Purine derivatives for use in the treatment of allergic, inflammatory and infectious diseases

[0001] The present disclosure relates to compounds, methods for their preparation, compositions containing them, their use in the treatment of various disorders, in particular allergic diseases and other inflammatory conditions, for example allergic rhinitis and asthma, infectious diseases, cancer, and as vaccine adjuvants.

[0002] Vertebrates are still at risk of microbial invasion and have developed immune defense mechanisms to eliminate infectious pathogens. In mammals, this immune system includes two branches; innate immunity and acquired immunity. The host's first line of defense is the innate immune system that works through macrophages and dendritic cells. Acquired immunity includes the elimination of pathogens in the late stages of infection, as well as allows the generation of immunological memory. Acquired immunity is highly specific due to the broad repertoire of lymphocytes with antigen-specific receptors that have undergone gene rearrangement.

[0003] The innate immune response was originally thought to be non-specific, but it is now known that this response is able to distinguish its own organism from a number of pathogens. The innate immune system recognizes microbes by a limited number of sexually encoded pattern recognition receptors (PRRs) that have several important characteristics.

[0004] Toll-like receptors (TLRs) are a family of ten pattern recognition receptors described in humans. TLRs are expressed mainly by innate immune cells, where their role is to monitor the environment for signs of infection and when activated, mobilize defense mechanisms aimed at eliminating invading pathogens. Early innate immune responses triggered by TLRs limit the spread of infection, while proinflammatory cytokines and chemokines that induce them lead to the recruitment and activation of antigen presenting cells, B lymphocytes and T lymphocytes. TLRs can modulate the nature of adaptive immune responses to achieve adequate protection by activating dendritic cells and cytokine release (Akira S., et al., Nat. Immunol., 2001: 2, 675-680). The observed response profile from different TLR agonists depends on the type of cell activated.

[0005] TLR7 is a member of the TLR subgroup (TLR 3, 7, 8 and 9), located in the endosomal compartment of cells that have specialized in the detection of foreign nucleic acids. TLR7 plays a key role in antiviral defense by ssRNA recognition (Diebold SS, et al., Science, 2004: 303, 1529-1531; and Lund J. M., et al., PNAS, 2004: 101, 5598-5603). TLR7 has a limited expression profile in humans and is expressed mainly by B lymphocytes and plasmacytoid dendritic cells (pDC) and to a lesser extent by monocytes. Plasmacytoid DCs are a unique population of dendritic cells of lymphatic origin (0.2-0.8% peripheral blood mononuclear cells (PBMC)), which are primary type I interferon producing cells that secrete high levels of interferon-alpha (IFNa) and interferon-beta ( IFNP) in response to viral infections (Liu YJ, Annu. Rev. Immunol., 2005: 23, 275-306).

[0006] Allergic diseases are associated with a Th2-shifted immune response to allergens. Th2 responses are associated with elevated levels of IgE, which through its action on mast cells, promotes hypersensitivity to allergens, causing symptoms visible, for example, in allergic rhinitis. In healthy individuals, the immune response to allergens is more balanced with a mixed response of Th2 / Th1 and regulatory T cells. TLR7 ligands have been shown to reduce Th2 cytokine release and increase Th1 cytokine release in vitro and ameliorate Th2 inflammatory responses in in vivo allergic lung models (Fili L., et al., J. All. Clin. Immunol., 2006: 118, 511-517; Moisan J., et al., Am. J. Physiol. Lung Cell Mol. Physiol., 2006: 290, L987-995; Tao et al., China. Med. J., 2006: 119, 640-648). Thus, TLR7 ligands have the potential to alter the balance of the immune response found in allergic individuals and lead to disease modification.

[0007] In generating an effective innate immune response in mammals, mechanisms that induce interferons and other cell-acting cytokines to induce many effects are essential. These effects may include activation of anti-infective gene expression, activation of antigen presentation in cells to stimulate strong antigen-specific immunity, and promote phagocytosis in phagocytic cells.

[0008] Interferon was first described as a substance that could protect cells against viral infection (Isaacs & Lindemann, J. Virus Interference. Proc. R. Soc. Lon. Cheese. B. Biol. Sci. 1957: 147, 258-267). In humans, type I interferons are a family of related proteins encoded by genes on chromosome 9 and encoding at least 13 interferon alpha (IFNa) isoforms and one interferon beta isoform (IFNP). Recombinant IFNa was the first approved biological drug and became an important therapy in viral infections and cancer. In addition to direct antiviral activity in cells, interferons are known to be potent modulators of the immune response by acting on cells of the immune system.

[0009] As a primary therapy against hepatitis C (HCV), interferon combinations can be highly effective in reducing viral load and in certain individuals in eliminating viral replication. However, many patients do not show a long-term virological response and in these patients the viral load is not controlled. In addition, injected interferon therapy may be associated with many unwanted side effects that have been shown to affect discipline (Dudley T., et al., Gut., 2006: 55 (9), 1362-3).

[0010] Administration of a small molecule compound that could stimulate an innate immune response, including the activation of type I interferons and other cytokines, could become an important strategy for treating or preventing 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 (Krieg., Curr. Oncol. Rep., 2004: 6 (2), 88-95), allergic diseases (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 Suppl), S32-7).

[0011] In animal models, imiquimod showed adjuvant or local activity (Adams S., et al., J. Immunol., 2008, 181: 776-84; Johnston D., et al., Vaccine, 2006, 24: 1958-65), or systemically (Fransen F. et al., Infect. Immun., 2007, 75: 593946). Resiquimod and other related TLR7 / 8 agonists have also been shown to have adjuvant activity (Ma R. et al., Biochem. Biophys. Res. Commun., 2007, 361: 537-42; Wille-Reece U., et al., Proc. Natl. Acad. Sci. USA, 2005, 102: 15190-4; WilleReece U., et al., US2006045885 A1).

[0012] The mechanisms leading to the induction of type I interferons are only partially understood. One of the mechanisms that can lead to the induction of interferon in many cell types is the recognition of double-stranded viral RNA by RIG-I and MDA5 RNA helicases. This mechanism is considered the basic mechanism by which interferons are induced by infection of cells with Sendai virus.

[0013] Further mechanisms of interferon induction act through TLR-dependent signaling events. In humans, plasmacytoid dendritic cells (pDCs) are specialized interferon-producing cells capable of producing large amounts of interferons in response to, for example, a viral infection. These pDCs have been shown to preferentially express TLR7 and TLR9 and stimulation of these receptors with viral RNA or DNA, respectively, can induce expression of interferon alpha.

[0014] Oligonucleotide agonists TLR7 and TLR9 and low molecular weight purine-based TLR7 agonists that can induce interferon alpha from these cell types in animals and humans have been described (Takeda K. et al., Annu. Rev. Immunol., 2003: 21, 335-76). TLR7 agonists include imidazoquinoline compounds such as imiquimod and resiquimod, oxoadenine analogs as well as nucleoside analogues such as loxoribine and 7-thia-8-oxoguanosine, which have long been known to induce interferon alpha. In international patent application with publication number WO 2008/114008 (AstraZeneca AB / Dainippon Sumitomo Pharma Co. Ltd.) 9-substituted-8-oxoadenine compounds are disclosed as TLR7 modulators.

[0015] WO2007 / 142755 describes purine analogue compounds that are TLR agonists and their use for the treatment of various diseases and disorders.

[0016] It is still unclear how small molecule purine-like compounds can induce type I interferons and other cytokines because the molecular targets of these known inducers have not been identified. However, a test strategy has been developed to characterize small molecule inducers of human interferon IFNa (regardless of mechanism), which is based on the stimulation of primary human donor cells with compounds and is disclosed herein.

Brief description of the invention [0017] Certain compounds of the invention have been shown to be inducers of human interferon and may exhibit an improved profile against known human interferon inducers, for example increased potency, and may show increased selectivity for IFNa against TNFa. For example, certain compounds of the invention exhibit greater than 1000 fold selectivity for IFNa induction compared to TNFa induction. Compounds that induce human interferon may be useful in the treatment of various disorders, for example the treatment of allergic diseases and other inflammatory conditions, for example allergic rhinitis and asthma, treatment of infectious diseases and cancer, and may also be useful as vaccine adjuvants.

[0018] Certain compounds of the invention are potent immunomodulators, and as a result, care should be taken when handling them.

Summary of the Invention [0019] In a first aspect, there are provided compounds of formula (I):

<img file="PL3000813T3_D0001.tif" />

wherein;

R¹ is (1S) -1-methylbutyloxy;

m is an integer from 3 to 6;

n is an integer from 0 to 4;

and their salts.

[0020] In a further embodiment, m is 3.

[0021] In a further embodiment, m is 4.

[0022] In a further embodiment, m is 5.

[0023] In a further embodiment, m is an integer from 4 to 6.

[0024] [0025] [0026] [0027] [0028] [0029] [0030] [0031]

In a further embodiment, m is 6.

In a further embodiment, n is 0.

In a further embodiment, n is 1.

In a further embodiment, n is 2.

In a further embodiment, n is 3.

In a further embodiment, n is 4.

In a further embodiment, n is an integer from 2 to 4.

In another aspect of the disclosure, compounds of formula (IA) are provided:

<img file="PL3000813T3_D0002.tif" />

<img file="PL3000813T3_D0003.tif" />

wherein;

R^1A is C1-6alkylamino or C1-6alkoxy; mA is an integer from 3 to 6; nA is an integer from 0 to 4;

and their salts.

[0032] In a further embodiment, R^1A is n-butyloxy.

[0033] In a further embodiment, R^1A is n-butylamino.

[0034] In a further embodiment, R^1A is (1S) -1-methylbutyloxy.

[0035] In a further embodiment, R^1A is (1S) -1-methylpropyloxy.

[0036] In a further embodiment, R^1A is (1S) -1-methylpentyloxy.

[0037] In a further embodiment, R^1A is 1-methylethyloxy.

[0038] In a further embodiment, R^1A is (1R) -1-methylbutylamino.

[0039] In a further embodiment, R^1A is (1S) -1-methylbutylamino.

[0040] In a further embodiment, mA is 4.

[0041] In a further embodiment, mA is 5.

[0042] In a further embodiment, mA is 6.

[0043] In a further embodiment, nA is 0.

[0044] In a further embodiment, nA is 1.

[0045] In a further embodiment, nA is 2.

[0046] In a further embodiment, nA is 3.

[0047] In a further embodiment, nA is 4.

[0048] In another aspect of the disclosure, compounds of formula (IA) and salts thereof as defined herein above are provided, wherein m is an integer from 4 to

6.

[0049] In another aspect of the disclosure, compounds of formula (IA) and salts thereof as defined herein above are provided, wherein 6-amino-2- (butyloxy) -9- [3- (1-pyrrolidinyl) propyl] -7 , 9-dihydro-8H-purin-8-one is excluded.

[0050] In a further aspect, there are provided compounds of formula (I) and their salts, wherein 6 amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-di-hydro-8Hpuryn-8-one and its salts are excluded.

[0051] In another aspect of the disclosure, compounds of formula (IA) and salts thereof as defined hereinabove are provided, wherein m is an integer from 4 to 6 and 6-amino-2 - {[(1S) - 1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one and its salts are excluded.

[0052] In another aspect of the disclosure, compounds of formula (IA) and salts thereof as defined hereinabove are provided, wherein 6-amino-2- (butyloxy) -9- [3- (pyrrolidinyl) propyl] - 7,9-dihydro-8H-purin-8-one and 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9- dihydro-8H-purin-8-one and its salts are excluded.

[0053] Examples of compounds of formula (I) are set out in the following list and constitute another aspect of this invention:

6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8 H -purin-8-one;

6-amino-9- [4- (hexahydro-1H-azepin-1-yl) butyl] -2 - {[(1S) -1-methylbutyl] oxy} -7,9-dihydro-8H-purin-8- he;

6-amino-9- [5- (hexahydro-1H-azepin-1-yl) pentyl] -2 - {[(1S) -1-methylbutyl] oxy} -7,9-dihydro-8H-purin-8- he;

6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [3- (1-piperidinyl) propyl] -7,9- dihydro-8H-purin-8-one; and their salts.

[0054] In one aspect of the disclosure, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine is provided -8-he or a salt thereof.

[0055] In a further embodiment of the disclosure, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin- 8-he or a pharmaceutically acceptable salt thereof.

[0056] In a further embodiment of the disclosure, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin- 8-on in the form of a free rule.

[0057] Thus, as a further aspect of the invention there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0058] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-one, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0059] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-on, in the form of the free base, for use in therapy.

[0060] It will be obvious that when a compound of formula (I) or a pharmaceutically acceptable salt thereof is used in therapy, it is used as a therapeutically active agent.

[0061] Therefore, there is also provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of allergic and other inflammatory diseases, infectious diseases and cancer.

[0062] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-one, or a pharmaceutically acceptable salt thereof, for use in the treatment of allergic and other inflammatory diseases, infectious diseases and cancer.

[0063] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-on, in the form of the free base, for use in the treatment of allergic and other inflammatory diseases, infectious diseases and cancer.

[0064] Therefore, there is also provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of allergic rhinitis.

[0065] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-one, or a pharmaceutically acceptable salt thereof, for use in the treatment of allergic rhinitis.

[0066] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-on, in the form of the free base, for use in the treatment of allergic rhinitis.

[0067] According to the disclosure, there is also provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of asthma.

[0068] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-one, or a pharmaceutically acceptable salt thereof, for use in the treatment of asthma.

[0069] According to the disclosure, therefore, 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H is also provided -purin-8-on, in the form of the free base, for use in the treatment of asthma.

[0070] According to the disclosure, therefore, there is also provided a vaccine adjuvant comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0071] According to the disclosure, there is further provided an immunogenic vaccine composition comprising an antigen or antigen composition and a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0072] According to the disclosure, there is further provided a vaccine composition comprising an antigen or antigen composition and a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0073] According to the disclosure, there is further provided a method of treating or preventing a disease comprising administering to a human subject suffering from or susceptible to the disease an immunogenic composition comprising an antigen or antigen composition and a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0074] According to the disclosure, there is further provided a method of treating or preventing a disease comprising administering to a human subject suffering from or susceptible to the disease a vaccine composition comprising an antigen or antigen composition and a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0075] According to the disclosure, there is further provided the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of an immunogenic composition comprising an antigen or antigen composition, for treating or preventing a disease.

[0076] According to the disclosure, there is further provided the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of a vaccine composition comprising an antigen or antigen composition, for treating or preventing a disease.

[0077] According to the disclosure, there is further 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 allergic and other inflammatory diseases, infectious diseases and cancer.

[0078] According to the disclosure, there is further provided the use of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-on, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of allergic and other inflammatory diseases, infectious diseases and cancer.

[0079] According to the disclosure, there is further provided the use of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-on, in the form of the free base, for the preparation of a medicament for the treatment of allergic and other inflammatory diseases, infectious diseases and cancer.

[0080] According to the disclosure, there is further 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 allergic rhinitis.

[0081] According to the disclosure, there is further provided the use of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-one, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of allergic rhinitis.

[0082] According to the disclosure, there is further provided the use of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-on, in the form of the free base, for the preparation of a medicament for the treatment of allergic rhinitis.

[0083] According to the disclosure, there is further 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 asthma. [0084] According to the disclosure, there is further provided the use of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-one, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of asthma.

[0085] According to the disclosure, there is further provided the use of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-on, in the form of the free base, for the preparation of a medicament for the treatment of asthma.

[0086] According to the disclosure, there is further provided a method of treating allergic and other inflammatory diseases, infectious diseases and cancer, the method comprising administering to a human subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable compound thereof salt.

[0087] According to the disclosure, there is further provided a method of treating allergic and other inflammatory diseases, infectious diseases and cancer, the method comprising administering to a human subject in need of such treatment a therapeutically effective amount of 6-amino-2 - {[(1S) - 1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one, or a pharmaceutically acceptable salt thereof.

[0088] According to the disclosure, there is further provided a method of treating allergic and other inflammatory diseases, infectious diseases and cancer, the method comprising administering to a human subject in need of such treatment a therapeutically effective amount of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one as the free base.

[0089] According to the disclosure, there is further provided a method of treating allergic rhinitis, which method comprises administering to a human subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0090] According to the disclosure, there is further provided a method of treating allergic rhinitis, the method comprising administering to a human subject in need thereof a therapeutically effective amount of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9 - [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one, or a pharmaceutically acceptable salt thereof.

[0091] According to the disclosure, there is further provided a method of treating allergic rhinitis, which method comprises administering to a human subject in need thereof a therapeutically effective amount of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9 - [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one as the free base.

[0092] According to the disclosure, there is further provided a method of treating asthma, which method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0093] According to the disclosure, there is further provided a method of treating asthma, which method comprises administering to a subject in need thereof a therapeutically effective amount of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [ 5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one, or a pharmaceutically acceptable salt thereof.

[0094] According to the disclosure, there is further provided a method of treating asthma, which method comprises administering to a human subject in need thereof a therapeutically effective amount of 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [ 5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one as the free base.

[0095] The disclosure further provides a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with at least one other therapeutically active agent.

[0096] The disclosure further provides a combination comprising 6-amino-2 {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine He, or a pharmaceutically acceptable salt thereof, together with at least one other therapeutically active agent.

[0097] The disclosure further provides a combination comprising 6-amino-2 {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-on, in the form of the free base, together with at least one other therapeutically active agent.

[0098] Also provided is a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents or carriers.

[0099] According to the disclosure, there is further provided a pharmaceutical composition comprising 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro -8H-purin-8-one, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents or carriers.

[0100] According to the disclosure, there is further provided a pharmaceutical composition comprising 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro -8H-purin-8-one, in the form of the free base, and one or more pharmaceutically acceptable diluents or carriers.

