6.84 g, 6.49 mmol) was subjected to azeotropic breakdown from pyridine (3 x 50 mL), leaving pyridine (40 mL) after the last concentration. To this solution under nitrogen, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinan 2-oxide (4.19 g, 22.71 mmol) was added in one portion. The reaction was stirred at room temperature under nitrogen for 30 minutes, after which LCMS indicated consumption of starting material, then the reaction was inhibited with the addition of water (4.09 mL, 227 mmol). Then 3H-benzo[c][1,2]dithiol-3-one (1.638 g, 9.73 mmol), and the mixture was stirred at room temperature for 5 minutes before being poured into a water solution ( 400 ml) containing sodium bicarbonate (27.3 g, 324 mmol). The mixture was stirred for 10 minutes, then EtOAc (200 mL) was added and the mixture was stirred for a further 10 minutes. The solution was transferred to a separatory funnel, and the aqueous layer was separated from the organic layer. The aqueous layer was further extracted with EtOAc (2 x 200 mL), then the combined organic layers were dried (Na2SO4), filtered and evaporated in vacuo until an orange oil was obtained. The material was stored under nitrogen at 4 °C until use in the next step.

[0462] After warming to room temperature, the crude material was diluted with toluene (50 mL) and evaporated to remove excess pyridine. This process was repeated twice more. The crude product was purified by chromatography (silica gel, ISCO Teledyne Gold, 220 g of silica). Gradient elution was performed from 0-10% methanol in dichloromethane over 15 minutes, followed by a 5 minute isocratic hold at 10% methanol in dichloromethane. Then the gradient increased from 10-20% methanol in dichloromethane over 15 minutes followed by a 5 minute isocratic hold at 20% methanol in dichloromethane. The desired fractions were combined and evaporated under vacuum to obtain an orange oil. The material was stored under nitrogen at 4 °C overnight.

[0463] After warming to room temperature, the material was purified again by chromatography (silica gel, ISCO Teledyne Gold, 120 g of silica). Gradient elution was performed from 0-10% methanol in dichloromethane over 15 minutes, followed by a 5 minute isocratic hold at 10% methanol in dichloromethane. Then the gradient increased from 10-20% methanol in dichloromethane over 10 minutes, followed by a 5 minute isocratic hold at 20% methanol in dichloromethane. The desired fractions were combined and evaporated in vacuo to obtain the crude title compound (2.81 g) as a yellow solid. LCMS indicated the presence of two main diastereomers with retention times of 1.09, 1.18 minutes, respectively. Two minor isomers were also observed. The product was stored under nitrogen at 4 °C until use in the next step without further purification. LCMS m/z 1068 (M+H).

[0464] Intermediate 5: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl )oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, salt bisammonium

[0465] To a solution of N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H-purin-9-yl)-18-[ (tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-oxo-12-sulfanyl-3-sulfanilidene-2,4,7,11,13,16-hexaoxa-3X5,12À5- diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide (1.0 g, 0.936 mmol) in ethanol (5 mL) was added methylamine (33% by weight in ethanol )(20 ml, 161 mmol) at room temperature. The mixture was stirred at room temperature for 30 minutes, after which the LCMS indicated consumption of the starting materials. The reaction mixture was concentrated in vacuo until a dark residue (tan) was obtained. The material was introduced into DMSO and purified using chromatography (reversed phase silica gel, RediSep Gold C18, 30 g). Gradient elution was performed at 100% water (with 0.1% NH4OH modifier) for 4 CV followed by 0-15% acetonitrile in water (with 0.1% NH4OH modifier) for 3 CV followed by 15 -25% acetonitrile in water (with 0.1% NH4OH modifier) to 6 CV followed by 25-90% acetonitrile in water (with 0.1% NH4OH modifier) to 3 CV followed by an isocratic hold at 90 % acetonitrile in water (with 0.1% NH4OH modifier) for 4 CV.

[0466] The purification fractions were combined and two main isomeric products were isolated: • Isomer 2 of the title compound as a bisammonium salt, with the exact stereochemistry at the two phosphorus centers being undetermined (28 mg, LCMS purity = 54% , 0.018 mmol, yield 1.921%) as colorless residue, LCMS m/z 807.1 (M+H), tRET = 0.80 min. • Isomer 3 of the title compound as a bisammonium salt, the exact stereochemistry at the two phosphorus centers being undetermined (46 mg, LCMS purity = 70%, 0.038 mmol, 4.09% yield) as a yellow residue, LCMS m/z 807.2 (M+H), tRET = 0.91 min.

[0467] Examples 8a and 8b: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-18 -hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa- 3À5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0468] Isomer 2 of intermediate 5, (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)- 8,17-bis(6-amino-9H-purin-9-yl)-18-[ (tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12- dione (25 mg, 0.031 mmol), obtained from the previous step, was suspended in pyridine (0.5 mL) and triethylamine (0.5 mL). The mixture was stirred and heated to 50°C, then triethylamine trihydrofluoride (0.5 mL, 3.07 mmol) was added and the mixture was stirred at 50°C for 2 hours, after which the LCMS indicated the total consumption of the starting material and the conversion to the desired product. The reaction mixture was evaporated under vacuum, and the flask was stored under nitrogen at 4 °C overnight.

[0469] After warming to room temperature, the residue was placed in water (~ 5 mL) and a precipitate was formed. The solids were separated from the mixture and the filtrate was adjusted to pH = 10 using ammonium hydroxide. The solution was purified using reverse phase HPLC (0-5% acetonitrile:water (with 0.1% NH4OH modifier), Gemini C18 50 x 30 mm column, 40 mL/min, 7 minute gradient, UV detection = 214 nm). The collected solids were placed in water (~2 mL) and methanol (~0.5 mL) and ammonium hydroxide was added to pH = 10. The solution was purified using reversed-phase HPLC (0-5% acetonitrile: water (with 0.1% NH4OH modifier), Gemini C18 column 50 x 30 mm, 40 mL/min, 7 minute gradient, UV detection = 214 nm).

[0470] The desired fractions from both purifications were combined and removed in vacuum to obtain a colorless residue. The residue was placed in water (2 mL) and lyophilized overnight to obtain the title compound (Example 8a, 7 mg) as a bisammonium salt as a single diastereomer, with the exact stereochemistry at the two undetermined phosphorus centers. The product was a white solid. LCMS m/z 693.3 (M+H). tRET = 0.29 min.1H NMR (400 MHz, DMSO-d6 with a drop of D2O) δ ppm 8.59-8.62 (m, 1H), 8,398.41 (m, 1H), 8.17 ( s, 1H), 8.13 (s, 1H), 6.19-6.27 (m, 1H), 6.05-6.10 (m, 1H), 5.705.87 (m, 1H), 5 .24-5.36 (m, 1H), 5.14-5.23 (m, 1H), 4.31 -4.36 (m, 1H), 4.23-4.29 (m, 1H) , 4.15-4.23 (m, 1H), 4.11-4.15 (m, 1H), 3.98-4.08 (m, 1H), 3.82-3.91 (m, 1H), 3,663.72 (m, 1H). 31P NMR (162 MHz, DMSO-d6 with a drop of D2O) δ ppm 53.66, 55.91. 19F NMR (376 MHz, DMSO-d6 with a drop of D2O) δ ppm -206.48.

[0471] Isomer 3 of intermediate 5, (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)- 8,17-bis(6-amino-9H-purin-9-yl)-18-[ (tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl- 2,4,7,11,13,16-hexaoxa-3À5,12À5-diphosfatricido[13.2.1.06,10]octadecan-3,12- dione (175 mg, 0.217 mmol), obtained from a different batch (LCMS purity = 50%), was suspended in pyridine (1 mL) and triethylamine (1 mL). The mixture was stirred and heated to 50°C, then triethylamine trihydrofluoride (1 mL, 6.14 mmol) was added and the mixture was stirred at 50°C for 3 hours, after which LCMS indicated consumption. complete process of starting material and conversion to the desired product. The mixture was allowed to cool to room temperature, then acetone (~10 mL) was added and a fine precipitate was formed. The precipitate was collected by vacuum filtration to obtain a gray residue, which was discarded. The filtrate was evaporated under vacuum and the flask was stored under nitrogen at 4 °C overnight.

[0472] After warming to room temperature, the residue was placed in methanol (~6 mL) and purified using reverse phase HPLC (0-15% acetonitrile: water (with 0.1% NH4OH modifier), Gemini column C18 50 x 30 mm, 47 mL/min, 8 minute gradient, UV detection = 214 nm). The desired fractions were combined and removed under vacuum to obtain a white solid, still contaminated by some impurities.

[0473] The solid was further purified using a preparative HILIC column (Luna HILIC, 5u 21 x 250 mm, 20 mL/min, UV detection = 254 nm) with an isocratic gradient of 20% aqueous ammonium formate and 80% acetonitrile. The desired fractions were combined and evaporated under 90% vacuum, then placed in water and added with acetonitrile and 5 drops of ammonium hydroxide at pH = 10. The material was frozen and freeze-dried overnight. This process was repeated two more times to obtain the title compound (Example 8b, 12 mg) as a bisammonium salt as a single diastereomer, with the exact stereochemistry at the two undetermined phosphorus centers. The product was a white solid. LCMS m/z 693.3 (M+H), tRET = 0.37 min.1H NMR (600 MHz, DMSO-d6 with a drop of D2O) δ ppm 8.50-9.29 (m, 1H), 8.43 (br s, 1H), 8.16 (s, 1H), 7.73-8.11 (m, 1H), 6.26 (br d, J=14.4 Hz, 1H), 6 .15 (br d, J=7.2 Hz, 1H), 5.72 (s, 1H), 5.29-5.41 (m, 1H), 5.17-5.29 (m, 1H) , 4.27-4.46 (m, 2H), 4.21 (br s, 1H), 4.01-4.15 (m, 1H), 3.86-3.91 (m, 1H), 3.81-3.86 (m, 1H), 3.77 (br d, J=10.6 Hz, 1H), 31P NMR (162 MHz, DMSO-d6 with a drop of D2O) δ ppm 54.27 , 49.69, 19F NMR (376 MHz, DMSO-d6 with a drop of D2O) δ ppm -204.90 (br,)

[0474] Examples 9a and 9b - Compounds 2a and 2b (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9 - fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0475] Intermediate 2: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl) tetrahydrofuran-3-yl hydrogen phosphonate

[0476] Intermediate 2 was produced, in general, according to the procedure described below. Small modifications may be used, such as those described for Intermediate 2 in other examples.

[0477] To a room temperature solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy )methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (4.01 g, 4.6 mmol) in acetonitrile (35 mL) and water (0.165 mL, 9.1 mmol) was added pyridine 2,2,2-(1.06 g, 5.5 mmol). The reaction was stirred for 10 minutes, then pure tert-butylamine (24.2 mL, 228 mmol) was added. The reaction was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The resulting white foam was subjected to azeotropic cracking with acetonitrile (2x), then the dry residue was placed in a mixture of dichloromethane (100 mL) and water (0.82 mL, 45.7 mmol) and treated with acid 2 Pure ,2-dichloroacetic acid (3.01 mL, 36.5 mmol). After 30 minutes, the reaction was quenched with pyridine (5.91 mL, 73.1 mmol), then concentrated under reduced pressure to an oily suspension. The material was subjected to azeotropic breakdown with acetonitrile (3x), then placed in anhydrous acetonitrile (60 mL) and concentrated to a volume of about 20 mL to obtain the impure title compound as a light orange suspension. LCMS m/z 437.9 (M+H). This mixture was used in the next step without further purification.