[0101] According to the disclosure, there is also provided a method of preparing a pharmaceutical composition which comprises mixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable diluents or carriers.

[0102] In accordance with the disclosure, there is also provided a method of preparing a pharmaceutical composition which comprises mixing 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7 , 9-dihydro-8H-purin-8-one, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable diluents or carriers.

[0103] In accordance with the disclosure, there is also provided a method of preparing a pharmaceutical composition which comprises mixing 6-amino-2 - {[(1S) -1-methylbutyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7 , 9-dihydro-8H-purin-8-one, in the form of the free base, with one or more pharmaceutically acceptable diluents or carriers. [0104] Compounds of formula (I) and their salts can be prepared according to the methodology described herein, which is another aspect of this disclosure.

[0105] According to the disclosure, there is provided a method for preparing a compound of formula (I), which method comprises deprotecting a compound of formula (II):

<img file="PL3000813T3_D0004.tif" />

in which R¹, min are as previously defined herein for the compound of formula (I) and R² is C1-6alkyl, and then, when required, to carry out one or more of the following possible steps:

(i). removal of any necessary protecting group;

(ii). salt formation of the compound thus formed.

[0106] The present disclosure includes all combinations of the embodiments and aspects described herein.

Detailed description of the invention [0107] The present invention is described using terms known and understood by those skilled in the art. In order to facilitate reference, certain terms are defined herein below. However, the fact that certain terms are defined should not be considered as indicating that the defined terms have been used in a manner inconsistent with the usual meaning or, alternatively, that any term which has not been defined is indefinite or has not been used in accordance with the usual and accepted meaning. However, it is believed that all terms used herein describe the invention in such a way that a person of ordinary knowledge will be able to appreciate the scope of the present invention. The following definitions are intended to clarify, but not to limit, defined terms.

[0108] References to "alkyl" include references to both straight chain and branched chain aliphatic isomers of their corresponding alkyl containing up to six carbon atoms, for example up to four carbon atoms or up to two carbon atoms. Such references to "alkyl" also apply when the alkyl group is part of another group, for example an alkylamino or alkoxy group. Examples of such alkyl groups and groups containing alkyl groups are C1-6alkyl, C1-6alkylamino and C1-6alkoxy.

[0109] References to "halogen" refer to iodine, bromine, chlorine or fluorine, typically bromine, chlorine or fluorine. References to "halo" refer to iodo, bromo, chloro or fluoro, typically bromo, chloro or fluoro. [0110] It should be understood that references herein to compounds of the invention mean a compound of formula (I) in the form of the free base, or in the form of a salt, for example a pharmaceutically acceptable salt.

[0111] Salts of compounds of formula (I) include pharmaceutically acceptable salts and salts that may not be pharmaceutically acceptable salts, but may be useful in the preparation of compounds of formula and pharmaceutically acceptable salts thereof. Salts may be derived from some inorganic or organic acids or some inorganic or organic bases.

[0112] The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of formula (I).

[0113] Examples of the salts are pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition and base addition salts. For review of suitable salts, see Berge et al., J. Pharm. Sci., 66: 1-19 (1977).

[0114] Examples of the pharmaceutically acceptable acid addition salts of the compound of formula (I) include the hydrobromide, hydrochloride, sulfate, p-toluenesulfonate, methanesulfonate, naphthalenesulfonate and phenylsulfonate salts.

[0115] Salts may be prepared using techniques well known in the art, for example by precipitation from solution followed by filtration or by evaporation of the solvent.

[0116] Typically, a pharmaceutically acceptable acid addition salt may be prepared by reacting a compound of formula (I) with an appropriate strong acid (such as hydrobromic, hydrochloric, sulfuric, p-toluenesulfonic, methanesulfonic or naphthalene sulfonic acids), optionally in a suitable solvent such like an organic solvent, to form a salt, which is usually isolated by crystallization and filtration, for example.

[0117] It should be understood that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvents with high boiling points and / or solvents with a high tendency to form hydrogen bonds, such as water, ethanol, isopropyl alcohol and N-methylpyrrolidinone can be used to prepare solvates. Methods for identifying solvates include, but are not limited to, NMR and microanalysis. Solvates of the compounds of formula (I) are within the scope of the invention. As used herein, the term solvate includes solvates of both the free base compound and any salt thereof.

[0118] Certain compounds of the invention may contain chiral atoms and / or multiple bonds, and therefore may exist in one or more stereoisomeric forms. The present invention includes all stereoisomers of the compounds of the invention, including optical isomers, either as individual stereoisomers or as mixtures thereof, including racemic forms. Any stereoisomer may contain less than 10% by weight, for example less than 5% by weight, or less than 0.5% by weight, of any other stereoisomer. For example, any optical isomer may contain less than 10% by weight, for example less than 5% by weight, or less than 0.5% by weight, of its antipode.

[0119] Certain compounds of the invention may exist in tautomeric forms. It should be understood that the present invention includes all tautomers of the compounds of the invention, either as individual tautomers or as mixtures thereof.

[0120] The compounds of the invention may be in crystalline or amorphous form. In addition, certain crystalline forms of the compounds of the invention may exist as polymorphs, all of which are within the scope of the present invention. The thermodynamically stable polymorphic form or forms of the compounds of the invention are of particular interest.

[0121] Polymorphic forms of the compounds of the invention can be characterized and distinguished using a variety of traditional analytical techniques, including, but not limited to, powder X-ray diffraction (XRPD), infrared spectroscopy (IR), Raman spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and solid state nuclear magnetic resonance (ssNMR).

[0122] It should be understood from the above that the scope of the invention includes solvates, hydrates, isomers and polymorphic forms of the compounds of formula (I) and their salts and solvates.

[0123] Examples of disease states in which the compounds of formula (I) and their pharmaceutically acceptable salts may have potentially beneficial effects include allergic diseases and other inflammatory conditions, for example allergic rhinitis and asthma, infectious diseases and cancer. The compounds of formula (I) and their pharmaceutically acceptable salts also have potential use as vaccine adjuvants.

[0124] As modulators of the immune response, compounds of formula (I) and pharmaceutically acceptable salts thereof may also be useful, alone or in combination as an adjuvant, in the treatment and / or prevention of immunologically mediated disorders, including but not limited to inflammatory or allergic diseases, such as asthma allergic rhinitis and rhinitis and conjunctivitis, food alergy, pulmonary hypersensitivity diseases, eosinophilic pneumonia, delayed type hypersensitivity disorders, atherosclerosis inflammation of the pancreas, gastritis, colitis osteoarthritis, psoriasis, sarcoidosis, pulmonary fibrosis respiratory distress syndrome, bronchiolitis Chronic obstructive pulmonary disease, sinusitis, cystic fibrosis, actinic keratosis, cutaneous dysplasia, chronic urticaria eczema and all types of dermatitis.

[0125] The compounds of formula (I) and their pharmaceutically acceptable salts may also be useful in the treatment and / or prevention of responses to respiratory infections, including but not limited to said viral respiratory tract irritation and tonsillitis. The compounds may also be useful in the treatment and / or prevention of autoimmune diseases including, but not limited to rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, Sjoegren's disease ankylosing spondylitis, scleroderma, dermatomyositis, diabetes, transplant rejection including graft versus host disease, inflammatory bowel disease including but not limited to Crohn's disease and ulcerative colitis.

[0126] The compounds of formula (I) and their pharmaceutically acceptable salts may also be useful in the treatment of infectious diseases including, but not limited to, caused by hepatitis viruses (e.g. hepatitis B virus, hepatitis C virus), human immunodeficiency virus, papilloma viruses, herpes viruses, respiratory viruses (e.g. influenza viruses, respiratory syncytial virus, rhinovirus, metapneumovirus, pseudo-influenza virus, SARS), and West Nile virus. The compounds of formula (I) and their pharmaceutically acceptable salts may also be useful in the treatment of microbial infections caused by, for example, bacteria, fungi or protozoa. These include, but are not limited to, tuberculosis, bacterial pneumonia, athlete's foot fungus, histoplasmosis, candidiasis, pneumocystosis, leprosy, chlamydia, cryptococcosis, cryptosporidosis, toxoplasmosis, leishmaniasis, malaria and trypanosomiasis.

[0127] The compounds of formula (I) and their pharmaceutically acceptable salts may also be useful in the treatment of various cancers, in particular the treatment of cancers that are known to respond to immunotherapy and include, but are not limited to, renal cell carcinoma, lung cancer , breast cancer, colorectal cancer, bladder cancer, melanoma, leukemia, lymphomas and ovarian cancer.

[0128] Those skilled in the art will understand that references to treatment or therapy may, depending on the condition, extend to prophylaxis as well as to treatment of established conditions. [0129] As mentioned herein, compounds of formula (I) and pharmaceutically acceptable salts thereof may be useful as therapeutic agents.

[0130] The compounds of formula (I) and pharmaceutically acceptable salts thereof can be formulated for administration in any convenient manner.

[0131] The compounds of formula (I) and their pharmaceutically acceptable salts may, for example, be formulated for oral, topical, inhalational, nasal, buccal, parenteral (for example intravenous, subcutaneous, intradermal or intramuscular) or rectal administration. In one aspect, compounds of formula (I) and pharmaceutically acceptable salts thereof are formulated for oral administration. In another aspect, the compounds of formula (I) and pharmaceutically acceptable salts thereof are formulated for topical administration, for example for intranasal or inhalational administration.

[0132] Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, for example syrup, gum arabic, gelatin, sorbitol, tragacanth, starch gruel, cellulose or polyvinylpyrrolidone; fillers, for example, lactose, microcrystalline cellulose, sugar, cornstarch calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch croscarmellose sodium or sodium starch glycolate; or wetting agents, such as sodium lauryl sulfate. The tablets may be coated according to methods well known in the art.

[0133] Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be formulated as a dry product for reconstitution with water or other suitable excipient before use. Such liquid preparations may contain traditional additives, such as suspending agents, for example, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxymethylcellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oil esters, propylene glycol or ethyl alcohol; or preservatives, for example, methyl or propyl p-hydroxybenzoates, or sorbic acid. The preparations may also contain buffer salts, flavoring, coloring and / or sweetening agents as needed (e.g. mannitol).

[0134] Compositions for nasal administration include aqueous compositions administered to the nose as drops or by means of a pressure pump. Suitable compositions contain water for this purpose as a diluent or carrier. 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 osmotic pressure regulating agents, one or more more co-solvents, and may include ingredients to control the pH of the composition, for example, a buffer system. In addition, the compositions may contain other excipients such as antioxidants, for example sodium metabisulfite, and taste masking agents. The compositions may also be administered to the nose or other areas of the respiratory tract by nebulization.

[0135] Intranasal compositions may allow the compound (s) of formula (I) or a pharmaceutically acceptable salt (s) thereof to be delivered to all areas of the nasal cavity (target tissue) and may further allow the compound (s) to remain. ) of formula (I) or a pharmaceutically acceptable salt (s) thereof in contact with the target tissue for a long time. A suitable dosage regimen for nasal compositions could be slow inhalation by the patient through the nose after cleansing the nasal cavity. During inhalation, the composition would be administered into one nostril while the other is manually clamped. This procedure would then be repeated for the other nostril. Typically, one or two injections on the nostril would be administered in the above procedure one, two or three times a day, preferably once a day. Particularly interesting are nasal compositions suitable for once daily administration.

[0136] The suspending agent (s), if present, will typically be present in an amount of 0.1 to 5% (w / w), such as 1.5% to 2.4% (w / w) wt.) 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.

[0137] Compositions for pulmonary or nasal administration may contain one or more excipients and may be protected against infection and growth of microorganisms or fungi by the addition of one or more preservatives. Examples of pharmaceutically acceptable antimicrobials or preservatives include, but not limited to quaternary ammonium compounds (for example benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, lauralkonium chloride and myristyl picolinium chloride), mercury agents (e.g. phenylmercuric nitrate, phenylmercuric acetate and thimerosal), alcoholic agents (for example chlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterial esters (for example, para-hydroxybenzoic acid esters), chelating agents, such as disodium edetate (EDTA) and other antimicrobials, such as chlorhexidine chlorocresol, sorbic acid and its salts (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, methyl paraben, ethyl paraben, propyl paraben and butyl paraben. The preservative (s), if present, may be present in an amount of 0.001 to 1% (w / w), such as 0.015% to 0.5% (w / w) based on the total weight of the composition.

[0138] Compositions (for example, wherein at least one compound is in suspension) may include one or more surfactants that act to facilitate dissolution of drug particles in the aqueous phase of the composition. For example, the amount of surfactant used is an amount that will not cause foaming during mixing. Examples of pharmaceutically acceptable surfactants include alcohols, esters and fatty ethers such as polyoxyethylene (20) sorbitan monooleate (Polysorbate 80), macrogol ethers and poloxamers. The surfactant may be present in an amount between about 0.01 to 10% (w / w), such as 0.01 to 0.75% (w / w), for example about 0.5% ( w / w), relative to the total weight of the composition.

[0139] One or more osmotic pressure regulating agents may be present to achieve tonicity with body fluids e.g. fluids in the nasal cavity, which reduces the level of irritation. Examples of pharmaceutically acceptable agents for regulating osmotic pressure include, but are not limited to, sodium chloride, dextrose, xylitol, calcium chloride, glucose, glycerin and sorbitol. The osmotic pressure regulating 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 5 , 0% (w / w), based on the total weight of the composition.

[0140] The compositions of the invention may be buffered by the addition of appropriate buffering agents such as sodium citrate, citric acid, trometamol, phosphates such as disodium phosphate (for example dodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodium phosphate and mixtures thereof.

[0141] The 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. [0142] Examples of taste masking agents include sucralose, sucrose, saccharin or their salt, fructose, dextrose, glycerol, corn syrup, aspartame, acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycerisinate, thaumatin, neotame, mannitol, menthol eucalyptus, camphor, natural flavoring, artificial flavoring, and combinations thereof.

[0143] One or more cosolvents may be added to aid the solubility of the therapeutic 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 (for example PEG300 or PEG400) and methanol. In one embodiment, the cosolvent is propylene glycol.

[0144] The co-solvent (co-solvents), if present, may be included in an amount of from 0.05 to 30% (w / w), such as from 1 to 25% (w / w), for example from 1 to 10% (w / w) based on the total weight of the composition.

[0145] Compositions for inhaled administration include aqueous, organic or aqueous / organic mixtures, dry powder or crystalline compositions administered to the respiratory tract by a pressurized pump or inhaler, for example, dry powder reservoir inhalers, unit dose dry powder inhalers, multiple metered dry powder inhalers doses, nasal inhalers or pressure aerosol inhalers, nebulizers or insufflators. Suitable compositions contain water for this purpose as a diluent or carrier and may be provided with conventional excipients such as buffering agents, osmotic pressure modifying agents and the like. Aqueous compositions can also be administered to the nose and other areas of the respiratory tract by spraying. Such compositions may be aqueous solutions or suspensions or aerosols delivered from pressurized packs, such as a metered dose inhaler, using a suitable liquefied propellant.

[0146] Compositions for topical administration to the nose (for example, for the treatment of rhinitis) or to the lungs include pressurized aerosol compositions and aqueous compositions delivered to the nasal cavities by a pressure pump. Particularly interesting are compositions that are not pressure and are suitable for topical administration to the nasal cavity. Suitable compositions contain water for this purpose as a diluent or carrier. Aqueous compositions for pulmonary or nasal administration may be provided with conventional excipients, such as buffering agents, osmotic pressure modifying agents and the like. The aqueous compositions can also be administered to the nose by spraying. [0147] A fluid dispenser may typically be used to deliver a fluid composition to the nasal cavity. The liquid composition may be aqueous or non-aqueous, but usually aqueous. Such a liquid dispenser may have a dispensing tip or dispensing opening through which a metered dose of the liquid composition is dispensed after a user applies force to the fluid dispenser pump mechanism. Such fluid dispensers are generally provided with a reservoir for multiple metered doses of the fluid composition, with the doses being dispensed the next time the pump is actuated. The dispensing tip or opening may be configured to enter the nostrils of a user for spraying dispensing a fluid composition into the nasal cavity. A fluid dispenser of the said type is described and illustrated in International Patent Application Publication No. WO 2005/044354 (Glaxo Group Limited). The dispenser has a housing housing a fluid discharge device having a pressure pump mounted on the container to contain the liquid composition. The housing has at least one finger operated side lever that is movable inward relative to the housing moving the container up in the housing with a cam, forcing the pump to compress and pump a metered dose of composition from the pump shaft through the nasal tip of the housing. In one embodiment, the fluid dispenser is of the general type illustrated in Figures 30-40 of WO 2005/044354.

[0148] Aqueous compositions containing a compound of formula (I) or a pharmaceutically acceptable salt thereof may also be provided by a pump disclosed in International Patent Application Publication No. WO2007 / 138084 (Glaxo Group Limited), for example as disclosed in reference to its Figures 22- 46, or as disclosed in British Patent Application No. GB0723418.0 (Glaxo Group Limited), for example as disclosed in reference to Figures 7-32 thereof. The pump may be actuator driven as disclosed in Figures 1-6 GB0723418.0.

[0149] Dry powder compositions for topical delivery to the lungs by inhalation may, for example, be provided in capsules and cartridges of, for example, gelatin, or blisters, for example, of laminated aluminum foil, for use in an inhaler or insufflator. The powder mix compositions generally contain a powder mix 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 ( for example lactose or starch). Dry powder compositions may also include, in addition to the drug and the carrier, a further excipient (for example a third agent such as a sugar ester, for example cellobiose octaacetate, calcium stearate or magnesium stearate).

[0150] In one embodiment, a composition suitable for inhaled administration may be introduced into a plurality of sealed dose containers provided on the drug package (s) attached inside the appropriate inhalation device. The containers may be torn, tearable, or otherwise open individually, and the doses of the dry powder composition administered by inhalation onto the mouthpiece of an inhalation device as known in the art. The drug packet can have many different forms, for example, the shape of a shield or an elongated strip. Representative inhalation devices are DISKHALER ™ and DISKUS ™ devices, marketed by GlaxoSmithKline.

[0151] The inhalable dry powder composition can also be provided as a collecting reservoir in an inhalation device, the device further being provided with a metering mechanism for measuring a dose of the composition from the reservoir to the inhalation channel, where the metered dose can be inhaled by the patient inhaling from the mouthpiece of the device. Examples of commercially available devices of this type are TURBUHALER ™ (AstraZeneca), TWISTHALER ™ (Schering) and CLICKHALER ™ (Innovata).

[0152] A further method of delivering an inhalable dry powder composition is metered doses of the composition delivered in capsules (one dose per capsule), which are then introduced into the inhalation device, typically by the patient as needed. The device has means for tearing, piercing or other opening of the capsule so that the dose can be carried into the patient's lungs when inhaled from the device's mouthpiece. Examples of such devices on the market include ROTAHALER ™ (GlaxoSmithKline) and HANDIHALER ™ (Boehringer Ingelheim).

[0153] Pressure aerosol compositions suitable for inhalation may be a suspension or solution and may contain a compound of formula (I) or a pharmaceutically acceptable salt thereof and a suitable propellant, such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, especially 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 additional excipients of the composition well known in the art, such as surfactants e.g. oleic acid, lecithin or oligomilic acid or a derivative thereof, e.g. as described in WO 94/21229 and WO 98/34596 (Minnesota Mining and Manufacturing Company) and cosolvents e.g. ethanol. Pressure compositions will generally be canned (e.g. aluminum can) closed by a valve (e.g. valve) and equipped with an actuator equipped with a mouthpiece.

[0154] Ointments, creams and gels may be, for example, formulated with an aqueous or oily base with the addition of a suitable thickening and / or gelling agent and / or solvents. Such substrates may thus, for example, include water and / or oil, such as liquid paraffin or vegetable oil, such as peanut oil or castor oil, or a solvent such as polyethylene glycol. Thickening agents and gelling agents that can be used according to the nature of the substrate include soft paraffin, aluminum stearate, cetostearyl alcohol, polyethylene glycols, sheep wool fat, beeswax, carboxypolymethylene and cellulose derivatives, and / or glyceryl monostearate and / or nonionic agents emulsifying.

[0155] Lotions may be formulated with an aqueous or oily base and will generally also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents or thickening agents.

[0156] Powders for external application may be made with the help of any suitable powder base, for example talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also containing one or more dispersing agents, solubilizing agents, suspending agents or preservatives.

[0157] The compounds of formula (I) and their pharmaceutically acceptable salts can be, for example, formulated for transdermal delivery by patching or other equipment (e.g., compressed gas devices) that deliver the active ingredient to the skin.

[0158] For buccal administration, the compositions may take the form of conventional tablets or lozenges.

[0159] The compounds of formula (I) and their pharmaceutically acceptable salts may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

[0160] The compounds of formula (I) and their pharmaceutically acceptable salts may also be formulated for parenteral administration by bolus injection or continuous infusion and may be provided in unit dosage form, for example as ampoules, vials, low-volume infusions or filled syringes, or in multi-dose containers with an added preservative. The compositions may take such forms as solutions, suspensions or emulsions in aqueous or non-aqueous vehicles, and may contain formulatory agents such as antioxidants, buffers, antimicrobials and / or agents that regulate osmotic pressure. Alternatively, the active ingredient may be in the form of a powder for reconstitution with a suitable excipient prior to use, e.g. sterile pyrogen-free water. The dry solid form can be prepared by introducing a sterile powder aseptically into individual sterile containers or by introducing a sterile solution aseptically into each container and lyophilizing.

[0161] The compounds of formula (I) and their pharmaceutically acceptable salts may also be formulated with vaccines as adjuvants to modulate their activity. Such compositions may contain the antibody (antibodies) or antibody fragment (s) or antigenic component comprising, but not limited to protein, GOUT, live or dead bacteria and / or viruses or virus-like particles, together with one or more ingredients with adjuvant activity including, but not limited to aluminum salts oil and water emulsions, 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.

[0162] The compounds of formula (I) and their pharmaceutically acceptable salts can be used alone or in combination with other therapeutic agents. The compounds of formula (I) and their pharmaceutically acceptable salts and other pharmaceutically active agent (s) may be administered together or separately, and when administered separately, administration may occur simultaneously or sequentially, in any order. The amounts of the compound of formula (I) or a pharmaceutically acceptable salt (s) thereof and other pharmaceutically active agent (s) and the relative times of administration will be chosen so as to achieve the desired combined therapeutic effect. Administration of a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof with other therapeutic agents can be accomplished by co-administration in a unitary pharmaceutical composition comprising both compounds, or in separate pharmaceutical compositions, each of which includes one of the compounds. Alternatively, the combination can be administered separately in a sequential manner in which one therapeutic agent is administered first and the next one as a second, or vice versa. Such subsequent administration may be near in time or distant in time.

[0163] The compounds of formula (I) and pharmaceutically acceptable salts thereof can be used in combination with one or more agents useful in the prevention or treatment of viral infections. Examples of such agents include, without limitation; polymerase inhibitors such as those disclosed in WO 2004/037818-A1 as well as those disclosed in WO 2004/037818 and WO 2006/045613; JTK-003, JTK-019, NM-283, HCV-796, R-803, R1728, R1626, as well as disclosed in WO 2006/018725, WO 2004/074270, WO 2003/095441, US2005 / 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 1065213, 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 BILN2061 protease inhibitors, VX-950, SCH503034; and similar means; nucleoside and nucleotide reverse transcriptase inhibitors, such as zidovudine didanosine, lamivudine, zalcitabine, abacavir, stavidine, adefovir adefovir dipivoxil, fozivudine, todoksil, emtricitabine alovudine, amdoxovir, elvucitabine and similar agents; non-nucleoside reverse transcriptase inhibitors (including an agent having antioxidant activity, such as an immunocal oltipraz etc.), such as nevirapine delavirdine, efavirenz, loviride, immunokal, oltipraz, capravirine, TMC-278, TMC-125, etravirine, and similar measures; entry inhibitors such as enfuvirtide (T-20), T1249, 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 measures; chemokine receptor inhibitors, such as wikriwirok (Sch-C), Sch-D, TAK779, Marawirok (UK-427.857), TAK449, as also disclosed 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 measures; neuraminidase inhibitors, such as CS-8958, zanamivir, oseltamivir, peramivir and similar agents; ion channel blockers, such as amantadine or rimantadine and similar agents; and interfering RNA and antisense oligonucleotides, and such as ISIS-14803 and similar agents; antiviral agents with unspecified mechanism of action, for example disclosed in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin, and similar measures. The compounds of formula (I) and their pharmaceutically acceptable salts can also be used in combination with one or more other agents that may be useful in the prevention or treatment of viral infections, for example immunotherapy (e.g. interferon or other cytokines / chemokines, cytokine / chemokine receptor modulators, cytokine agonists or antagonists, and similar agents); and therapeutic vaccines, anti-fibrotic agents, anti-inflammatory agents such as corticosteroids or NSAID (nonsteroidal anti-inflammatory agents) and similar agents.

[0164] The compounds of formula (I) and 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 an allergic disease, inflammatory disease, autoimmune disease, for example; for antigenic immunotherapy, antihistamines, steroids, NSAIDs, bronchodilators (e.g. beta 2 agonists, adrenergic agonists, anticholinergics, theophylline), methotrexate, leukotriene modulators and similar agents; for therapy with monoclonal antibodies such as anti-IgE, anti-TNF, anti-IL-5, anti-IL-6, anti-IL-12, anti-IL-1 and similar agents; for receptor therapies e.g. entanercept and similar means; for non-specific antigenic immunotherapies (e.g. interferon or other cytokines / chemokines, cytokine / chemokine receptor modulators, cytokine agonists or antagonists, TLR agonists and similar agents).

[0165] The compounds of formula (I) and their pharmaceutically acceptable salts can be used in combination with one or more other agents, which may be useful in preventing or treating cancer, for example chemotherapeutic agents, such as alkylating agents, topoisomerase inhibitors, antimetabolites anti-mitotic agents, kinase inhibitors and similar agents; monoclonal antibody therapy such as trastuzumab, gemtuzumab and other similar agents; and hormone therapy like tamoxifen, goserelin and similar agents.

[0166] The pharmaceutical compositions of the invention may also be used alone or in combination with at least one other therapeutic agent in other therapeutic fields, for example gastrointestinal disease. The compositions of the invention may also be used in combination with gene replacement therapy. [0167] The disclosure includes, in a further aspect, a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with at least one other therapeutically active agent.

[0168] The combinations indicated above may conveniently be provided for use in the form of a pharmaceutical composition, and thus pharmaceutical compositions comprising the combination as defined above together with at least one pharmaceutically acceptable diluent or carrier thereof are another aspect of the disclosure.

[0169] A therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof will depend on a number of factors. For example, the factors taken into account are the species, age and weight of the recipient, the exact condition requiring treatment and its severity, the nature of the composition and the route of administration. The therapeutically effective amount should ultimately depend on the attending physician. Regardless, an effective amount of a compound of the present invention for treating people suffering from the disease in general should be in the range of 0.0001 to 100 mg / kg body weight of the recipient per day. More often, the effective amount should be in the range of 0.001 to 10 mg / kg body weight per day. So, for an adult weighing 70 kg, some example of an actual daily amount would normally be 7 to 700 mg. For intranasal and inhalational routes, typical doses for an adult weighing 70 kg should be in the range of 1 microgram to 1 mg per day. This amount may be administered in a single daily dose or in multiple (such as two, three, four, five or more) sub-doses per day so that the total daily dose is the same. The effective amount of a pharmaceutically acceptable salt of the compound of formula (I) can be determined as a proportion relative to the effective amount of the compound of formula (I) or relative to its pharmaceutically acceptable salt per se. Similar dosages should be appropriate for the treatment of other conditions mentioned herein. [0170] Compounds of formula (I) and pharmaceutically acceptable salts thereof may also be administered at any appropriate frequency, e.g. 1-7 times a week. The exact dosage regimen will of course depend on factors such as the therapeutic indication, the age and condition of the patient, and the particular route of administration chosen.

[0171] Pharmaceutical compositions may be offered in unit dosage forms containing a predetermined quantity of active ingredient per unit dose. Such a unit may contain, as a non-limiting example, 0.5 mg to 1 g of a compound of formula (I) or a pharmaceutically acceptable salt thereof, depending on the condition being treated, the route of administration, and the age, weight and condition of the patient. Preferred unit dosage compositions are those containing the daily dose or sub-dose, as mentioned above, or a corresponding portion thereof, of the active ingredient. Such pharmaceutical compositions can be prepared by any of the methods well known in the pharmaceutical art.

[0172] Thus, there is further provided a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable diluents or carriers.

[0173] According to the disclosure, there is also provided a method of making such a pharmaceutical composition which comprises mixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable diluents or carriers.

[0174] Throughout the description and the claims that follow it, unless the context otherwise requires, the word "include / include" and variations such as "includes / includes" and "including / containing" will be understood to mean the inclusion of a specific number an integer or stage or group of integers and not as exclusion of any other integer or stage or group of integers or stages.

[0175] The compounds of formula (I) and their salts can be prepared according to the methodology described below, which is a further aspect of this disclosure.

[0176] According to the disclosure, there is provided a method for preparing a compound of formula (I), which method comprises deprotecting a compound of formula (II):

<img file="PL3000813T3_D0005.tif" />

in which R¹, min are as previously defined herein for the compound of formula (I) and R² means C1-6alkyl, and then, when required, carrying out one or more of the following optional steps:

(i). removal of any necessary protecting group;

(ii). salt formation of the compound thus formed.

[0177] For example, the compound of formula (II) is dissolved in a suitable solvent in the presence of a solution of a suitable acid, for example a solution of hydrogen chloride in

1,4-dioxane and mixed at a suitable temperature, e.g., ambient temperature, for a suitable period of time, e.g., 12-24 hours. The solvent is removed under reduced pressure and the residue is dissolved in a suitable solvent, e.g. methanol, and added to an ion exchange cartridge, e.g. an aminopropyl cartridge for SPE. The cartridge is eluted with a suitable solvent, for example methanol, and the solvent removed to give the compound of formula (I).

[0178] The compound of formula (II) can be produced by reacting the compound of formula (III):

<img file="PL3000813T3_D0006.tif" />

in which R¹ and m are as previously defined for the compound of formula (I), R² has the meaning as previously defined herein for the compound of formula (II) and X is a leaving group, for example a halogen group such as bromine or chlorine, with a compound of formula (IV):

<img file="PL3000813T3_D0007.tif" />

wherein n is as defined for a compound of formula (I).

[0179] For example, a compound of formula (III), a compound of formula (IV) and a suitable base, for example N, N-diisopropylethylamine, is dissolved in a suitable solvent, for example DMF, and heated at a suitable temperature, for example -60 ° C for a suitable period of time, e.g. 46-50 hours. If desired, additional compound of formula (IV) and base are added and the reaction mixture is heated at a suitable temperature, e.g. 50-60 ° C for a suitable period of time, e.g. 46-50 hours. The product is then extracted from the reaction mixture using traditional means, for example, by partitioning between the appropriate organic solvent and water, followed by isolation of the organic phase and removal of the solvent, and purification as required.

[0180] The compound of formula (III) can be produced by reacting a compound of formula (V), for example a salt of a compound of formula (V), such as a trifluoroacetate salt:

<img file="PL3000813T3_D0008.tif" />

where R¹ has the meaning as hereinbefore defined for the compound of formula (I) and R² has the meaning as hereinbefore defined for the compound of formula (II), with the compound of formula (VI):

^ΒΓΧ^χ (VI) wherein m is as previously defined herein for a compound of formula (I) and X is as previously defined for a compound of formula (III).

[0181] For example, the trifluoroacetate salt of the compound of formula (V) and a suitable base, e.g. potassium carbonate, are suspended in a suitable solvent, e.g. DMF, and heated to a suitable temperature, e.g. 50-60 ° C, suitable atmosphere, e.g. nitrogen atmosphere, for a suitable period of time, e.g. 20-120 minutes. The mixture is cooled to a suitable temperature, e.g. ambient temperature, and the compound of formula (VI) is added and stirring is continued at ambient temperature for a suitable period of time, e.g. 18-24 hours. The solvent is evaporated under reduced pressure and the residue is partitioned between a suitable solvent, for example DCM, and water. The crude product is then isolated from the organic phase and purified by traditional techniques such as column chromatography.

[0182] Alternatively, a compound of formula (II) can be prepared by reacting a compound of formula (V), for example a salt of a compound of formula (V), such as a trifluoroacetate salt, of a compound of formula (VI) wherein X is bromine , and the compound of formula (IV) as a "one-pot" process.

[0183] For example, the trifluoroacetate salt of the compound of formula (V) is dissolved in a suitable solvent, for example DMF, and a suitable base, for example potassium carbonate, is added. The reaction mixture is stirred at a suitable temperature, e.g. 45-60 ° C, in a suitable atmosphere, e.g. nitrogen, for a suitable period of time, e.g. 1-2 hours, and then cooled to a suitable temperature, e.g. ambient temperature. The compound of formula (VI) is then added, wherein X is bromine and, after stirring for a suitable period of time, e.g. 40-60 minutes, the compound of formula (IV) and the corresponding base, e.g. triethylamine, are added in a suitable solvent, e.g. DMF. The reaction mixture is then stirred for a suitable period of time, e.g. 12-24 hours. The solvent is removed and the residue is partitioned between a suitable organic solvent, for example dichloromethane, and water. The crude product of formula (II) is isolated using traditional means and purified by, for example, chromatography.

[0184] A salt of the compound of formula (V) can be prepared by deprotecting a compound of formula (VII):

<img file="PL3000813T3_D0009.tif" />

in which R¹ has the meaning as hereinbefore defined for the compound of formula (I), R² has the meaning as hereinbefore defined for the compound of formula (II) and P is a protecting group, e.g. tetrahydro-2H-pyran-2-yl, in the presence of a suitable acid, e.g. trifluoroacetic acid.

[0185] For example, a suitable acid, for example trifluoroacetic acid, is added to a solution of the compound of formula (VII) in a suitable solvent, for example methanol. The mixture is stirred at a suitable temperature, e.g. ambient temperature, for a suitable period of time, e.g. 48-72 hours. The reaction mixture is then concentrated under reduced pressure before dilution with a suitable solvent, for example ethyl acetate. The resulting mixture is filtered and washed with a small amount of a suitable solvent, for example ethyl acetate, until the filtrate becomes colorless. The residue is air-dried and then under reduced pressure to give a salt of the compound of formula (V). The filtrate can be concentrated and this concentrate is diluted with a small amount of a suitable solvent, for example ethyl acetate, then filtered and dried to give a second crop of the salt of the compound of formula (V).

[0186] A salt of a compound of formula (V), for example a trifluoroacetate salt, can also be prepared by reacting a compound of formula (IX):

<img file="PL3000813T3_D0010.tif" />

in which R¹ has the meaning as hereinbefore defined for the compound of formula (I) and P is as defined hereinbefore for the compound of formula (VII) with a suitable halogenating agent, e.g. N-bromosuccinimide, followed by reaction with an alkoxide anion, e.g. methanol anion followed by isolation in the presence of a suitable acid, for example trifluoroacetic acid.