[0478] Intermediate 3: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2R, 3R, 4R, 5R)-2 -(6-benzamido-9H-purin-9-yl)-4- ((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl) -4-fluorotetrahydrofuran-3-yl

[0479] Intermediate 3 was produced, in general, according to the procedure described below. Small modifications may be used, such as those described for Intermediate 3 in other examples.

[0480]A (2R, 3R, 4R, 5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- ((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (5.9 g, 5.9 mmol) was subjected to azeotropic breakdown with acetonitrile (2x) then placed in 40 mL of acetonitrile anhydrous, approximately half concentrated, then stored under nitrogen on 3Å molecular sieves. After 1 hour, this solution was added to the previously prepared crude mixture (Intermediate 2) of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro- 2-(hydroxymethyl)tetrahydrofuran-3-yl (2.0 g, 4.6 mmol) under nitrogen. The reaction was stirred at room temperature for 1 hour, then treated with 3-(dimethylaminomethylidene)amino)-3H-1,2,4-dithiazol-3-thione (1.03 g, 5.0 mmol), stirred for 30 minutes and concentrated under reduced pressure. The residue was placed in a mixture of dichloromethane (60 mL) and water (0.823 mL, 45.7 mmol) and then treated with 2,2-dichloroacetic acid (4.5 mL, 54.8 mmol). This was stirred at room temperature for 15 minutes before inhibition with pyridine (25 mL, 309 mmol) and concentration under reduced pressure. The oily concentrate was subjected to azeotropic breakdown from pyridine, then placed in anhydrous pyridine (60 ml) and concentrated under reduced pressure to about 20 ml to obtain the impure title compound as a dark orange colored oil. LCMS m/z 1054.2 (M+H). This mixture was used in the next step without further purification.

[0481] Intermediate 6: N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H-purin-9-yl)-18-[( tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-12-oxo-3-sulfanylidene-2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricycle [13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide

[0482] A crude hydrogen phosphonate solution of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2R, 3R, 4R, 5R) -2-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy) methyl)-4-fluorotetrahydrofuran-3-yl (Intermediate 3, 4.8 g, 4.6 mmol) in pyridine (20 mL) under nitrogen was added 2-chloro-5,5-dimethyl-2-oxide. 1,3,2-dioxaphosphinan (3.0 g, 16.0 mmol). The reaction was stirred at room temperature under nitrogen for 30 minutes, then quenched with the addition of water (2.9 mL, 160 mmol) followed by iodine (1.5 g, 5.9 mmol). After 10 minutes, the mixture was poured into a solution of sodium bisulfite (0.95 g, 9.1 mmol) in water (300 ml). After 5 minutes, the reaction mixture was treated portionwise with solid sodium bicarbonate (19.2 g, 229 mmol). The resulting golden brown suspension was extracted with EtOAc (3 x 200 mL), then the extracts were washed with saturated aqueous sodium bicarbonate, dried over Na2SO4 and concentrated to an oil. One of the following two purification methods was then used to purify the product from different batches.

[0483]Method A: The oil was subjected to azeotropic breakdown with toluene to remove excess pyridine, then purified by chromatography on silica (Biotage-100g column) eluting with successive gradients of 0-10% MeOH in DCM (10 min), 10% MeOH in DCM (10 minutes), 10-20% MeOH in DCM (10 min) and finally 20-40% MeOH in DMC (10 min). Fractions of interest identified by LCMS were combined and concentrated to obtain the crude title compound (1.03 g, 0.979 mmol) as a light orange solid. Two isomers were observed by LCMS [m/z 1052.3 (M+H)] in an approximate 1:1 ratio with retention times of 1.00, 1.09 minutes, respectively. The product was stored under a nitrogen atmosphere at 4 °C and used in the next step without further purification.

[0484]Method B: The oil was subjected to azeotropic breakdown with toluene to remove excess pyridine, then purified by chromatography on silica (Teledyne ISCO Gold -120g) eluting with successive gradients of 100% DCM (5 min), 0-10% MeOH in DCM (5 min), 10% MeOH in DCM (10 min), and 10-40% MeOH in DMC (20 min). The fractions of interest were combined and concentrated to obtain an approximately 1:1 mixture of diastereomers as a dark yellow solid. The mixture of diastereomers was separated by reversed-phase HPLC (Gemini C-18 column: 30 x 50 mm; 45-60% CH3CN w/ 0.1% TFA/ water w/ 0.1% TFA), 12 min run, collection at 214 nm. The fractions of interest were combined, treated with saturated aqueous sodium bicarbonate, then concentrated to remove acetonitrile. The aqueous concentrates were then extracted with EtOAc. The extracts were dried (over Na2SO4) and concentrated to obtain the individual diastereomers. • Diastereomer 1 of the title compound (42 mg) as a white solid, the exact stereochemistry at the chiral phosphorus center being undetermined. LCMS m/z 1052.7 (M+H), tRET = 1.00 min. • Diastereomer 2 of the title compound (43 mg) as a white solid, the exact stereochemistry at the chiral phosphorus center being undetermined. LCMS m/z 1052.7 (M+H). tRET = 1.09 min.