For example, to a solution of a crude compound of formula (IX) in a suitable anhydrous solvent, e.g. anhydrous chloroform, at a suitable temperature, e.g. ambient temperature, a suitable halogenating agent, e.g. N-bromosuccinimide, is added portionwise for a suitable period of time for example 5 minutes. The solution is stirred at a suitable temperature, e.g. ambient temperature, for a suitable period of time, e.g. 25-35 minutes. The reaction mixture is then washed with water and the organic phase is dried by, for example, passing through a hydrophobic frit and concentrating under reduced pressure. The resulting solid is dissolved in a suitable anhydrous solvent, e.g. anhydrous methanol, and a suitable alcoholate, e.g., a solution of sodium methoxide in methanol, is added at a suitable temperature, e.g., ambient temperature, under an inert atmosphere, e.g., nitrogen. The reaction mixture is heated at a suitable temperature, e.g. 60-70 ° C, with an attached condenser, for a suitable period of time, e.g. 12-18 hours. The reaction mixture is then cooled and concentrated under reduced pressure. The residue is taken up in a suitable solvent, e.g. ethyl acetate, and poured into a suitable aqueous medium, e.g. saturated aqueous ammonium chloride solution. The organic layer is separated and washed further with water, dried, e.g. over magnesium sulfate, filtered and concentrated under reduced pressure. To a solution of this substance in a suitable anhydrous solvent, such as anhydrous methanol, a suitable acid, e.g. trifluoroacetic acid, is added at a suitable temperature, e.g. ambient temperature. The reaction mixture is stirred for a suitable period of time, e.g. 25-35 hours, and concentrated under reduced pressure to obtain a compound of formula (V).

[0188] The compound of formula (VII) can be produced by reacting the compound of formula (VIII):

<img file="PL3000813T3_D0011.tif" />

in which R¹ has the meaning as hereinbefore defined for the compound of formula (I), P is as defined hereinbefore for the compound of formula (VII) and Q is a halogen atom, for example a bromine atom, with an alkoxide anion, for example a methanolate anion.

For example, a solution of the compound of formula (VIII) in a suitable solvent, e.g. methanol, is heated to reflux with a solution of the appropriate alkoxide, e.g. sodium methoxide, in a suitable solvent, e.g. methanol, for a suitable period time, for example 4-5 hours. The reaction mixture is concentrated under reduced pressure and partitioned between a suitable organic solvent, e.g. ethyl acetate, and a suitable aqueous medium, e.g. saturated aqueous ammonium chloride solution. The organic phase is separated, washed, for example with brine, and dried, for example by passing through a hydrophobic frit. The solvent is then removed under reduced pressure to give a compound of formula (VII).

[0190] The compound of formula (VIII) can be produced by reacting the compound of formula (IX) with a suitable halogenating agent such as N-bromosuccinimide.

[0191] For example, the compound of formula (IX) is dissolved in a suitable solvent, for example chloroform, and cooled to a suitable temperature, for example, 00.5 ° C. A suitable halogenating agent, such as N-bromosuccinimide, is added to this solution, keeping the temperature below about 3 ° C. The solution is stirred at a suitable temperature, e.g. 2-3 ° C, for a suitable period of time, e.g. 3045 minutes, then allowed to warm to a suitable temperature, e.g., ambient temperature, and mixed for a suitable period of time, e.g., 5-7 hours. The reaction mixture is then washed with water and the organic phase is dried and separated from the aqueous phase using, for example, a hydrophobic frit. Then the organic solvent is removed and the crude product is purified by, for example, chromatography to give a compound of formula (VIII).

[0192] A compound of formula (IX) wherein R¹ is C1-6alkoxy can be prepared by reacting a compound of formula (X):

<img file="PL3000813T3_D0012.tif" />

wherein P is as previously defined herein for a compound of formula (VII) and T is a suitable leaving group, e.g. a halogen atom, e.g. a chlorine atom or a fluorine atom, with a solution of the compound of formula (XIII):

R¹-M (XIII) in which R¹ is C1-6alkoxy and M is a ligand of the corresponding alkali metal, such as sodium, prepared in the solvent of formula (IIIS):

R¹-H (XIIIS) in which the R group¹ in the compound of formula (XIII) is the same as group R¹ in the solvent of formula (XIIIS).

[0193] For example, a compound of formula (XIII), such as sodium t-butoxide, is added to the solvent of formula (XIIIS). The mixture is stirred until homogeneous, then the compound of formula (VII) is added. The reaction mixture is heated to a suitable temperature, e.g. 100 ° C, for a suitable period of time, e.g. 1218 hours. The solvent is generally removed under reduced pressure and the residue is partitioned between a suitable solvent, for example diethyl ether and water. The organic phase is separated off and the aqueous phase is again extracted with another solvent. The organic layers are then separated, combined, dried with a suitable drying agent, for example anhydrous magnesium sulfate. The drying agent is removed by filtration and the product removed under reduced pressure from the product to give a compound of formula (IX) in which R¹ is C1-6alkoxy.

[0194] A compound of formula (IX) wherein R¹ a C1-6alkylamino group can be prepared by reacting a compound of formula (X) with a compound of formula (XIV):

R¹-H (XIV) in which R¹ is a C1-6alkylamino group.

[0195] For example, a compound of formula (XIV) is added to a solution of a compound of formula (X) in a suitable anhydrous solvent, for example anhydrous ethylene glycol, at a suitable temperature, for example ambient temperature, in a suitable inert atmosphere, for example a nitrogen atmosphere . The reaction mixture is heated at a suitable temperature, e.g. 110-130 ° C, for a suitable period of time, e.g. 12-18 hours. The reaction mixture is then cooled to a suitable temperature, e.g. ambient temperature, diluted with a suitable solvent, e.g. ethyl acetate, and washed with water. The organic layer is dried with a suitable drying agent, for example anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give a compound of formula (IX) in which R¹ is a C1-6alkylamino group.

[0196] The compound of formula (X) can be produced by reacting the compound of formula (XI): v ^p (XI) wherein P is as previously defined herein for a compound of formula (VII) and T is as previously defined for a compound of formula (X) and V is a suitable leaving group, e.g. for example a chlorine atom with an alcoholic ammonia solution, for example a solution of ammonia in isopropyl alcohol.

[0197] For example, the compound of formula (XI) is heated with an alcoholic ammonia solution, for example a 2M solution of ammonia in isopropyl alcohol, at a suitable temperature, for example 50-60 ° C, for a suitable period of time, for example 5-6 hours. The reaction mixture is then allowed to stand at a suitable temperature, e.g. ambient temperature, for a suitable period of time, e.g. 12-18 hours. Another amount of an alcoholic ammonia solution, for example a 2M solution of ammonia in isopropyl alcohol, is added to break the resulting cake and the reaction mixture is heated for a further period of time, e.g. 8-10 hours, until the reaction is complete. Water is added to the reaction mixture and the solid is removed by filtration, washed with a suitable washing agent, e.g. a mixture of isopropyl alcohol and water, and then dried, e.g. by air-drying under suction, to give a first crop of formula (X). The filtrate is allowed to stand for a further period of time, e.g. 12-18 hours, and the resulting second crop of the compound of formula (X) is isolated by filtration and dried. [0198] The compound of formula (X) can also be produced by reacting the compound of formula (XII):

<img file="PL3000813T3_D0013.tif" />

wherein T is as previously defined for a compound of formula (X) and V is as previously defined for a compound of formula (XI) herein with a compound of formula (XV):

P^AT-H (XV) in which P^AT is a suitable precursor for the protecting group P, for example 3,4-dihydro-2H-pyranyl, followed by reaction with an alcoholic ammonia solution, for example a solution of ammonia in isopropyl alcohol.

[0199] For example, p-toluenesulfonic acid monohydrate is added to a solution of the compound of formula (XII) in a suitable anhydrous solvent, for example anhydrous ethyl acetate. The reaction mixture is heated to a suitable temperature, e.g. 50-60 ° C, and the compound of formula (XV) is added. The reaction mixture is stirred at a suitable temperature, e.g. 50-60 ° C, for a suitable period of time, e.g. 1-2 hours, and the solvent removed under reduced pressure. A suspension of the resulting solid in an alcoholic ammonia solution, e.g. a 2M solution of ammonia in isopropyl alcohol, is heated in a suitable inert atmosphere, e.g. nitrogen, at a suitable temperature, e.g. 60-70 ° C, for a suitable period of time, e.g. 4 -5 hours with connected cooler. The reaction mixture is poured into water and allowed to cool for a suitable period of time, e.g. 12-18 hours. The resulting precipitate is isolated by filtration and dried to give the compound of formula (X).

[0200] The compound of formula (X) can also be produced by reacting the compound of formula (XIA):

<img file="PL3000813T3_D0014.tif" />

wherein T is a fluorine atom, with a suitable safener, for example a silylating agent such as N, O-bis (trimethylsilyl) acetamide, followed by reaction of the protected compound of formula (XIA) with a compound of formula (XVE):

P^AT-E (XVE) in which P^AT is a suitable precursor for the protecting group P, for example 3,4-dihydro-2H-pyranyl, and E is an acyloxy group, for example acetate.

For example, a suitable safener, e.g. N, O-bis (trimethylsilyl) acetamide, is added with stirring to a suspension of a compound of formula (XIA), e.g. 2-fluoro-1H-purin-6-amine, in a suitable anhydrous solvent, e.g. anhydrous acetonitrile, and the resulting mixture is heated to reflux for a suitable period of time, e.g. 2-3 hours. The reaction mixture is then cooled to a suitable temperature, e.g. 0-5 ° C. A solution of the compound of formula (XVE), e.g. tetrahydropyranyl acetate, in a suitable anhydrous solvent, e.g. anhydrous acetonitrile, is then slowly added, followed by dropwise addition of Lewis acid, e.g. trimethylsilyl trifluoromethanesulfonate. The reaction temperature is adjusted to a suitable temperature, e.g. 8-15 ° C, and stirring is maintained for a further period of time, e.g. 1-2 hours. The mixture is then mixed with 1 M sodium carbonate. The organic layer is cooled while stirring to 0 ° C. The precipitated solid is then collected by, for example, filtration and dried.

[0202] The compound of formula (XI) can be produced by reacting the compound of formula (XII) with the compound of formula (XV).

[0203] For example, a suitable organic solvent, for example ethyl acetate, and then p-toluenesulfonic acid are added to the compound of formula (XII). The mixture is heated to a suitable temperature, e.g. 50-60 ° C, and then added

3,4-dihydro-2H-pyran. The reaction mixture is then heated to a suitable temperature, e.g. 50-60 ° C, for a suitable period of time, e.g. 4-5 hours. The solvent is then removed from the reaction mixture under reduced pressure to give a compound of formula (XI).

Abbreviations [0204] The following list gives definitions of some of the abbreviations used herein. It should be understood that the list is not exhaustive, but the meaning of the abbreviations not defined below will be apparent to those skilled in the art.

DCM

DMF

DMSO

EtOAc

Et 2 O

HCI

HPLC

ISCO Companion

MDAP HPLC

SPE

MeOH min

The stripped

TFA iPr t-Bu

ms

Ac n-Bu

ph

tp

Dichloromethane N, N-Dimethylformamide Dimethyl sulfoxide Ethyl acetate Diethyl ether Hydrochloric acid High performance liquid chromatography

Automated flash chromatography device with fraction analysis by UV absorption available from Presearch Limited, Basingstoke, Hants., RG24 8PZ, UK

Reverse phase HPLC on a C18 column using a two-solvent gradient and electrospray mass spectrometry analysis

Solid phase extraction. Methanol minutes

Removal of solvent under reduced pressure. Trifluoroacetic acid iso-Propyl tert-Butyl Mesyl

Acetyl n-Butyl Phenyl room temperature [0205] The synthesis methods described herein above are shown in Scheme 1.

Diagram 1

<img file="PL3000813T3_D0015.tif" />

[0206] Typical reaction conditions for each of the synthetic steps in Scheme 1 are as follows:

A Dihydropyran / paratoluene sulfonic acid, e.g. 50 ° C for 3-6 hours.

A1: Dihydropyran / paratoluene sulfonic acid, e.g. 50 ° C for 1 hour, then ammonia / iPrOH, e.g. 60 ° C for 4 hours, then add water and cool to ambient temperature over 12-18 hours.

A2 BSA in MeCN, heat to reflux, cool to 0 ° C, then THP acetate in MeCN, heat to 10 ° C, then NaHCO3 (aq.).

B Ammonia / iPrOH, e.g. 50 ° C for 5 hours, then ambient temperature for 12-18 hours, then 50 ° C for 9 hours.

C For X = NH, R^AND = C1-6alkyl: R.^ANDNH2 / ethylene glycol e.g. 120 ° C for 12-18 hours. For Z = O, R^AND = C1-6alkyl: R.^ANDONa / BuOH / dimethoxyethane e.g. 93-110 ° C for 12-18 hours.

C1 NBS in CHCl3 e.g. 0-5 ° C for 30 minutes, then ambient temperature for 0.5-1 hour, then e.g. NaOMe / methanol under N2 / 60-70 ° C / 12-18 hours, then TFA / MeOH e.g. ambient temperature for 18-65 hours.

D NBS in CHCl3 e.g. 0-5 ° C for 30 minutes, then ambient temperature for 3648 hours.

E NaOMe / MeOH e.g. heat to reflux 4-6 hours.

F TFA / MeOH e.g. ambient temperature for 18-65 hours.

G K2CO3 / DMF, then 50 ° C for 1-1.5 hours, then add (VI), stir minutes, then add (IV) / Et3N, then ambient temperature for 18 hours.

G1 K2CO3 / DMF, then 50 ° C under N2 atmosphere for 30 minutes, then ambient temperature, add (VI), stir for 20 hours.

G2: Solution in DMF with N, N-diisopropylethylamine, then 50 ° C for 48 hours, then add more (IV), then a further 50 ° C for 48 hours.

H: HCl / methanol, then ambient temperature for 18 hours.

[0207] Compounds of formulas (IV), (VI), (XIA), (XII), (XIII), (XIV) and (XV) are either known in the literature or are commercially available, for example from Sigma-Aldrich , UK, or may be prepared by analogy with known procedures, for example disclosed in standard references of synthetic methodology, such as J. March, Advanced Organic Chemistry, 6th Edition (2007), WileyBlackwell, or Comprehensive Organic Synthesis (Trost BM and Fleming I ., (ed.), Pergamon Press, 1991).

[0208] Examples of other protecting groups that can be used in the synthetic routes described herein and methods for their removal can be found in TW Greene "Protective Groups in Organic Synthesis", 4th edition, J. Wiley and Sons, 2006.

[0209] Traditional heating and cooling methods may be used for any of the previously described reactions or processes, for example temperature controlled oil baths or temperature controlled heating blocks, and ice / salt or dry ice / acetone baths. Traditional isolation methods may be used, for example extraction from or into aqueous or non-aqueous solvents. Traditional methods for drying organic solvents, solutions or extracts, such as shaking with anhydrous magnesium sulfate or anhydrous sodium sulfate, or passing through a hydrophobic frit can be used. Traditional purification methods, for example crystallization and chromatography, for example silica chromatography or reverse phase chromatography can be used as required. Crystallization can be carried out using traditional solvents such as ethyl acetate, methanol, ethanol or butanol, or aqueous mixtures thereof. It should be understood that specific reaction temperature times can typically be determined by reaction monitoring techniques, for example thin layer chromatography and LC-MS.

[0210] Where appropriate, the individual isomeric forms of the compounds of the invention can be prepared as individual isomers using traditional procedures such as fractional crystallization of diastereomeric derivatives or chiral high performance liquid chromatography (chiral HPLC).

[0211] The absolute stereochemistry of the compounds can be determined using traditional methods such as X-ray crystallography.

[0212] Aspects of the invention are illustrated by reference to the following Examples, while they are not in any way limited by these examples.

General experimental details [0213] The compounds were named using chemical naming software

ACD / Name PRO 6.02 from Advanced Chemistry Developments Inc., Toronto, Ontario, M5H2L3, Canada.

[0214] The experimental details of the LCMS AD systems mentioned here are as follows:

System A [0215]

Column: 50mm x 2.1mm diameter internal, 1.7m Acquity UPLC BEH C18 Flow rate: 1 ml / min Temp .: 40 ° C

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using electrospray ionization in positive and negative modes with alternative scanning

Solvents: A: 0.1% v / v formic acid in water B: 0.1% v / v formic acid in acetonitrile

<td>Gradient:</td><td>Time (min)</td><td>AND%</td><td>B%</td>

<td></td><td>0</td><td>97</td><td>3</td>

<td></td><td>0.1</td><td>97</td><td>3</td>

<td></td><td>1.4</td><td>0</td><td>100</td>

<td></td><td>1.9</td><td>0</td><td>100</td>

<td></td><td>2.0</td><td>97</td><td>3</td>

System B [0216]

Column: 30 mm x 4.6 mm diameter internal, Sunfire Ci8 3.5 μm column

Flow rate: 3 ml / min

Temp: 30 ° C

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using electrospray ionization in positive and negative mode with alternative scanning

Solvents: A: 0.1% v / v solution of formic acid in water

B: 0.1% v / v solution of formic acid in acetonitrile

<td>Gradient:</td><td>Time (min)</td><td>AND%</td><td>B%</td>

<td></td><td>0</td><td>97</td><td>3</td>

<td></td><td>0.1</td><td>97</td><td>3</td>

<td></td><td>4.2</td><td>0</td><td>100</td>

<td></td><td>4.8</td><td>0</td><td>100</td>

<td></td><td>4.9</td><td>97</td><td>3</td>

<td></td><td>5.0</td><td>97</td><td>3</td>

System C [0217]

Column: 50 mm x 2.1 mm diameter internal, 1.7 μm Acquity UPLC BEH Ci8 Flow rate: 1 ml / min Temp: 40 ° C

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using electrospray ionization in positive and negative modes with alternative scanning

Solvents: A: 10 mM ammonium bicarbonate in water with a pH adjusted at 10 with ammonia solution

Gradient:

<td colspan="3">B: acetonitrile</td>

<td>Time (min)</td><td>AND%</td><td>B%</td>

<td>0</td><td>99</td><td>1</td>

<td>1.5</td><td>3</td><td>97</td>

<td>1.9</td><td>3</td><td>97</td>

<td>2.0</td><td>0</td><td>100</td>

System D [0218]

Column: 50 mm x 4.6 mm diameter internal, 3.5 μm XBridge Ci8 column Flow rate: 3 ml / min Temp: 30 ° C

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using electrospray ionization in positive and negative modes with alternative scanning

Solvents: A: 10 mM ammonium bicarbonate in water with a pH adjusted at 10 with ammonia solution

B: acetonitrile

<td>Gradient:</td><td>Time (min) 0</td><td>AND% 99</td><td>B% 1</td>

<td></td><td>0.1</td><td>99</td><td>1</td>

<td></td><td>4.0</td><td>3</td><td>97</td>

<td></td><td>5.0</td><td>3</td><td>97</td>

[0219] Chromatographic purification was typically carried out using pre-packed silica gel cartridges. Flashmaster II is an automated multi-user flash chromatography system available from Argonaut Technologies Ltd that uses disposable Solid Phase Extraction (SPE) cartridges (2 g to 100 g) in a normal phase arrangement. Provides four-component online mixing of solvents to enable gradient processes. Samples are placed in order using multifunctional open access software that manages solvents, flow rates, gradient profile and collection conditions. The system is equipped with a Knauer UV detector with variable wavelength and two Gilson FC204 fraction collectors enabling automated peak cutting, collection and tracking. [0220] Solvent removal using a nitrogen stream was carried out at 30-40 ° C on a GreenHouse Blowdown system available from Radleys Discovery Technologies Saffron Walden, Essex, CB11 3AZ, UK.

[0221] Spectra ¹H NMR was recorded in CDCl3 or DMSO-d6 on a Bruker DPX 400 or Bruker Avance DRX spectrometer, or Varian Unity 400 operating at 400 MHz. The internal standard used was tetramethylsilane or residual protonated solvent at 7.25 ppm for CDCl3 or 2.50 ppm for DMSO-d6.

[0222] Autopreparative HPLC coupled to the mass spectrometer was run under the conditions below. UV detection was an average signal from 210 nm to 350 nm and mass spectra were recorded on a mass spectrometer using electrospray ionization in positive and negative mode with alternative scanning.

Method A [0223] Method A was carried out on an XBridge C18 column (typically 150 mm x 19 mm ID, fill diameter 5 μm) at ambient temperature. The solvents used were:

A = 10 mM ammonium bicarbonate aqueous solution with a pH adjusted at 10 ammonia solution.

B = acetonitrile.

Method B [0224] Method B was carried out on a Sunfire C18 column (typically 150 mm x 30 mm diameter.

internal, filling diameter 5 μm) at ambient temperature. The solvents used were:

A = 0.1% v / v solution of formic acid in water B = 0.1% v / v solution of formic acid in acetonitrile.

Method C [0225] Method C was carried out on a Sunfire C18 column (typically 150 mm x 30 mm ID, fill diameter 5 μm) at ambient temperature. The solvents used were:

A = 0.1% v / v solution of trifluoroacetic acid in water B = 0.1% v / v solution of trifluoroacetic acid in acetonitrile.

Method D [0226] Method D was carried out on an Atlantis C18 column (typically 100 mm x 30 mm ID, fill diameter 5 μm) at ambient temperature. The solvents used were:

A = 0.1% v / v solution of formic acid in water B = 0.1% v / v solution of formic acid in acetonitrile.

Method E [0227] Method E was carried out on a Supelcosil ABZ + Plus column (typically 100 mm x 21.2 mm ID, packing diameter 5 μm) at ambient temperature. The solvents used were:

A = 0.1% v / v solution of formic acid in water B = acetonitrile: water 95: 5 + 0.05% formic acid

Examples

Intermediate 1: 2,6-Dichloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purine [0228]

<img file="PL3000813T3_D0016.tif" />

[0229] To 2,6-dichloropurine (25.0 g) (commercially available, for example, from Aldrich, UK) was added ethyl acetate (260 ml) followed by p-toluenesulfonic acid (0.253 g). The mixture was heated to 50 ° C and then 3,4-dihydro-2H-pyran (16.8 g) was added. The reaction mixture was then heated at 50 ° C for 4 hours. The reaction mixture was evaporated in vacuo to give the title compound as a yellow solid (36.9 g).

1H NMR (CDCl3): 8.35 (1H, s), 5.77 (1H, dd), 4.20 (1H, m), 3.79 (1H, m), 2.20-1.65 ( 6H,

m).

Intermediate 2: 2-Chloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0230]

<img file="PL3000813T3_D0017.tif" />

2,6-Dichloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purine (36.9 g) was heated with 2 M ammonia in isopropanol (250 ml) at 50 ° C for 5 ° C hours. After standing at ambient temperature overnight, another 2 M ammonia in isopropanol (100 mL) was added to break the resulting cake and the reaction mixture was heated for a further 9 hours to complete the reaction. Water (70 ml) was added to the reaction mixture and the yellow solid was filtered off. The solid was washed with isopropyl alcohol: water (5: 1 (v / v), 60 ml) and then air dried with suction to give a first crop. The filtrate was again filtered after standing overnight to separate the precipitate and both solids were dried in vacuo. The first wave was clean and the second wave material showed very little pollution (a separate 3.5 ppm wide signal not visible in the first wave) but was otherwise identical. First crop in the form of a solid (28.4 g), second crop in the form of a solid (3.42 g).

1H NMR (CDCl3): 8.01 (1H, s), 5.98 (2H, broad s), 5.70 (1H, dd), 4.16 (1H, m), 3.78 (1H, m ), 2.15-1.60 (6H, overlapping m).

Intermediate 2 (alternative method): 2-Chloro-9 - ('tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0232]

<img file="PL3000813T3_D0018.tif" />

[0233] To a solution of 2,6-dichloropurine (25 g) (commercially available, for example, from Aldrich, UK) in anhydrous ethyl acetate (200 ml) was added p-toluenesulfonic acid monohydrate (235 mg). The reaction mixture was heated to 50 ° C and 3,4-dihydro-2H-pyran (18.1 mL) was added once. The reaction mixture was allowed to stir at 50 ° C for 1 hour and the solvent removed under reduced pressure. A yellow solid was obtained. A suspension of this solid (~ 36 g) in 2.0 M ammonia in isopropanol (460 ml) was heated under nitrogen at 60 ° C for 4 hours with a condenser attached. The reaction mixture was poured into water (50 mL) and allowed to cool overnight. The precipitate was filtered off and dried on a rotary evaporator (60 ° C) for 30 minutes to give the title compound as an off-white solid, 31 g (93%, 2 steps).

MS calculated for (C10H12ClN5O)⁺ = 254, 256

MS found (electrospray): (M)⁺ = 254, 256 (3: 1) ¹H NMR ((CD3) 2SO): δ 8.43 (1H, s), 7.82 (2H, s), 5.55 (1H, dd), 4.00 (1H, m), 3.69 ( 1H, m), 2.21 (1H, m), 1.95 (2H, m), 1.74 (1H, m), 1.56 (2H, m).

Intermediate 3: 2- (Butyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0234]

<img file="PL3000813T3_D0019.tif" />

[0235] Sodium tert-butoxide (15.2 g) was added in portions to butan-1-ol (76 ml) (Note: the reaction mixture becomes warm). The above mixture was stirred until homogeneous (about 15 min), then 2-chloro-9 (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (10.0 g) was added to the resulting pale yellow solution. The reaction mixture was then heated to 100 ° C overnight. The reaction mixture was stripped of the solvent to remove as much of butan-1-ol as possible and then partitioned between diethyl ether and water. The diethyl ether phase was separated and the aqueous phase re-extracted with diethyl ether. The combined organic layers were dried over magnesium sulfate (anhydrous). The magnesium sulfate was filtered off and the filtrate was stripped of solvent to give a viscous brown oil which was azeotroped with toluene (3 times) and placed under high vacuum overnight, transferred to a new flask with dichloromethane and stripped off the solvent, placed under high vacuum to give the title compound as a brown glass (9.45 g).

1H NMR (CDCl3): 7.85 (1H, s), 5.92 (2H, broad s), 5.64 (1H, d), 4.32 (2H, t), 4.14 (1H, m ), 3.75 (1H, m), 2.10-1.95 (3H, overlapping m), 1.81-1.58 (5H, overlapping m),

1.50 (2H, m), 0.97 (3H, t).

Intermediate 4: 8-Bromo-2- (hutoxyl) -9- (tetrahydro-2-pyran-2-yl) -9-purple-6-amine [0236]

<img file="PL3000813T3_D0020.tif" />

2- (Butyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (9.45 g) was dissolved in chloroform (50 ml) and cooled to 0 ° C ( ice bath). To this solution, N-bromosuccinimide (6.07 g) was added in portions keeping the temperature below 3 ° C. This dark-green solution was obtained, stirred at 2.5 ° C for 30 min, after which it was allowed to warm to room temperature and then stirred for 6 hours. The reaction mixture was then washed with water (100 ml, twice). The organic phase was dried / separated using a hydrophobic frit and evaporated, and a dark brown gum was obtained, which was purified by silica chromatography (120 g) (ISCO) using a gradient elution of 0-50% ethyl acetate: cyclohexane, a pale yellow solid (8.37 g). 1H NMR (CDCl3): 5.61 (1H, dd), 5.49 (2H, broad s), 4.32 (2H, m), 4.17 (1H, m), 3.71 (1H, m ), 3.04 (1H, m), 2.11 (1H, wide d), 1.89 - 1.45 (6H, overlapping m), 1.50 (2H, m), 0.97 (3H , t).

Intermediate 5: 2- (Bioxy) -8- (ethyloxy) -9- (tetrahydro-2 / - / - pyran-2-yl) -9 / - pyrazine-6-amine [0238]

<img file="PL3000813T3_D0021.tif" />

[0239] 8-Bromo-2- (butyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6-amine (8.37 g) was heated to reflux with 25% sodium methoxide in methanol (14.4 ml) and methanol (65 ml) for 4.5 hours The reaction mixture was concentrated under reduced pressure and partitioned between ethyl acetate and saturated ammonium chloride solution. The organic phase was separated and the extraction into ethyl acetate repeated. The organic phases were combined and washed with brine (twice). After separation of the aqueous phase, the organic phase was passed through a sintered hydrophobic frit and evaporated, and a light brown gum was obtained, which was placed under a high vacuum, in which foam (7.52 g) which had dropped to give residual pressure (7.34 g) was generated at ambient pressure. and solidified over night, resulting in the formation of a yellow compound in the form of a yellow amorphous solid.

MS calculated for (C15H23N5O3)⁺ = 321

MS found (electrospray): (M + H)⁺ = 322

1H NMR (CDCl3): 5.50 (1H, dd), 5.17 (2H, broad s), 4.29 (2H, t), 4.12 (3H, s and 1H, m),

3.70 (1H, m), 2.77 (1H, m), 2.05 (1H, m), 1.82-1.63 (6H, overlapping m), 1.50 (2H, m) , 0.97 (3H, t).

Intermediate 6: 2- (Hi-oxvoxy) -8- (methyloxy) -9 // - purine-6-avinyl trifluoroacetate salt [0240]

<img file="PL3000813T3_D0022.tif" />

OH

F [0241] To a solution of 2- (butyloxy) -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-puri-6-amine (7.34 g) in methanol (100 ml ) trifluoroacetic acid (10 ml) was added. The mixture was stirred at ambient temperature over the weekend to give a suspension. The reaction mixture was concentrated to a small volume (thick suspension), then diluted with ethyl acetate (50 mL). The resulting suspension was filtered and washed with a small amount of ethyl acetate until the filtrate became colorless. The remaining solid was air dried and then in vacuo to give the title compound as a white solid (6.20 g). The previously obtained filtrate was concentrated to give a slurry, which was diluted with a small amount of ethyl acetate (10 ml), then filtered and dried as above. A second crop was isolated as a white solid (0.276 g). Both views were NMR identical.

MS calculated for (C10H15N5O2)⁺ = 237

MS found (electrospray): (M + H)⁺ = 238

1H NMR (CD3OD): 4.47 (2H, t), 4.15 (3H, s), 1.80 (2H, m), 1.50 (2H, m), 0.99 (3H, t) (exchangeable NH2, NH protons and COOH were not observed).

Intermediate 7: N²-Butyl-9- (tetrahydro-2H-pyran-2-yl) -9H-purine-2,6-diamine [0242]

<img file="PL3000813T3_D0023.tif" />

[0243] To a solution of 2-chloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (10 g) in anhydrous ethylene glycol (50 ml) at room temperature and under nitrogen in one n-butylamine (16 mL) was added in portions. The reaction mixture was heated at 120 ° C overnight. The reaction mixture was cooled to room temperature before it was diluted with ethyl acetate (150 ml) and washed with water (2 x 50 ml). The organic layer was dried over MgSO4, filtered and concentrated in vacuo. This gave the title compound as a green viscous oil (10.2 g), which was used in the next step without further purification.

MS calculated for (C14H22N6O)⁺ = 290

MS found (electrospray): (M + H)⁺ = 291 ¹H NMR ((CD3) 2SO): δ 7.8 (1H, s), 6.6 (2H, s), 6.2 (1H, t), 5.4 (1H, dd), 4.0 (1H, m), 3.6 (1H, m), 3.2 (2H, m), 2.2 (1H, m), 1.9 (1H, m), 1.8 (1H, m) , 1.7 (1H, m), 1.5 (2H, m), 1.4 (2H, m), 1.3 (2H, m), 0.9 (3H, t).

Intermediate 8: Salt of trifluoroacetic acid with N²-butyl-8- (methyloxy) -9H-purine-2,6-diamine [0244]

<img file="PL3000813T3_D0024.tif" />

[0245] For crude solution N²-butyl-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-2,6-diamine (about 10.2 g) in anhydrous chloroform (100 ml) at room temperature, N-bromosuccinimide (6, 3 g) for 5 min. The dark solution was allowed to stir at room temperature for 30 min. The reaction mixture was stirred

N '"O was washed with water (20 ml). The organic phase was passed through a hydrophobic frit and concentrated in vacuo. This resulted in a beige solid, which was dissolved in anhydrous methanol (100 mL) and a solution of sodium methoxide (25% by weight) was added in one portion at room temperature under nitrogen atmosphere. in methanol, 24 ml). The reaction mixture was heated at 65 ° C, with a condenser attached, overnight. The reaction mixture was cooled and concentrated in vacuo. The resulting orange residue was taken up in ethyl acetate (150 mL) and poured into a saturated aqueous solution of ammonium chloride (50 mL). The organic layer was separated and then washed with water (50 ml). The organic layer was dried over MgSO4, filtered and concentrated in vacuo. To this substance in anhydrous methanol (70 ml) at room temperature was added trifluoroacetic acid (7 ml) in one portion. The reaction mixture was stirred for 30 hours and concentrated in vacuo to give a dark brown solid. This was taken up in diethyl ether (20 mL) and triturated. The solid was filtered off to give the title compound as a beige solid (3.3 g, 35%, 4 steps).

MS calculated for (C10H16N6O)⁺ = 236

MS found (electrospray): (M + H)⁺ = 237 ¹H NMR ((CD3) 2SO): δ 13.3-12.3 (1H, br.m), 8.6-7.3 (2H, m), 4.05 (3H, s), 3.28 (2H, m),

1.52 (2H, m), 1.33 (2H, m), 0.89 (3H, t) (other interchangeable blurred protons). Intermediate 9: 2 - {[(1S) -1-Methylbutyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0246]

N ΐ

.....ABOUT ·!

Method A [0247] Sodium tert-butoxide (48.5 g, 505 mmol) was added portionwise to (S) -2-pentanol (185 mL) (commercially available, for example from Julich Chiral Solutions, Germany) at room temperature and mixed until homogeneous (Note: the reaction is exothermic). 2-chloro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (32 g, 126 mmol) was added and the reaction mixture was heated at 70 ° C for 72 hours. The reaction mixture was cooled to room temperature and partitioned between ethyl acetate (500 ml) and water (500 ml). The organic phase was washed with saturated sodium chloride solution (100 ml), dried (MgSO 4), filtered and evaporated. The residue was triturated with ether and the solid filtered off. The precipitate was washed again with ether and the filtrates combined and evaporated. The crude material (approximately 30 g) was dissolved in DMSO: methanol (1: 1) and purified by reverse phase column chromatography (C18) (330 g) using a 25-65% acetonitrile (+ 0.1% TFA) gradient ) -water (+ 0.1% TFA) through 8 column volumes, fractions were immediately neutralized with saturated aqueous sodium carbonate solution. The appropriate fractions were combined and partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution. The organic phase was dried by passing through a hydrophobic frit, filtered and evaporated to give the title compound as a pale cream foam (14.97 g).

LCMS (System B): tRET = 2.21 min; MH⁺ 306

Method B [0248] / Sodium m-butoxide (206 g, 2.144 mole) was added to (S) -2-pentanol (720 mL, 6.58 mole) (commercially available, for example from Julich Chiral Solutions, Germany) in 2 l round-bottom flask. The mixture was stirred at 50 ° C to dissolve all sodium / butoxide. 2-Fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (130 g, 548 mmol) was then added in portions over 5 minutes. After 3 hours, LCMS analysis indicated complete consumption of the starting material and the mixture was poured into ice / water (3 L), followed by extraction with methyl / eryl butyl ether. This caused an emulsion, and the mixture was filtered through Celite, and the organic phase was separated. The aqueous layer was then treated with solid NaCl and then re-extracted with tert-butyl methyl ether. The organic extracts were combined and washed with brine, dried over magnesium sulfate, filtered and then evaporated to give the title compound as a pale brown gum (158.59 g).