[0485] Examples 9a and 9b: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-8,17-bis(6-amino-9H-purin-9-yl)-9-fluoro-12 ,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0486] A solution of intermediate 6 (purified by method A), N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H-purin- 9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-12-oxo-3-sulfanilidene- 2,4,7,11,13, 16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide (1.03 g, 0.979 mmol) in methylamine at 33% by weight in EtOH (40 mL, 321 mmol) was stirred at room temperature for 4 hours after which LCMS indicated complete conversion of the starting material and the presence of the desired O-TBS protected intermediate. Only a slightly broad peak for the intermediate was noted, indicating at least one diastereomer was present. The reaction was concentrated until a dark orange residue was obtained, which was purified by reverse phase HPLC (Gemini C-18 column 30 x 50 mm; 10-60% acetonitrile w/ 0.1% TFA/water p / 0.1% TFA), 12-minute gradient, with detection at 254 nm. The fractions of interest were combined and concentrated under reduced pressure. At this point, LCMS indicated the loss of the silyl protecting group. The aqueous phase was further concentrated to approximately 3 to 5 mL and then methanol was added (25 mL). The resulting suspension was filtered and the solids were washed with MeOH and then diethyl ether, then dried by suction to obtain 75 mg of the desired crude diTFA salt product as a white solid. LCMS m/z 677.2 (M+H).

[0487] The product was further purified by pre-preparative chromatography (Luna HILIC 3u: 4.6 x 150 mm; isocratic elution with 30 mM aqueous HCO2NH4 (20%), CH3CN (80%)). The fractions of interest were combined and concentrated until obtaining a residue that was lyophilized from water (5 mL) and 3 drops of ammonium hydroxide). To remove residual ammonium formate, the freeze-drying process was repeated 4 more times to obtain the title compound (Example 9a, 50 mg) as a bisammonium salt as a single diastereomer, with exact stereochemistry at the chiral phosphorus center. undetermined. The titled product was a white solid. LCMS m / z 677.6 (M+H), tRET = 0.11 min1H NMR(600 MHz, DMSO-d6 with a drop of D2O): δ ppm 8.48 (s, 1H), 8.39 (s , 1H), 8.17 (s, 1H), 8.12 (s, 1H), 6.23 (dd, J =15.1, 3.0 Hz, 1H), 6.09 (d, J = 8.3 Hz, 1H), 5.69 (br dt, J =52.1, 3.4 Hz, 1H), 5.16 (ddd, J =8.1, 6.6, 4.2 Hz, 1H), 5.02-5.10 (m, 1H), 4.35 (d, J=4.2Hz, 1H), 4.24 (br s, 1H), 4.11-4.18 ( m, 1H), 4.09 (br s, 1H), 3.97 (br d, J =10.6 Hz, 1H), 3.89-3.95 (m, 1H), 3.72 (br d, J =12.5 Hz, 1H).13C NMR(150 MHz DMSO-d6, with a drop of D2O): δ ppm 156.0, 155.8, 153.0, 152.8, 150.3, 148.9, 119.1, 118.4, 92.4, 85.4, 84.0, 83.3, 81.0, 77.9, 72.3, 71.4, 65.9, 62, 6.31P NMR (162 MHz, DMSO-d6, with a drop of D2O) δ ppm 55.67 and -2.51.19F NMR (376 MHz, DMSO-d6, with a drop of D2O) δ ppm -205.16 ,

[0488] It is noted that Example 9a can also be prepared using the procedure described below for Example 9b.

[0489] A solution of Diastereomer 2 of Intermediate 6 (purified by method B), N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(6-benzamido-9H -purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-12-oxo-3-sulfanilidene-2,4,7,11 ,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide (43 mg, 0.041 mmol), in 33% methylamine in EtOH (8.0 mL, 64.3 mmol) was stirred at room temperature for 30 minutes and then concentrated. The resulting dark orange residue was placed in anhydrous pyridine (0.50 mL) and triethylamine (0.50 mL), heated to 50 °C and then treated with triethylamine trihydrofluoride (0.50 mL, 3.07 mmol). . After 1 hour, the reaction was complete. The mixture was cooled and concentrated to a dark oil which was placed in water (7.5 mL) and ammonium hydroxide (10 drops). The resulting suspension (pH ~ 3) was filtered and the solids were dissolved in a mixture of water (2 mL) and ammonium hydroxide (1 mL) and then purified by reversed-phase HPLC (Gemini C-18 column: 30 x 50 mm; 0-10% acetonitrile / water with 0.1% NH4OH; 214 nm). The fractions of interest were combined and concentrated under reduced pressure until a wet residue was obtained, which was placed in water (5 ml) and 5 drops of ammonium hydroxide. then lyophilized to obtain the title compound (Example 9b, 7.0 mg) as a bisammonium salt as a single diastereomer, the exact stereochemistry at the chiral phosphorus center being undetermined. The product was a white solid. LCMS m/z 677.2 (M+H), tRET = 0.32 min.1H NMR (600MHz, DMSO-d6 with a drop of D2O): δ ppm 8.65-9.35 (m, 1H), 8.44 (br s, 1H), 7.77-8.29 (m, 2H), 6.25 (br d, J =14.4 Hz, 1H), 6.11-6.19 (m, 1H), 5.53-5.73 (m, 1H), 5.18-5.44 (m, 1H), 4.96-5.08 (m, 1H), 4.40-4.54 ( m, 1H), 4.33 (br s, 2H), 4.23 4.30 (m, 1H), 4.16 (br s, 1H), 3.98-4.06 (m, 1H), 3.81 (br s, 1H).31P NMR (162 MHz, DMSO-d6 with a drop of D2O) δ ppm 49.48 and -2.94. 19F NMR (376 MHz, DMSO-d6 with a drop of D2O) δ ppm -206.44 (br).

[0490] Examples 10a and 10b - Compounds 28a and 28b (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin -9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa- 3À5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione

[0491] Intermediate 7: (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-(((((2R, 3R, 4R, 5R)- 4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)oxy)(2- cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl

[0492](2R, 3R.4R, 5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6 -oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite (4.0 g, 4.12 mmol) was subjected to azeotropic breakdown three times with acetonitrile (20 mL). After the last concentration, 15 mL of acetonitrile was kept in the reaction flask and 3Å molecular sieves (~ 20 spheres) were added to the clean solution. The solution was left standing on the molecular sieves under nitrogen for ~1 hour.