LCMS (System D): tRET = 2.65 min; MH⁺ 306

Intermediate 10: 8-Bromo-2 - {[(1S) -1-methylbutyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0249]

NH_?

N ^N

..: I, ..... / Br '' O '··! ^N [0250] N-Bromosuccinimide (12.16 g, 68.3 mmol) was added portionwise with stirring over 5 minutes to a solution of 2 - {[(1S) -1-methylbutyl] oxy} -9- (tetrahydro-2H- pyran-2-yl) -9H-purin-6-amine (14.9 g, 48.8 mmol) in chloroform (80 ml) at &lt;5 ° C under nitrogen. The reaction mixture was stirred at &lt;5 ° C for 5 hours, then washed with saturated sodium bicarbonate solution (80 ml), then water (80 ml). The foam was dissolved in DCM (50 ml) and washed with water (50 ml) followed by brine (50 ml). The combined aqueous phases were washed with DCM (50 mL). The combined organic layers were dried by passing through a hydrophobic frit and the solvent removed in vacuo to give the title compound as an orange foam (18.5 g). LCMS (System D): tRET = 3.06 min; MH⁺ 384/386

Intermediate 11: 2 - {[(1S) -1-Methylbutyl] oxy} -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0251] nh₂

N '^J:- ...^N . 1 J.^; °

HE ^h [0252] 8-Bromo-2 - {[(1S) -1-methylbutyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (7.1 g, 18 , 48 mmol) was dissolved in anhydrous methanol (70 ml) and a solution of sodium methoxide (25%) in methanol (8 ml) was added dropwise under a nitrogen atmosphere. The solution was heated to reflux at 90 ° C for 4 hours under nitrogen. Additional sodium methoxide in methanol (25% solution, 3 mL) was added and the reaction mixture was stirred at 60 ° C for a further 16 hours. An additional portion of sodium methoxide in methanol (25% solution, 5 mL) was added and the reaction mixture was stirred at 90 ° C for a further 7 hours. The solvent was removed by rotary evaporation and the crude product was partitioned between EtOAc (75 mL) and saturated ammonium chloride solution (75 mL). The organic layer was washed with brine (75 mL). The solvent was removed by rotary evaporation to give the title compound as a pale orange foam (6 g).

LCMS (System C): tRET = 1.14 min; MH⁺ 336, 337

Intermediate 12: 2 - {[(1S) -1-methylbutyl] oxy} -8- (methyloxy) -9H-purin-6-amine trifluoroacetate salt [0253]

<img file="PL3000813T3_D0025.tif" />

ΝΗ_Ζ

Ε ^Ν| '1' '' 'Ο Ν' [0254] 2 - {[(1S) -1-Methylbutyl] oxy} -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-Purin-6 -amine (6 g, 17.89 mmol) was dissolved in methanol (50 mL). Trifluoroacetic acid (20.67 mL, 268 mmol) was added dropwise and the mixture was stirred at 20 ° C for 72 hours under nitrogen. The solvent was removed in vacuo and the resulting solid was washed with ethyl acetate and filtered. The filtrate was stripped off and the residue was washed with ethyl acetate. The combined solid residue was dried in a vacuum oven for 2 hours to give the title compound as an off-white solid (5.3 g).

LCMS (System C): tRET = 0.76 min; MH⁺ 252, 253

Intermediate 13: 2- (Butyloxy) -9- (3-chloropropyl) -8- (methyloxy) -9H-purin-6-amine [0255]

ΝΗ, »! ».1" "" 'Ό' "'ν' Ν '

CI [0256] 2- (Butyloxy) -8- (methyloxy) -9H-purin-6-amine trifluoroacetate (4.7 g, 13.38 mmol) and potassium carbonate (4.62 g, 33.4 mmol) in anhydrous DMF (50 mL) was stirred and heated at 50 ° C, under nitrogen, for 75 min. The mixture was allowed to cool to room temperature then cooled to 0 ° C and 1-bromo-3-chloropropane (2.106 g, 13.38 mmol) was added. The mixture was stirred at 0 to 10 ° C for about 5 hours and then allowed to warm to room temperature and stirred for about 40 consecutive hours when LCMS showed about 70% of the desired product. The mixture was allowed to stand and the supernatant was pipetted and the solvent was evaporated on a rotary evaporator using a high vacuum at about 23 ° C. Chloroform and water were added to the combined residues while stirring, and the phases were separated using a hydrophobic sinter. The aqueous layer was extracted again with further portions of chloroform and the combined chloroform extracts evaporated under high vacuum at 23 ° C to give a yellow solid (2.798 g). This crude material was combined with a similar material obtained from two similar production methods (0.56 g and 0.995 g) and purified by column chromatography on silica using 2: 1 ethyl acetate / chloroform as the eluent to give the title compound as an off-white solid . (3.011 g).

LCMS (System D): tRET = 2.79 min; MH⁺ 314, 316

Intermediate 14: 2- (Butyloxy) -9- (4-chlorobutyl) -8- (methyloxy) -9H-purin-6-amine [0257] [0258] 2- (Butyloxy) -8- (methyloxy) - trifluoroacetate 9H-purin-6-amine (2 g, 5.69 mmol) and potassium carbonate (1.967 g, 14.23 mmol) were suspended in DMF (20 mL) and heated to 50 ° C under nitrogen for 30 min . The mixture was cooled to room temperature, 1-bromo-4-chlorobutane (0.656 mL, 5.69 mmol) was added and stirring was continued at room temperature for 20 hours. The solvent was evaporated under reduced pressure and the residue was partitioned between DCM (40 ml) and water (40 ml). The layers were separated using a hydrophobic frit and the aqueous layer was washed with DCM (10 mL). The combined organic extracts were concentrated in vacuo to give a crude material which was purified by silica chromatography using FlashMaster (70 g cartridge) using a gradient of 0-100% cyclohexane: ethyl acetate as eluent over 30 min. The product-containing fractions were combined and evaporated to give the title compound as a white solid (1.4 g).

LCMS (System D): tRET = 2.92 min; MH⁺ = 328, 330

Intermediate 15: 2- (Butyloxy) -9- (5-chloropentyl) -8- (methyloxy) -9H-purin-6-amine [0259] nh₃

Ν '.........^N.

&lt;O.

Cl [0260] 2- (Butyloxy) -8- (methyloxy) -9H-purin-6-amine trifluoroacetate (2 g, 5.69 mmol) and potassium carbonate (1.967 g, 14.23 mmol) were suspended in DMF (20 ml) and heated to 50 ° C, under nitrogen for 1 hour. The mixture was cooled to room temperature, 1-bromo-5-chloropentane (0.75 mL, 5.69 mmol) was added and stirring was continued at room temperature for 18 hours. The reaction mixture was partitioned between DCM (40 ml) and water (40 ml) and the layers were separated using a hydrophobic frit. The aqueous layer was re-extracted with DCM (10 mL) and the combined organics were washed with saturated lithium chloride solution, separated (hydrophobic frit) and concentrated in vacuo to give the title compound as a yellow oil (1.946 g).

LCMS (System B): tRET = 2.58 min; MH⁺ = 342, 344

Intermediate 16: 2- (Butyloxy) -9- (5-chlorohexyl) -8- (methyloxy) -9H-purin-6-amine [0261]

NH | l | ⁰ ·_ο· _K -isj

Cl [0262] To a solution of 2- (butyloxy) -8- (methyloxy) -9H-purine-6amine trifluoroacetate salt (3 g, 8.54 mmol) in DMF (30 mL) was added potassium carbonate (2.95 g, 21 , 35 mmol) and the mixture was stirred at 60 ° C for 1 hour under nitrogen. The mixture was then cooled to room temperature and 1-bromo-6-chlorohexane (1.27 mL, 8.54 mmol) was added and the reaction mixture was heated to 50 ° C and stirred overnight under nitrogen. The reaction mixture was diluted with water (about 50 ml) and extracted with ethyl acetate (2 x 70 ml). The combined organic extracts were dried (MgSO 4), filtered and the filtrate concentrated to give an orange oil (about 3.5 g). This material was dissolved in dichloromethane and purified using Flashmaster II (70 g aminopropyl cartridge) using a gradient of 0-100% ethyl acetate in cyclohexane over 60 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a yellow oil which solidified to a pale yellow solid (1.2 g).

LCMS (System D): tRET = 3.59 min; MH⁺ = 356, 358

Intermediate 17: A.²-Butyl-9- (3-chloropropyl) -8- (methyloxy) -9H-purine-2,6-diamine [0263]

<img file="PL3000813T3_D0026.tif" />

[0264] Trifluoroacetate N²-butyl-8- (methyloxy) -9H-purine-2,6-diamine (701 mg, 2.001 mmol) and potassium carbonate (690 mg, 4.99 mmol) were suspended in DMF (10 mL) and the mixture was heated at 50 ° C under nitrogen for 2 hours. The mixture was allowed to cool and then 1-bromo-3-chloropropane (198 μΐ, 2.002 mmol) was added and the reaction mixture was stirred at ambient temperature overnight. After 16 hours, the reaction mixture was partitioned between water and DCM (25 mL each). The aqueous phase was extracted with further DCM (2 x 20 mL). The combined DCM extracts were dried over magnesium sulfate and concentrated in vacuo to give the impure title compound as a pale yellow oil with a small amount of solid (0.76 g) present which was used without further purification. LCMS (System D): tRET = 2.75 min; MH⁺ = 313, 315

Intermediate 18: A²-Butyl-9- (4-chlorobutyl) -8- (methyloxy) -9H-purine-2,6-diamine [0265]

<img file="PL3000813T3_D0027.tif" />

[0266] Trifluoroacetate N²-butyl-8- (methyloxy) -9H-purine-2,6-diamine (5 g, 14.27 mmol) and potassium carbonate (4.93 g, 35.7 mmol) were suspended in DMF (40 mL ) and heated to 50 ° C under nitrogen for 30 min. The mixture was cooled to room temperature, 1-bromo-4-chlorobutane (1.645 mL, 14.27 mmol) was added and stirring was continued at room temperature for 20 hours. The solvent was concentrated in vacuo and the residue was partitioned between DCM (100 ml) and water (100 ml). The layers were separated using a hydrophobic frit and the aqueous phase reextracted with DCM (100 mL). The combined organic extracts were concentrated in vacuo and the residue purified by chromatography using a FlashMaster apparatus (silica cartridge 100 g) and a gradient of 0-25% DCM: methanol over 40 min. The desired fractions were combined and concentrated in vacuo to give the impure title compound as a yellow oil (5.1 g).

LCMS (System D): tRET = 2.88 min; MH⁺ = 327, 329

Intermediate 19: 9- (5-Chloropentyl) -2 - {[(1S) -1-methylbutyl] oxy} -8- (methyloxy) 9H-purin-6-amine [0267]

<img file="PL3000813T3_D0028.tif" />

[0268] 2- {[(1S) -1-methylbutyl] oxy} -8- (methyloxy) -9H-purin-6-amine trifluoroacetate (600 mg, 1.642 mmol) and potassium carbonate (567 mg, 4.11 mmol ) was stirred at temperature

60 ° C in DMF (10 ml) for 1 hour under nitrogen. The reaction mixture was cooled to room temperature and 1-bromo-5-chloropentane (0.216 mL, 1.642 mmol) and triethylamine (0.334 mL, 2.464 mmol) were added and the mixture was stirred at 20 ° C under nitrogen for 16 hours. The mixture was then diluted with water (10 mL) and brine (10 mL) and extracted with DCM (2 x 10 mL). The combined organic extracts were evaporated and the residue dissolved in DCM and purified by column chromatography using Flashmaster II (70 g aminopropyl cartridge) in a gradient of 0-100% ethyl acetate in cyclohexane over 40 minutes. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a yellow gum (430 mg).

LCMS (System D): tRET = 4.15 min; MH⁺ = 356, 358

Intermediate 20: 9- [3- (1-Azetidinyl) propyl] -2- (butyloxy) -8- (methyloxy) -9H-purin-6-amine [0269] nh₂

N ¹ 'Ί .....-^N.

and O '' o 'n "' '^N at^N [0270] 2- (Butyloxy) -8- (methyloxy) -1H-purin-6-amine trifluoroacetate (100 mg, 0.285 mmol) was dissolved in DMF (1 mL) and potassium carbonate (98 mg, 0.712 mmol) was added. The reaction mixture was stirred at 50 ° C under nitrogen for 1 hour and then cooled to room temperature. 1,3-dibromopropane (0.029 mL, 0.285 mmol) was added and after stirring for another 40 min. azetidine (0.038 mL, 0.569 mmol) and triethylamine (0.079 mL, 0.569 mmol) in DMF (1 mL) were added. The reaction mixture was then stirred for another 18 hours. The solvent was removed and the residue was partitioned between dichloromethane (2 mL) and water (2 mL). The layers were separated using a hydrophobic frit and the aqueous phase reextracted with DCM (2 mL). The combined organic extracts were concentrated and the residue was dissolved in 1: 1 MeOH: DMSO (1 mL) and purified by MDAP (Method A). The product-containing fractions were evaporated under a stream of nitrogen to give the title compound as a white solid (13 mg).

LCMS (System B): tRET = 1.07 min; MH⁺ = 335

Intermediate 21: 2- (Butyloxy) -8- (methyloxy) -9- [3- (1-pyrrolidinyl) propyl] -9H-purin-6-amine [0271] nh₂

N '¹/ .....^N . ... ..I⁰ '..... o "N' N / '- N [0272] Prepared as intermediate 20 from 2- (butyloxy) -8 (methyloxy) -1H-purin-6-amine trifluoroacetate, 1,3- dibromopropane and pyrrolidine.

LCMS (System C): tRET = 0.60 min; MH⁺ = 349

Intermediate 22: 2- (Butyloxy) -9- [3- (hexahydro-1H-azepin-1-yl) propyl] -8- (methyloxy) -9H-purin-6-amine [0273] νη₂ h ' ^Ν

I °

OK N and ^N [0274] Prepared like Intermediate 20 from 2- (butyloxy) -8 (methyloxy) -1H-purin-6-amine trifluoroacetate, 1,3-dibromopropane and hexahydro-1H-azepine.

LCMS (System B): tRET = 1.24 min; MH⁺ = 377

Intermediate 23: 9- [4- (1-Azetidinyl) butyl] -2- (butyloxy) -8- (methyloxy) -9H-purin-6-amine [0275]

NH ^

N ..

[0276] 2- (Butyloxy) -9- (4-chlorobutyl) -8- (methyloxy) -9H-purin-6-amine (100 mg, 0.305 mmol), azetidine (0.021 mL, 0.305 mmol) and N, N diisopropylethylamine (0.107 mL, 0.610 mmol) was dissolved in DMF (2 mL) and heated at 50 ° C for 48 hours. LCMS analysis showed that the reaction was not complete and additional azetidine (0.021 mL, 0.305 mmol) and N, N-diisopropylethylamine (0.107 mL, 0.610 mmol) were added and the reaction mixture was heated at 50 ° C for another 48 hours. The mixture was then partitioned between DCM (4 mL) and water (4 mL) and the layers were separated using a hydrophobic frit. The aqueous phase was re-extracted with DCM (4 mL) and the combined organic extracts were concentrated and the residue purified by MDAP (Method A). The product containing fractions was evaporated under a stream of nitrogen to give the title compound as a transparent gum (7.6 mg).

LCMS (System B): tRET = 1.15 min; MH⁺ = 349

Intermediate 24: Formic acid salt with 2- (butyloxy) -8- (methyloxy) -9- [4- (1-pyrrolidinyl) butyl] -9H-purin-6-amine [0277]

<img file="PL3000813T3_D0029.tif" />

[0278] Similarly to Intermediate 20, 2- (butyloxy) -8 (methyloxy) -1H-purin-6-amine, 1,4-dibromobutane and pyrrolidine trifluoroacetate, but using the mass spectrometer coupled autopreparative Method D.

LCMS (System B): tRET = 1.19 min; MH⁺ = 363

Intermediate 25: Formic acid salt with 2- (butyloxy) -8- (methyloxy) -9- [4- (1-piperidinyl) butyl] -9H-purin-6-amine [0279]

NH,

J .............

· 'Ο

HCO₂H

AND--.

[0280] Similarly to Intermediate 20 from 2- (bVyloxv) -8 (methyloxy) -1H-purine-6-amino, 1,4-dibromobutane and pipervidium trifluoroacetate, but with the subsequent use of an autopreparative mass spectrometer, Method A and then Metodv D.

LCMS (System B): tRET = 1.22 min; MH⁺ = 377

Intermediate 26: 2- (Butyloxy) -9-14- (hexahvdro-1 ^ -a / epin-1-yl) hutvolol-8- (methyloxy) -9 // - purine-6-ainine [0281]

NH,

And about

[0282] Similar to Intermediate 20 from 2- (butyloxy) -810 (methyloxy) -1H-purine-6-amino, 1,4-dibromobutane and hexahydro-1H-azepinv trifluoroacetate.