[0493] In a round-bottom flask separated from the previously prepared crude mixture (Intermediate 2) of (2R, 5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2- hydrogen phosphonate (hydroxymethyl)tetrahydrofuran-3-yl in acetonitrile (10 mL) was added, by syringe, to the above pre-dried solution of (2R, 3R.4R, 5R)-5-((bis(4-methoxyphenyl)(phenyl) methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite in acetonitrile (~15 mL). After 30 minutes of stirring at room temperature, (E)-N,N-dimethyl-N'-(3-oxo-3H-1,2,4-dithiazol-5-yl) formimidamide (DDTT) was added (780 mg, 3.80 mmol). The mixture was stirred at room temperature for 30 minutes, followed by vacuum evaporation of the acetonitrile. Diplomethane (DCM)(50 mL) and Water (650 μL) were then added to the residue, followed by the addition of 2,2-dichloroacetic acid (3.5 mL, 42.4 mmol). This was stirred at room temperature for 30 minutes before being quenched with pyridine (20 ml). The mixture was concentrated in vacuo to obtain the crude title compound as an orange oil. LCMS m/z 1036.2 (M+H). The crude product was used in the next step without further purification.

[0494] Intermediate 8: N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9 -fluoro-12-hydroxy-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-3-sulfanilidene-2,4, 7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide

[0495] A crude solution of hydrogen phosphonate of (2R, 3R, 4R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-(((((2R, 3R, 4R, 5R )-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)oxy)( 2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl (Intermediate 7, 3.55 g, 3.43 mmol) in pyridine (60 mL) was added 2-chloro-5-2-oxide ,5-dimethyl-1,3,2-dioxaphosphinan (DMOCP)(2.2 g, 11.92 mmol), and the mixture was stirred under nitrogen at room temperature for 30 minutes. The reaction was inhibited with water (2.2 mL, 10 equiv. of DMOCP), followed by addition of iodine (1.2 g, 4.73 mmol). The mixture was stirred for 10 minutes, then poured into a solution of water (400 mL) and sodium bisulfite (NaHSO3) (1.0 g, 9.61 mmol). After 5 minutes of stirring, sodium bicarbonate (NaHCO3)(14.4 g, 171 mmol) was added slowly in portions as a solid (caution: gas evolution). The product was extracted with 1:1 diethyl ether:EtOAc (300 mL x 2) and the combined extracts were dried over sodium sulfate, filtered and concentrated in vacuo. Excess pyridine was removed by concentration with toluene (2 x 100 mL). The crude material was purified by chromatography on silica (100 gram column) using a gradient of 0-20% MeOH/DCM, then remaining at 20% MeOH/DCM until all of the desired product had been eluted. of the column. The desired fractions were combined and concentrated to obtain two isomeric products: • Isomer 1 of the more polar title compound as an impure mixture (1.39 g, LCMS purity ~ 33% together with ~ 28% of Isomer 2) . LCMS m/z 1034.1 (M+H), tRET = 0.98 min. • Isomer 2 of the title compound, less polar, as an impure mixture (230 mg, LCMS purity ~ 33%). LCMS m/z 1034.2 (M+H), tRET = 1.09 min.

[0496] Intermediates 9a and 9b: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl )-8-(6-amino-9H-purin-9-yl)-9-fluoro-12-hydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[ 13.2.1.06,10]octadecan-3,12-dione

[0497] Isomer 1 of Intermediate 8, N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2- cyanoethoxy)-9-fluoro-12-hydroxy-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-3-sulfanilidene-2 ,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide (1.39 g, 1.34 mmol), was stirred in methanamine (33% by weight in EtOH)(10.0 mL, 80 mmol) under nitrogen at room temperature for 3 hours. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC, using a gradient of 10-50% ACN/H2O (0.1% TFA)), to obtain Isomer 1 of the compound of title (Intermediate 9a, impure, 280 mg) as a dark solid. LCMS m/z 807.1 (M+H), tRET = 0.80 min.

[0498] Following the same procedure for the preparation of Intermediate 9a, Isomer 2 of Intermediate 8, N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-18- [(terc -butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro-12-hydroxy-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl ]-12-oxo-3-sulfanilidene- 2,4,7,11,13,16-hexaoxa-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin- 6-yl }benzamide (230 mg, 0.22 mmol), was stirred in methanamine (33% by weight in EtOH)(2.0 mL, 16 mmol) under nitrogen at room temperature for 3 hours. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC, using a gradient of 10-50% ACN/H2O (0.1% TFA)), to obtain Isomer 2 of the compound title (Intermediate 9b, impure, 60 mg) as a dark solid. LCMS m/z 807.1 (M+H), tRET = 0.85 min.

[0499] Examples 10a and 10b: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl )-8-(6-amino-9H-purin-9-yl)-9-fluoro-12,18-dihydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione

[0500] A suspension of Isomer 1 of (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9 -yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12-hydroxy-3-sulfanyl-2,4,7,11,13,16-hexaoxa-3À5,12À5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione (Intermediate 9a, 280 mg, 0.35 mmol) in pyridine (2 mL) and triethylamine (2 mL) at 50 °C was added triethylamine trihydrofluoride ( 1.5 mL, 9.21 mmol). The mixture was stirred at 50°C for 4 hours. LCMS indicated that some starting material had not yet been consumed, and the reaction was allowed to stir at room temperature for 16 hours. Then, the reaction mixture was diluted with acetone (~25 mL) and precipitation was formed. After 15 minutes of stirring, the reaction was filtered. The solid was rinsed with acetone (~10 mL) and dried. The filtrate was concentrated in vacuo and then toluene was added to remove any remaining pyridine. The crude filtrate and solid filtrate were individually purified by reverse phase HPLC using a 0 to 20% ACN:H2O (0.1% NH4OH) gradient and combined. The 19F NMR spectra showed residual TFA present in the samples. A second purification by reversed-phase HPLC, using a gradient of 0-10% ACN/H2O (0.1% NH4OH), gave the title compound (Example 10a, 4 mg) as a bisammonium salt as a diastereomer. unique, with the exact stereochemistry at the phosphorus center being undetermined. The product was a white solid. LCMS m/z 693.0 (M+H), tRET = 0.11 min.1H NMR (600 MHz, DMSO-d6 with a drop of D2O): δ ppm 8.30 (s, 1H), 8.19 (s, 1H), 7.97 (s, 1H), 6.26 (dd, J = 15.9, 2.3 Hz, 1H), 5.86 (d, J = 8.3 Hz, 1H) , 5.59-5.76 (m, 1H), 5.30 (br s, 1H), 5.06 (br d, J = 15.5 Hz, 1H), 4.35 (d, J = 3 .8Hz, 1H) 4.25 (br s, 1H), 4.02-4.07 (m, 1H), 4.01-4.13 (m, 2H), 3.88-3.99 ( m, 2H).19F NMR (376 MHz, DMSO-d6 with a drop of D2O): δ ppm -203.83. 31P NMR (162 MHz, DMSO-d6 with a drop of D2O): δ ppm 55.73, -2.66.

[0501] Following the same procedure for the preparation of Example 10a, the reaction of Isomer 2 of (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6 ,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-12-hydroxy-3-sulfanyl- 2,4,7,11, 13,16-hexaoxa-3À5,12À5-diphosfatricido[13.2.1.06,10]octadecan-3,12-dione (Intermediate 9b, 60 mg, 0.35 mmol) gave the title compound (Example 10b, 7 mg) as a bisammonium salt as a single diastereomer, the exact stereochemistry at the phosphorus center being undetermined. The product was a white solid. LCMS m/z 693.0 (M+H), tRET = 0.37 min.1H NMR (400 MHz, DMSO-d6 with a drop of D2O): δ ppm 8.50 (br s, 1 H), H ) 8.22 (br s, 1H), 7.94 (br s, 1H), 6.35 (d, J = 14 Hz, 1H), 5.87 (d, J = 7.86 Hz, 1H ), 5.54-5.67 (m, 1H), 4.98 (br, J = 15.5 Hz, 1H), 4.39 (br s, 1H), 4.33 (d, J = 6 .84 Hz, 1 H), 4.24 (br s, 1H), 4.13 (br s, 1H), 4.00-4.10 (m, 2H), 3.89-3.98 (m , 2H). 19F NMR (376 MHz, DMSO-d6 with a drop of D2O): δ ppm -205.00. 31P NMR (162 MHz, DMSO-d6 with a drop of D2O): δ ppm 49.15, -2.90.

[0502] Examples 11a and 11b - Compounds 27a and 27b (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin - 9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa- 3À5,12À5-diphosphatricide[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt 27a - Isomer 1 27b - Isomer 2

[0503] Intermediate 10: N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9 -fluoro-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-12-sulfanyl-3-sulfanilidene-2,4,7 ,11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide

[0504] To a crude hydrogen phosphonate solution of (2R, 5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,5R)-4 -((terc -butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy )methyl)-4-fluorotetrahydrofuran-3-yl (Intermediate 7, 2.58 g, 2.49 mmol) in pyridine (50 mL) was added 2-chloro-5,5-dimethyl-1-2-oxide, 3,2-dioxaphosphinan (DMOCP)(1.70 g, 9.21 mmol), and the mixture was stirred under nitrogen at room temperature for 30 minutes. Then, the reaction was inhibited with water (1.6 mL, 89 mmol), followed by the addition of 3H-benzo[c][1,2]dithiol-3-one (660 mg, 3.92 mmol). The mixture was stirred for 10 minutes, then poured into a beaker containing water (350 ml) and sodium bicarbonate (NaHCO3)(10 g, 119 mmol). The yellow slurry was stirred for 10 minutes then transferred to a separatory funnel. The product was extracted with 1:1 diethyl ether:EtOAc (2 x 300 mL). The combined extracts were dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by chromatography on silica (100 grams column) eluting with 0-10% MeOH/DCM then maintaining 10% MeOH/DCM. The desired fractions were combined and concentrated to obtain the crude title compound (1.1 g) as a tan solid. Two main isomers apparently make up ~77% of the mixture by LCMS. LCMS m/z 1050.1 (M+H), tRET = 1.09 and 1.20 min, respectively.

[0505] Intermediate 11: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)- 8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa -3X5,12X5- diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione

[0506]N-{9-[(1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-18-[(tert-butyldimethylsilyl)oxy]-3-(2-cyanoethoxy)-9-fluoro- 17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-12-oxo-12-sulfanyl-3-sulfanilidene-2,4,7,11, Impure 13,16-hexaox-3X5,12X5-diphosphatricyclo[13.2.1.06,10]octadecan-8-yl]-9H-purin-6-yl}benzamide (Intermediate 10, 0.77 g, 0.73 mmol) was stirred in methanamine (33% by weight in EtOH)(10.0 mL, 80 mmol) under nitrogen at room temperature for 2 hours. The crude LCMS indicated 4 isomers ((M+H)+ = 823) with tRET = 0.74, 0.83, 0.90 and 0.94 min, in a crude ratio of 20:3: 13:19 (but the ratio could be significantly affected by possible overlaps with the impurity peaks).Volatile compounds were removed under vacuum. The crude material was purified by reverse phase HPLC using a gradient of 10-60% ACN:H2O (0.1% TFA). Two main isomers have been separated. • Isomer 1 of the title compound (120 mg, 64% purity by LCMS together with 8% deprotected product from TBS) as a dark solid, the exact stereochemistry at two phosphorus centers being undetermined. LCMS m/z 823.1 (M+H). tRET = 0.74 min. • Isomer 2 of the title compound (130 mg, 50% purity by LCMS along with 18% deprotected product from TBS) as a dark solid, with the exact stereochemistry at two phosphorus centers undetermined. LCMS m/z 823.1 (M+H). tRET = 0.96-1.00 min as a broad peak with peak prolongation.

[0507] Examples 11a and 11b: (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl )-8-(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3X5,12X5- diphosfatricyclo[13.2.1.06,10]octadecan-3,12-dione, bisammonium salt

[0508] A suspension of Isomer 1 of Intermediate 11, (1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H- purin-9-yl)-8-(6-amino-9H-purin-9-yl)-18-[(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7, 11,13,16-hexaoxa-3À5,12À5-diphosphatricyclo[13.2.1.06,10]octadecan-3,12-dione (120 mg, 0.146 mmol), in pyridine (2 mL) and triethylamine (2 mL) at 50° Triethylamine trihydrofluoride (700 μL, 4.30 mmol) was added and the mixture was stirred and heated at 50 °C for 4 hours. The LCMS indicated that some starting material had not yet been consumed. The reaction mixture was left stirring for 16 hours at room temperature. Then, acetone (~25 mL) was added and the solid was precipitated. This was left stirring at room temperature for ~30 minutes, filtered and rinsed with acetone. The filtered solid did not contain any desired product, as determined by LCMS, and was discarded. The filtrate, containing the desired product, was concentrated in vacuo and toluene was added to remove any remaining additional pyridine. The residue was purified by reverse phase HPLC, using a gradient of 0-10% ACN: H2O (0.1% NH4OH), to obtain the product which was not very pure and appeared to be contaminated with trifluoroacetate . Thus, the solid was absorbed into ~2 mL of water and several drops of 30% aqueous NHOH were added. It was further purified by reverse phase HPLC using a gradient of 0-10% ACN:H2O (0.1% NH4OH) to give the title compound (Example 11a, 13 mg) as a salt of bisammonium as a single diastereomer, the exact stereochemistry at two phosphorus centers being undetermined. The product was a white solid. LCMS m/z 708.9 (M+H). tRET = 0.17 min. 1H NMR (600MHz, DMSO-d6 with a drop of D2O): δ ppm 8.31 (s, 1H), 8.21 (s, 1H), 8.11 (br s, 1H), 6.25 (dd , J = 15.1, 2.6 Hz, 1H), 5.84 (d, J = 8.3 Hz, 1H), 5.68 (d, J = 51.7 Hz, 1H), 5.27 -5.37 (m, 1H), 5.16-5.25 (m, 1H), 4.32 (d, J = 4.2 Hz, 1H), 4.26 (br s, 1H), 4 .01-4.17 (m, 3H), 3.90-3.96 (m, 1H), 3.81 (br d, J = 11.7 Hz, 1H). 19F NMR (376 MHz, DMSO-d6 with a drop of D2O) δ ppm -205.30 (br). 31P NMR (162 MHz, DMSO-d6 with a drop of D2O) δ ppm 55.77, 54.01.

[0509] Following the same procedure for the preparation of Example 11a, except that the first purification used a gradient of 0-20% ACN: H2O (0.1% NH4OH), the reaction of Isomer 2 of Intermediate 11, ( 1R, 6R, 8R, 9R, 10R, 15R, 17R, 18R)-17-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino- 9H-purin-9-yl)-18- [(tert-butyldimethylsilyl)oxy]-9-fluoro-3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3 [13.2.1.06,10]octadecan-3,12-dione (130 mg, 0.158 mmol), produced the title compound (Example 11b, 16 mg) in the form of a bisammonium salt as a single diastereoisomer, the stereochemistry being exact in two indeterminate phosphorus centers. The product was a white solid. LCMS m/z 708.9 (M+H). tRET = 0.42 min.1H NMR (600MHz, DMSO-d6 with a drop of D2O): δ ppm 8.22 (br s, 1H), 8.18 (s, 1H), 8.05 (br s, 1H), 6.27 (dd, J = 15.3, 2.1 Hz, 1H), 5.82 (br d, J = 8.3 Hz, 1H), 5.60 (d, J = 49, 9 Hz, 1H), 5.27-5.46 (m, 1H), 5.12-5.27 (m, 1H), 4.42-4.59 (m, 1H), 4.30 (br s, 1H), 4.14 (br d, J = 2.3 Hz, 1H), 4.11 (br d, J = 5.7 Hz, 2H), 4.06 (br d, J = 9, 1 Hz, 1H), 3.82 (br d, J = 11.0 Hz, 1H). 19F NMR (376 MHz, DMSO-d6 with a drop of D2O) δ ppm -205.05.31P NMR (162 MHz, DMSO-d6 with a drop of D2O) δ ppm 53.85, 47.48.

ABBREVIATIONS

[0510] The following list provides definitions of certain abbreviations used here. It is understood that the list is not exhaustive, but the meaning of abbreviations that are not defined below will be readily understood by those skilled in the art. DCMDichloromethane DMFN,N-Dimethylformamide DMSODimethylsulphoxide DMTrDimethoxytrityl THFTetrahydrofuran EtOAcEthyl acetate MeOHMethanol EtOHEthanol MeCNAcetonitrile HClHCl hydrochloric acidHigh performance liquid chromatography MDAPHPLC Mass-directed self-preparation SPESolid phase extraction MeOHMethanol TBDMSterc-Butyldimethylsilyl TB MEtert-Butyl methyl ether TFADIPEAN trifluoroacetic acid,N-diisopropylethylamine

NOMENCLATURE

[0511] Compounds were named from the structure using the Chem Draw (CambridgeSoft) or Marvin Sketch (ChemAxon) naming tool.

[0512] Example 12 - Injectable composition

[0513] An injectable form for administration of the present invention is produced by stirring 1.7% by weight of Compound #2 in a 0.9% saline solution.