LCMS (System B): tRET = 1.30 min; MH⁺ = 391

Intermediate 27: 9-15- (1-Ά / ctvdvnvlo) pcntvlol-2- (hιιtvloksv) -8- (Inctvloksv) -9 // - pιιrvno-6-amine [0283]

<img file="PL3000813T3_D0030.tif" />

ON — N [0284] 2- (Butyloxy) -9- (5-chloropentyl) -8- (methyloxy) -9 ^ -purin-6-amine (100 mg,

0.293 mmol), azethine (0.020 mL, 0.293 mmol) and N, N-diisopropylethylamine (0.102 mL, 0.585 mmol) were dissolved in DMF (2 mL) and heated at 50 ° C for 72 hours. The solvent was removed in vacuo and the residue was partitioned between DCM (5 mL) and water (5 mL) and the layers were separated using a high-performance sinter. The aqueous phase was again extracted with DCM (5 ml) and the combined organic extracts concentrated and the residue dissolved in 1: 1 MeOH: DMSO (1 ml) and purified by MDAP (Method A). The fractions containing the product were evaporated under a stream of nitrogen, whereby the resulting compound was obtained in the form of a transparent resin (6.8 mg).

LCMS (System B): tRET = 1.26 min; MH⁺ = 363

Intermediate 28: 2- (Biitvloxv) -8- (inctvloxv) -9-15- (1-pyrrolidinyl) pcntvol-9 // - purine-6-amine [0285]

<img file="PL3000813T3_D0031.tif" />

[0286] Prepared like Intermediate 27 from 2- (butyloxy) -9- (5-chloropentyl) -8- (methyloxy) -9H-purin-6-amine and pyrrolidine.

LCMS (System B): tRET = 1.27 min; MH⁺ = 377

Intermediate 29: 2- (Butyloxy) -8- (methyloxy) -9- [5- (1-piperidinyl) pentyl] -9H-purin-6-amine [0287]

NHL ż-O - ·_Ό '·'_N "-N [0288] Prepared like Intermediate 27 from 2- (butyloxy) -9- (5-chloropentyl) -8- (methyloxy) -9H-purin-6-amine and piperidine.

LCMS (System B): tRET = 1.33 min; MH⁺ = 391

Intermediate 30: 2- (Butyloxy) -9- [5- (hexahydro-1H-azepin-1-yl) pentyl] -8- (methyloxy) -9H-purin-6-amine [0289]

NH,

........ »

[0290] Prepared like Intermediate 27 from 2- (butyloxy) -9- (5-chloropentyl) -8- (methyloxy) -9H-purin-6-amine and hexahydro-1H-azepine, but in succession MDAP purification using Method A followed by Method E.

LCMS (System B): tRET = 1.38 min; MH⁺ = 405

Intermediate 31: 2- (Butyloxy) -9- [5- (hexahydro-1 (2H) -azocinyl) pentyl] -8- (methyloxy) -9H-purin-6-amine [0291]

<img file="PL3000813T3_D0032.tif" />

[0292] Prepared like Intermediate 38 from 2- (butyloxy) -9- (5-chloropentyl) -8- (methyloxy) -9H-purin-6-amine and octahydroazocine.

LCMS (System B): tRET = 1.45 min; MH⁺ = 419

Intermediate 32: 2- (Butyloxy) -8- (methyloxy) -9- [6- (1-pyrrolidinyl) hexyl] -9H-purin-6-amine [0293]

<img file="PL3000813T3_D0033.tif" />

[0294] Prepared like Intermediate 38 from 2- (butyloxy) -9- (6-chlorohexyl) -8- (methyloxy) -9H-purin-6-amine and pyrrolidine.

LCMS (System D): tRET = 2.97 min; MH⁺ = 391

Intermediate 33: 2- (Butyloxy) -8- (methyloxy) -9- [6- (1-piperidinyl) hexyl] -9H-purin-6-amine [0295]

<img file="PL3000813T3_D0034.tif" />

[0296] Prepared like Intermediate 38 from 2- (butyloxy) -9- (6-chlorohexyl) -8- (methyloxy) -9H-purin-6-amine and piperidine.

LCMS (System D): tRET = 3.12 min; MH⁺ = 405

Intermediate 34: 2- (Butyloxy) -9- [6- (hexahydro-1H-azepin-1-yl) hexyl] -8- (methyloxy) -9H-purin-6-amine [0297]

<img file="PL3000813T3_D0035.tif" />

[0298] Prepared like Intermediate 38 from 2- (butyloxy) -9- (6-chlorohexyl) -8- (methyloxy) -9H-purin-6-amine and hexahydro-1H-azepine.

LCMS (System D): tRET = 3.20 min; MH⁺ = 419

Intermediate 35: U²-Butyl-8- (methyloxy) -9- [4- (1-piperidinyl) butyl] -9H-purine2,6-diamine [0299]

<img file="PL3000813T3_D0036.tif" />

[0300] Trifluoroacetate N²-butyl-8- (methyloxy) -3H-purine-2,6-diamine (192 mg, 0.547 mmol) and potassium carbonate (189 mg, 1.368 mmol) were suspended in DMF (3 mL) and heated to 60 ° C for 1 hour. The reaction mixture was cooled to room temperature, 1-bromo-4-chlorobutane (0.063 mL, 0.547 mmol) was added and the reaction mixture was stirred for another 18 hours. Piperidine (0.054 mL, 0.547 mmol) and triethylamine (0.076 mL, 0.547 mmol) were added and the reaction mixture was heated to 60 ° C for 72 hours. The solvent was removed in vacuo and the residue was partitioned between DCM (2 mL) and water (2 mL). The aqueous phase was re-extracted with DCM (2 mL) and the combined organic extracts were concentrated. The residue (about 200 mg) was dissolved in 1: 1 MeOH: DMSO (1 ml) and purified by MDAP (Method A). The product-containing fractions were evaporated in vacuo to give the impure title compound as a yellow gum (106 mg) which was used without further purification.

LCMS (System B): tRET = 1.11 min; MH⁺ = 376

Indirect relationship 36: U²-Butyl-9- [4- (hexahydro-1H-azepin-1-yl) butyl] -8- (methyloxy) -9H-purine-2,6-diamine [0301]

<img file="PL3000813T3_D0037.tif" />

[0302] Trifluoroacetate N²-butyl-8- (methyloxy) -3H-purine-2,6-diamine (192 mg, 0.547 mmol) and potassium carbonate (189 mg, 1.368 mmol) were suspended in DMF (3 mL) and heated to 60 ° C for 1 hour. The reaction mixture was cooled to room temperature, 1-bromo-4-chlorobutane (0.063 mL, 0.547 mmol) was added and the reaction mixture was stirred for another 18 hours. Hexahydro-1H-azepine (54.2 mg, 0.547 mmol) and triethylamine (0.076 mL, 0.547 mmol) were added and the reaction mixture was heated to 60 ° C for 18 hours. The solvent was removed in vacuo and the residue was partitioned between DCM (5 mL) and water (5 mL). The aqueous phase was extracted again with DCM (5 mL) and the combined organic extracts were concentrated in vacuo. The residue was dissolved in 1: 1 MeOH: DMSO (2 mL) and purified in 2 injections by MDAP (Method B). The result was a substance (74 mg) that was still contaminated and which was again purified by MDAP (Method A). The product-containing fractions were evaporated under a stream of nitrogen to give the title compound as a transparent gum (13 mg).

LCMS (System B): tRET = 1.12 min; MH⁺ = 390

Intermediate 37: 9- [4- (Hexahydro-1H-azepin-1-yl) butyl] -2 - {[(1S) -1-methylbutyl] oxy} -8- (methyloxy) -9H-purine-6- amine [0303] [0304] Prepared like Intermediate 36 from 2 - {[(1S) -1-methylbutyl] oxy} -8- (methyloxy) -1H-purin-6-amine trifluoroacetate, 1-bromo-4-chlorobutane and hexahydro-1H-azepine, but using three consecutive MDAP procedures using Method B followed by Method A (x2).

LCMS (System B): tRET = 1.41 min; MH⁺ = 405

Intermediate 38: 2 - {[(1S) -1-Methylbutyl] oxy} -8- (methyloxy) -9- [5- (1-piperidinyl) pentyl] -9H-purin-6-amine [0305]

NH,. J.

; oo N '^J '[0306] 9- (5-Chloropentyl) -2 - {[(1S) -1-methylbutyl] oxy} -8- (methyloxy) -9H-purin-6amine (80 mg, 0.225 mmol), triethylamine (0.031 ml , 0.225 mmol) and piperidine (0.045 mL, 0.45 mmol) were suspended in DMF (3 mL) and the mixture was heated to 70 ° C for 18 hours. The solvent was removed and the residue was partitioned between DCM (4 mL) and saturated sodium bicarbonate (4 mL). The aqueous phase again with an additional portion of DCM and the combined organic extracts were concentrated and the residue dissolved in a 1: 1 MeOH: DMSO (1 mL) mixture and purified by MDAP (Method A). The product-containing fractions were combined and evaporated under a stream of nitrogen to give the title compound (47.2 mg).

LCMS (System D): tRET = 3.11 min; MH + = 405

Intermediate 39: 2-Fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0307] νη₂

Ν Τ ............

JI,. / '

And Ν 'Ν / -ο [0308] N, O-bis (trimethylsilyl) acetamide (975 mL, 3.988 mol) was added to the suspension of 2-fluoro-1H-purin-6-amine (200 g, 1.306 mmol) (available from, on example, AlliedSignal, US) in anhydrous acetonitrile (4 L) while stirring in a 10 L automated laboratory reactor and the resulting mixture was heated to reflux and held at that temperature for 2 hours. The circulator was reprogrammed and the reaction mixture was cooled to 0 ° C. A solution of tetrahydropyranyl acetate was then slowly added via a dropping funnel (preparation described in Tetrahedron Letters 2006, 47 (27), 4741) (282 g, 1.959 mol) in anhydrous acetonitrile (500 mL), followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (28 mL) , 1.567 mole). No significant exotherm was observed. The circulator temperature was again set to 10 ° C and stirring continued for another 1 hour. The mixture was then quenched by the addition of 1 M sodium carbonate (4 L). Solid precipitation was observed and the pH was checked basic. Additional water (1 L) was added to the suspension and the layers separated while standing, the aqueous layer containing a significant amount of inorganic solids. Most of the aqueous and inorganic solids were separated. The organic layer still contained a significant amount of solid and during stirring it was cooled to 0 ° C to promote further precipitation. The solid was collected by filtration and the cartridge was thoroughly washed with water and then dried under vacuum at 40 ° C overnight to give the title compound as a cream-colored solid (152.8 g).

LCMS (System D): tRET = 1.71 min; MH⁺ = 238

Intermediate 40: 2 - {[(1S) -1-Methylpropyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0309]

<img file="PL3000813T3_D0038.tif" />

[0310] To (2S) -2-butanol (10 g, 135 mmol) was added portionwise with stirring sodium tert-butoxide (3.24 g, 33.7 mmol). To the resulting suspension, 2-fluoro-9 (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (2 g, 8.43 mmol) was added and the mixture was heated to 50 ° C for 6 hours, when LCMS showed the reaction was completed. After cooling, the mixture was diluted with ethyl acetate (100 mL) and washed with water (50 mL) and the aqueous layer was extracted again with ethyl acetate (50 mL). The combined organic extracts were washed with brine, dried using a hydrophobic frit and evaporated in vacuo (at 62 ° C to remove excess alcohol). The residue (2.52 g) was dissolved in dichloromethane and purified on an aminopropyl cartridge (110 g) using a Flashmaster II apparatus and eluting with a gradient of 0-100% ethyl acetate in cyclohexane over 60 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a white solid (1.935 g).

LCMS (System D): tRET = 2.41 min; MH⁺ = 292

Intermediate 41: 8-Bromo-2 - {[(1S) -1-methylpropyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0311]

<img file="PL3000813T3_D0039.tif" />

Ν

br

Ν &lt;:

[0312] To a solution of 2 - {[(1S) -1-methylpropyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (1.935 g, 6.64 mmol) in chloroform (50 ml) at 0-5 ° C N-bromosuccinimide (1.182 g, 6.64 mmol) was added in portions. The resulting green solution was stirred at 05 ° C for 1 hour, during which time it turned red, then the mixture was allowed to warm to room temperature and stirred overnight. The resulting green solution was washed with water (2x20 mL), separated using a hydrophobic frit and concentrated. The residue was dissolved in dichloromethane and purified by silica gel chromatography (100 g cartridge) using a Flashmaster II apparatus and a 0100% ethyl acetate-cyclohexane gradient over 60 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a yellow foam (1.79 g).

LCMS (System B): tRET = 2.58 min; MH⁺ = 370/372

Intermediate 42: 8- (Methyloxy) -2 - {[(1S) -1-methylpropyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0313]

of NH

<img file="PL3000813T3_D0040.tif" />

O [0314] 8-Bromo-2- {[(1S) -1-methylpropyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (1.79 g, 4.83 mmol) was dissolved in methanol (15 mL) and 25% sodium methoxide in methanol (3.2 mL, 4.83 mmol) was added and the mixture was heated to reflux for 2.5 hours. The reaction mixture was allowed to stand at room temperature overnight, then concentrated in vacuo and the residue partitioned between dichloromethane (40 ml) and saturated ammonium chloride solution (40 ml). The layers were separated using a hydrophobic frit and the aqueous phase reextracted with dichloromethane (40 mL). The combined organic extracts were concentrated in vacuo to give the title compound as a yellow foam (1.65 g).

LCMS (System B): tRET = 2.11 min; MH⁺ = 322

Intermediate 43: 8- (Methyloxy) -2 - {[(1S) -1-methylpropyl] oxy} trifluoroacetate} -1H-Purin-6-amine [0315]

NH₂

Ϊ 1 ..1&gt; ' ⁰

ONN

OH FI

F \, ·! &lt;,.

" - ' ABOUT

F [0316] Prepared like Intermediate 12 using 8- (methyloxy) -2 {[(1S) -1-methylpropyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purine- 6-amine.

LCMS (System B): tRET = 1.19 min; MH⁺ = 238

Intermediate 44: 9- (4-Chlorobutyl) -8- (methyloxy) -2 - {[(1S) -1-methylpropyl] oxy} 9H-purin-6-amine [0317] nh₂

ΓΊ

Ο Ν

-N υ

..... N

CI [0318] Prepared like Intermediate 18 from 8- (methyloxy) -2 {[(1S) -1-methylpropyl] oxy} -1H-purin-6-amine trifluoroacetate and 1-bromo-4-chlorobutane with purification over aminopropyl (NH2) cartridge using a 0-100% ethyl acetate - cyclohexane gradient.

LCMS (System D): tRET = 2.83 min; MH⁺ = 328/330

Intermediate 45: 2 - {[(1S) -1-Methylpentyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0319]

<img file="PL3000813T3_D0041.tif" />

[0320] Sodium t-butoxide (4.86 g, 50.6 mmol) was added portionwise with stirring to a mixture of (S) -2-hexanol (12 g, 117 mmol) and 1,2-dimethoxyethane (12 mL). The resulting mixture was heated to 50 ° C under a nitrogen atmosphere, followed by the addition of 2-fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (3 g, 12.65 mmol). The resulting mixture was held at 50 ° C for 20 hours when LCMS showed the reaction was complete. The mixture was cooled to room temperature and partitioned between ethyl acetate (100 mL) and water (100 mL). The organic phase was washed with water (100 ml) then saturated brine (50 ml), dried over anhydrous magnesium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane and purified on an aminopropyl (NH2) cartridge (100 g) with a gradient elution of 0-100% ethyl acetate in cyclohexane over 40 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a white foam (1.665 g).

LCMS (System D): tRET = 2.88 min; MH⁺ = 320

Intermediate 46: 8-Bromo-2 - {[(1S) -1-methylpentyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0321]

NH₂

- Ν '' '' '..........^N . . . And .- ^^br

·. about_N N, O [0322] N-Bromosuccinimide (1.504 g, 8.45 mmol) was added portionwise with stirring to the 2 - {[(1S) -1-methylpentyl] oxy} -9- (tetrahydro-2H-pyran-2) solution -yl) -9H-purin-6-amine (2.453 g, 7.68 mmol) in chloroform (40 mL), under nitrogen, cooled in an ice bath. After 3 hours, LCMS analysis showed that the reaction was 80% complete and more N-bromosuccinimide (0.68 g) was added and stirring continued for another 2 hours. Water (40 ml) was added and the phases were separated using a hydrophobic frit. The organic phase was evaporated and the residue dissolved in dichloromethane and purified on an aminopropyl (NH2) cartridge (100 g) using a gradient of 0-100% ethyl acetate in cyclohexane, followed by a gradient of 0-20% methanol (+ 1% triethylamine) for 60 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a white foam (2.38 g).

LCMS (System D): tRET = 3.24 min; MH⁺ = 398/400

Intermediate 47: 8- (Methyloxy) -2 - {[(1S) -1-methylpentyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0323]

NH₂ [0324] For 8-bromo-2 - {[(1S) -1-methylpentyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine solution (2.368 g, 5.95 mmol) in methanol (10 mL), a solution of sodium methoxide in methanol (0.5 M, 20 mL, 10 mmol) was added and the mixture was heated under reflux for 5 hours. More sodium methoxide in methanol (4 mL, 2 mmol) was added and the mixture was heated at reflux for another 2 hours, then cooled and evaporated. The residue was partitioned between ethyl acetate (100 ml) and water (100 ml). The organic phase was separated, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane and purified on an aminopropyl (NH2) cartridge (100 g) using a gradient of 0-100% ethyl acetate in cyclohexane over 40 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a white foam (1.725 g).

LCMS (System D): tRET = 3.06 min; MH⁺ = 350

Intermediate 48: 8- (methyloxy) -2 - {[(1S) -1-methylpentyl] oxy} trifluoroacetate} -1H-Purin-6-amine [0325]

<img file="PL3000813T3_D0042.tif" />

[0326] For 8- (methyloxy) -2- {[(1S) -1-methylpentyl] oxy} -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine solution (1.479 g , 4.23 mmol) in methanol (25 ml) trifluoroacetic acid (2.3 ml, 3.40 g, 29.9 mmol) was added with stirring. The resulting mixture was stirred for 66 hours under nitrogen, then evaporated and dried in vacuo to give the title compound as a white solid (1.65 g).