[0514] Test

[0515] Compounds are tested in a STING binding assay similar to that described by Li et al. (Nature Chemical Biology, 10, 1043-1048, (2014)).

[0516] Biological activity

[0517] The compounds of the invention were tested in a STING binding assay similar to that described in Li et al. (Nature Chemical Biology, 10, 1043-1048, (2014)). The compounds of the invention were tested in a Fluorescence Resonance Energy Transfer (FRET) binding assay. Li et al. used a binding assay - Scintillation Proximity Assay (SPA).

[0518] The STING activity for the compounds of the invention is presented in Table 1 below. Table 1

[0519] While the preferred embodiments of the invention are illustrated by the foregoing description, it should be understood that the invention is not limited to the precise instructions disclosed herein, and that the right to all modifications comprised within the scope of the following claims is reserved.

Claims (25)

1. Compound CHARACTERIZED by the fact that it presents Formula (IV): where: X31 is S; X32 is O; R31 is OH and R32 is NH2 or R31 is NH2 and R32 is H; R33 is OH and R34 is NH2 or R33 is NH2 and R34 is H; R35 is selected from: F, OH and OC(O)R37; R36 is selected from: F, OH and OC(O)R37; provided that: at least one of R35 and R36 is F; and R38 and R39 are independently selected from: H, CH2OC(O)R37, CH2OCO2R37, CH2CH2SC(O)R37 and CH2CH2SSCH2R37; wherein R37 is selected from: aryl, heteroaryl, heterocycloalkyl, cycloalkyl, C1-20 alkyl and C1-20 alkyl substituted with one to five substituents independently selected from: aryl, cycloalkyl, hydroxy and F; or a pharmaceutically acceptable salt thereof. 2. Compound, according to claim 1, or a pharmaceutically acceptable salt thereof, CHARACTERIZED by the fact that: R35 is selected from: F and OH; and R36 is selected from: F and OH; provided that: at least one of R35 and R36 is F. 3. Compound, according to claim 1, or a pharmaceutically acceptable salt thereof, CHARACTERIZED by the fact that: R35 is OH; and R36 is F. 4. Compound, according to claim 1, CHARACTERIZED by the fact that it is selected from: or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof; a pharmaceutically acceptable salt of: a pharmaceutically acceptable salt of: a pharmaceutically acceptable salt of: or a pharmaceutically acceptable salt thereof; It is or a pharmaceutically acceptable salt thereof. 5. Pharmaceutical composition CHARACTERIZED by the fact that it comprises a compound, as defined in claim 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. 6. Use of a compound of formula (IV), or a pharmaceutically acceptable salt thereof, as defined in claim 1, CHARACTERIZED by the fact that it is for the manufacture of a medicament for the treatment of a disease state selected from of: inflammation, allergic diseases, autoimmune diseases, human immunodeficiency virus (HIV), infectious diseases, cancer and precancerous syndromes in a mammal in need of the same. 7. Use, according to claim 6, CHARACTERIZED by the fact that the mammal is a human. 8. Immunogenic composition CHARACTERIZED by the fact that it comprises an antigen or antigenic composition and a compound, as defined in claim 1, or a pharmaceutically acceptable salt thereof. 9. Use of a compound, or a pharmaceutically acceptable salt thereof, as defined in claim 1, CHARACTERIZED by the fact that it is for the manufacture of a medicament for the treatment of a disease selected from: HBV, HCV, influenza , skin warts, multiple sclerosis and allergic inflammation in a human in need of the same. 10. Compound, according to claim 1, CHARACTERIZED by the fact that it has the following structure: or a pharmaceutically acceptable salt thereof. 11. Compound, according to claim 1, CHARACTERIZED by the fact that it is: 12. Pharmaceutically acceptable salt CHARACTERIZED by the fact that it is of a compound, as defined in claim 10, which is: 13. Compound, according to claim 10, CHARACTERIZED by the fact that it is: or a pharmaceutically acceptable salt thereof. 14. Compound, according to claim 1, CHARACTERIZED by the fact that it is: 15. Pharmaceutically acceptable salt CHARACTERIZED by the fact that it is of a compound, as defined in claim 10, which is: 16. Compound, according to claim 10, CHARACTERIZED by the fact that it is: or a pharmaceutically acceptable salt thereof. 17. Compound, according to claim 1, CHARACTERIZED by the fact that it is: 18. Pharmaceutically acceptable salt CHARACTERIZED by the fact that it is of a compound, as defined in claim 10, which is: 19. Pharmaceutical composition CHARACTERIZED by the fact that it comprises a compound, as defined in claim 10, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. 20. Use of a compound, or a pharmaceutically acceptable salt thereof, as defined in claim 10, CHARACTERIZED by the fact that it is for the manufacture of a medicament for the treatment of a disease state selected from: inflammation, diseases allergic diseases, autoimmune diseases, human immunodeficiency virus (HIV), infectious diseases, cancer and pre-cancerous syndromes in a mammal in need thereof. 21. Use, according to claim 20, CHARACTERIZED by the fact that the mammal is a human. 22. Immunogenic composition CHARACTERIZED by the fact that it comprises an antigen or antigenic composition and a compound, as defined in claim 10, or a pharmaceutically acceptable salt thereof. 23. Use of a compound, or a pharmaceutically acceptable salt thereof, as defined in claim 10, CHARACTERIZED by the fact that it is for the manufacture of a medicament for the treatment of a disease selected from: HBV, HCV, influenza , skin warts, multiple sclerosis and allergic inflammation, in a human in need of the same. 24. Compound, according to claim 1, CHARACTERIZED by the fact that it is selected from: or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof; It is or a pharmaceutically acceptable salt thereof. 25. Compound, according to claim 1, or a pharmaceutically acceptable salt thereof, CHARACTERIZED by the fact that R38 and R39 are H.