LCMS (System D): tRET = 2.14 min; MH⁺ = 266

Intermediate 49: 9- (4-Chlorobutyl) -8- (methyloxy) -2 - {[(1S) -1-methylpentyl] oxy} 9H-purin-6-amine [0327]

Cl [0328] Prepared like Intermediate 44 from 8- (methyloxy) -2 {[(1S) -1-methylpentyl] oxy} -1H-purin-6-amine trifluoroacetate and 1-bromo-4-chlorobutane.

LCMS (System D): tRET = 3.22 min; MH⁺ = 356/358

Intermediate 50: 2 - [(1-Methylethyl) oxy] -9- (tetrahydro-2H-pyran-2-yl) -9H-purin6-amine [0329] ϋ Ν 'Ί

[0330] To 2-propanol (16.95 mL, 220 mmol) was added portionwise with stirring for 5 min sodium t-butoxide (1.30 g, 13.53 mmol). 2-Fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (2 g, 8.43 mmol) was added and the reaction mixture was heated and stirred at 50 ° C for 4 hours and then allowed to stand to cool to room temperature. The reaction mixture was then diluted with ethyl acetate (75 mL), washed with water (3x25 mL) and the combined aqueous layers re-extracted with ethyl acetate (2x25 mL). The combined organic layers were dried by passing through a hydrophobic frit, filtered and evaporated to give an off-white solid (2.30 g). This material was dissolved in dichloromethane and purified using an aminopropyl SPE cartridge (70 g) with a gradient elution of 0-100% ethyl acetate in cyclohexane. The appropriate fractions were combined and evaporated to give a white solid (1.6 g), which was again purified by column chromatography using reverse phase (C18) Flashmaster II by introducing 1: 1 MeOH / DMSO and using a gradient of 0 as eluent -50% acetonitrile (+ 0.1% TFA) in water (+ 0.1% TFA) for 40 min collecting fractions into vials containing about 2 ml of saturated aqueous sodium bicarbonate solution. The appropriate fractions were combined and extracted with dichloromethane (3x100 mL). The combined organic extracts were dried by passing through a hydrophobic frit and evaporated to give the title compound as a white solid (888 mg).

LCMS (System B): tRET = 1.76 min; MH⁺ = 278

Intermediate 51: 8-Bromo-2 - [(1-methylethyl) oxy] -9- (tetrahydro-2H-pyran-2-yl) 9H-purin-6-amine [0331]

NH,

N ^N

... J ^br

HE ^N

[0332] N-Bromosuccinimide (604 mg, 3.39 mmol) was added to the solution of 2 - [(1-methylethyl) oxy] -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6- amines (888 mg, 3.20 mmol) in chloroform (30 ml) at 0-5 ° C under nitrogen. The mixture was stirred at 0-5 ° C for 1 hour, during which time it became reddish brown, then warmed to room temperature and stirred for another 4 hours. LCMS analysis showed that the reaction was not complete and more N-bromosuccinimide (114 mg, 0.641 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with chloroform (30 mL), washed with water (2 x 20 mL) and the layers were separated using a hydrophobic frit and the organic layer was evaporated to give a red solid (1.16 g). This material was dissolved in dichloromethane and purified by silica gel chromatography on an SPE cartridge (50 g) using a gradient of 0-100% ethyl acetate in cyclohexane as eluent. The appropriate fractions were combined and evaporated to give the title compound as a pale yellow solid of 712 mg.

LCMS (System B): tRET = 2.36 min; MH⁺ = 356/358

Intermediate 52: 2 - [(1-Methylethyl) oxy] -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0333]

<img file="PL3000813T3_D0043.tif" />

[0334] For a suspension of 8-bromo-2 - [(1-methylethyl) oxy] -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (690 mg, 1.937 mmol) in methanol (15 ml) sodium methoxide (30% w / v) was added with stirring. methanol (2.4 mL) and the reaction mixture was heated at 50 ° C for 2 hours. The reaction mixture was then heated to 70 ° C and stirred for 2.5 hours. The solvent was evaporated and the residue was partitioned between saturated aqueous ammonium chloride (15 ml) and ethyl acetate (20 ml). The layers were separated, the aqueous phase extracted with additional ethyl acetate (2x10 mL) and the organic extracts combined, dried by passing through a hydrophobic frit and evaporated to give the title compound as a yellow solid (573 mg).

LCMS (System B): tRET = 1.92 min; MH⁺ = 308

Intermediate 53: 2 - [(1-methylethyl) oxy] -8- (methyloxy) -1H-purin-6amine trifluoroacetate [0335]

<img file="PL3000813T3_D0044.tif" />

[0336] Trifluoroacetic acid (1 mL, 12.98 mmol) was added with stirring to a solution of 2 - [(1-methylethyl) oxy] -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6amine (568 mg, 1.848 mmol) in methanol (10 mL) and the mixture was stirred at room temperature overnight. More trifluoroacetic acid (0.2 ml) was added and the reaction mixture was stirred at room temperature for another 1.5 hours and then evaporated in vacuo. The remaining solid was triturated with ethyl acetate, collected by filtration, washed with ethyl acetate and dried in vacuo overnight to give the title compound as a white solid (405 mg).

LCMS (System B): tRET = 1.02 min; MH⁺ = 224

Intermediate 54: 9- (5-Chloropentyl) -2 - [(1-methylethyl) oxy] -8- (methyloxy) -9H-purin-6-amine [0337]

<img file="PL3000813T3_D0045.tif" />

[0338] Prepared like Intermediate 44 from 2 - [(1-methylethyl) oxy] -8- (methyloxy) -1H-purin-6-amine trifluoroacetate and 1-bromo-5-chloropentane.

LCMS (System A): tRET = 0.93 min; MH⁺ = 328/330

Intermediate 55: 2- (Cyclobutyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6amine [0339]

NH, μ Ν η ΐ; ι

Ο ..... Ν Ν / - 'Ο [0340] To cyclobutanol (10 ml) was added sodium batchoxide (3.31 g, 34.2 mmol) in portions at room temperature. The mixture became very thick and heated to 50 ° C. 2-Fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (2 g, 8.43 mmol) was added, followed by 1,2-dimethoxyethane (3 mL) and the mixture was stirred at 50 ° C for 90 min, then cooled and partitioned between ethyl acetate (50 ml) and water (50 ml). The precipitate which did not dissolve in any phase was removed by filtration. The organic phase was separated, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated to give a creamy foam. This material was dissolved in dichloromethane and purified on an aminopropyl (NH2) cartridge (110 g) using a gradient of 0-100% ethyl acetate in cyclohexane followed by a gradient of 0-20% methanol (+ 1% triethylamine) for 40 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as an off-white solid (0.655 g).

LCMS (System B): tRET = 1.98 min; MH⁺ = 290

Intermediate 56: 8-Bromo-2- (cyclobutyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0341]

<img file="PL3000813T3_D0046.tif" />

ο [0342] N-Bromosuccinimide (1.152 g, 6.47 mmol) was added with stirring to a solution of 2- (cyclobutyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine ( 1.248 g, 4.31 mmol) in chloroform (15 mL) at 0 ° C. The mixture was warmed to room temperature and left overnight when water (15 ml) was added and the phases separated. The aqueous layer was extracted with dichloromethane and the organic extracts combined, washed with brine, dried over anhydrous magnesium sulfate and evaporated to give the title compound as an orange foam (1.79 g).

LCMS (System D): tRET = 2.72 min; MH⁺ = 368/370

Intermediate 57: 2- (Cyclobutyloxy) -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) 9H-purin-6-amine [0343]

<img file="PL3000813T3_D0047.tif" />

[0344] 8-Bromo-2- (cyclobutyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (1.79 g, 4.86 mmol) was dissolved in anhydrous methanol ( 25 ml) and 25% sodium methoxide in methanol (2.274 ml, 9.72 mmol) was added under a nitrogen atmosphere. The mixture was heated at 67 ° C for 24 hours and then cooled to room temperature. Ethyl acetate and water were added and the layers were separated. The aqueous layer was extracted twice more with ethyl acetate and the organic extracts were combined, washed with brine, dried over anhydrous magnesium sulfate and evaporated to give the title compound as a cream-colored foam (1.27 g).

LCMS (System D): tRET = 2.53 min; MH⁺ = 320

Intermediate 58: 2- (Cyclobutyloxy) -8- (methyloxy) -1H-purin-6amine trifluoroacetate [0345]

<img file="PL3000813T3_D0048.tif" />

[0346] Trifluoroacetic acid (3 mL, 38.9 mmol) was added to a solution of 2- (cyclobutyloxy) 8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine ( 1.27 g, 3.98 mmol) in methanol (50 mL) and the mixture was stirred at 20 ° C under nitrogen for 21 hours. The solvent was removed in vacuo and the remaining solid was triturated with 1,1-dimethylethyl ether and then collected by filtration and dried in vacuo to give the title compound as a cream-colored solid (1.0922 g).

LCMS (System D): tRET = 1.17 min; MH⁺ = 236

Intermediate 59: 9- (4-Chlorobutyl) -2- (cyclobutyloxy) -8- (methyloxy) -9H-purin-6amine [0347]

<img file="PL3000813T3_D0049.tif" />

ci [0348] Prepared like Intermediate 44 from 2- (cyclobutyloxy) -8- (methyloxy) -1H-purin-6-amine trifluoroacetate and 1-bromo-4-chlorobutane.

LCMS (System D): tRET = 2.76 min; MH⁺ = 326/328

Intermediate 60: 2- (Cyclopentyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6amine [0349]

<img file="PL3000813T3_D0050.tif" />

O 'Ν' '......... N

C [0350] Cyclopentanol (25 mL, 275 mmol) was added to sodium tert-butoxide (4.05 g, 42.2 mmol) to give a thick suspension, which was diluted with 1,2-dimethoxyethane (35 mL) and heated up to 50 ° C. 2-Fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6amine (2.5 g, 10.54 mmol) was added to the resulting solution, which was then stirred under nitrogen at 50 ° C for 20 hours. The mixture was cooled and water and ethyl acetate were added. The layers were separated and the aqueous layer was washed again with ethyl acetate. The organic extracts were combined, washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40 ° C. The residue was applied in cyclohexane (50 ml) to a 330 g silica cartridge and eluted using first a gradient of 0100% ethyl acetate in cyclohexane over 10 column volumes followed by a gradient of 030% methanol in ethyl acetate. The product-containing fractions were combined and evaporated to give the title compound as a white foam (2.51 g).

LCMS (System D): tRET = 2.51 min; MH⁺ = 304

Intermediate 61: 8-Bromo-2- (cyclopentyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0351]

<img file="PL3000813T3_D0051.tif" />

[0352] Prepared like Intermediate 56 from 2- (cyclopentyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine.

LCMS (System D): tRET = 2.88 min; MH⁺ = 382/384

Intermediate 62: 2- (Cyclopentyloxy) -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) 9H-purin-6-amine [0353]

<img file="PL3000813T3_D0052.tif" />

[0354] Prepared like Intermediate 57 from 8-bromo-2- (cyclopentyloxy) -9 (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine.

LCMS (System C): tRET = 1.11 min; MH⁺ = 334

Intermediate 63: 2- (Cyclopentyloxy) -8- (methyloxy) -1H-purin-6amine trifluoroacetate [0355]

<img file="PL3000813T3_D0053.tif" />

[0356] Prepared like Intermediate 58 from 2- (cyclopentyloxy) -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine.

LCMS (System B): tRET = 1.27 min; MH⁺ = 250

Intermediate 64: 9- (4-Chlorobutyl) -2- (cyclopentyloxy) -8- (methyloxy) -9H-purin6-amine [0357]

<img file="PL3000813T3_D0054.tif" />

[0358] Prepared like Intermediate 44 from 2- (cyclopentyloxy) -8- (methyloxy) -1H-purin-6-amine trifluoroacetate and 1-bromo-4-chlorobutane.

LCMS (System D): tRET = 2.90 min; MH⁺ = 340/342

Intermediate 65: 2- (Cyclohexyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6amine [0359]

<img file="PL3000813T3_D0055.tif" />

[0360] To cyclohexanol (15 ml) was added portionwise at room temperature sodium tert-butoxide (3.29 g, 34.2 mmol). The mixture became very thick and more cyclohexanol (10 ml) was added and the mixture was heated to 50 ° C. 2-Fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (2 g, 8.43 mmol) was added and the mixture was heated at 50 ° C for 1 hour and then heated to 60 ° C and heated for another 2 hours at which time LCMS analysis showed the reaction was complete. The mixture was cooled to room temperature and partitioned between ethyl acetate (150 ml) and water (150 ml). The organic phase was separated, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated using a water bath at 60 ° C. The residue was dissolved in dichloromethane and purified on a 70 g aminopropyl (NH2) cartridge using a gradient of 0-100% ethyl acetate in cyclohexane, followed by a gradient of 0-20% methanol (+ 1% triethylamine) for 30 min. Some product-containing fractions were contaminated with cyclohexanol and were again purified on a 70 g silica cartridge using a 0-100% ethyl acetate-cyclohexane gradient over 40 min. The product-containing fractions from the two purification steps were combined and evaporated in vacuo to give the title compound as a pale yellow foam (1.59 g).

LCMS (System D): tRET = 2.65 min; MH⁺ = 318

Intermediate 66: 8-Bromo-2- (cyclohexyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine [0361]

- 0 'N^{; N}

C [0362] N-Bromosuccinimide (0.214 g, 1.2 mmol) was added with stirring to a solution of 2- (cyclohexyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine ( 0.254 g, 0.80 mmol) in chloroform (5 mL) at 0 ° C. The resulting mixture was stirred at 0 ° C for

1.5 hours, then warmed to room temperature and stirred for another 2 hours. Water (5 ml) was added and the phases were separated using a hydrophobic frit. The organic phase was evaporated and the residue was dissolved in dichloromethane and purified on a 70 g aminopropyl (NH2) cartridge using a gradient elution of 0-100% ethyl acetate in cyclohexane over 40 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as a white solid (0.252 g). LCMS (System B): tRET = 2.83 min; MH⁺ = 396/398

Intermediate 67: 2- (Cyclohexyloxy) -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) 9H-purin-6-amine [0363]

NU

<img file="PL3000813T3_D0056.tif" />

[0364] Prepared like Intermediate 57 from 8-bromo-2- (cyclohexyloxy) -9 (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine.

LCMS (System D): tRET = 2.86 min; MH⁺ = 348

Intermediate 68: 2- (Cyclohexyloxy) -8- (methyloxy) -1H-purin-6amine trifluoroacetate [0365]

.... N / O - N νη₂

ί ...... '] ϊ /

Ο Ν

ΟΗ

F

F '·.

q

F [0366] Prepared like Intermediate 58 from 2- (cyclohexyloxy) -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine.

LCMS (System B): tRET = 1.43 min; MH⁺ = 264

Intermediate 69: 9- (4-Chlorobutyl) -2- (cyclohexyloxy) -8- (methyloxy) -9H-purin6-amine [0367] nh₂

Ν '....._r- \

II and 1 ° 'ON ^N

Cl [0368] Prepared like Intermediate 44 from 2- (cyclohexyloxy) -8- (methyloxy) -1H-purin-6-amine trifluoroacetate and 1-bromo-4-chlorobutane.

LCMS (System D): tRET = 3.05 min; MH⁺ = 354/356

Intermediate 70: A²- [(1R) -1-Methylbutyl] -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-2,6-diamine [0369]

<img file="PL3000813T3_D0057.tif" />

[0370] A crude (2R) -2-pentanamine sample containing dichloromethane (11.12 g containing about 3.1 g, 35.6 mmol of amine) was added to the suspension of 2-fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purin-6-amine (5.00 g, 21.08 mmol) in ethylene glycol (50 mL). The mixture was heated at 110 ° C for 20 hours and then cooled to room temperature and partitioned between water (200 ml) and ethyl acetate (200 ml). The organic phase was separated, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane and purified on a 110 g aminopropyl (NH2) cartridge using a gradient of 0-100% ethyl acetate cyclohexane over 40 min. The appropriate fractions were combined and evaporated in vacuo and the residue was triturated with diethyl ether and some insoluble starting material was removed by filtration. Evaporation of the ether filtrate gave the compound as an off-white foam (2.34 g).

LCMS (System D): tRET = 2.63 min; MH⁺ = 305

Intermediate 71: 8-Bromo-A²- [(1R) -1-methylbutyl] -9- (tetrahydro-2H-pyran-2-yl) 9H-purine-2,6-diamine [0371]

<img file="PL3000813T3_D0058.tif" />

[0372] N-Bromosuccinimide (2.08 g, 11.69 mmol) was added portionwise with stirring to the N solution²- [(1R) -1-methylbutyl] -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-2,6-di57 amine (2.27 g, 7.46 mmol) in chloroform (30 ml) at 0 ° C under nitrogen. The reaction mixture was allowed to stir for 1.5 hours, when chloroform (20 ml) and water (50 ml) were added. After stirring, the layers were separated using a hydrophobic frit, the aqueous layer was washed with an additional portion of chloroform and the combined organic extracts were evaporated. The residue was dissolved in dichloromethane and purified on a 110 g aminopropyl (NH2) cartridge using a gradient of 0-100% ethyl acetate in cyclohexane over 40 min. The appropriate fractions were combined and evaporated in vacuo to give the title compound as an off-white foam (0.846 g).

LCMS (System D): tRET = 3.05 Min; MH⁺ = 383/385

Intermediate 72: A²- [(1R) -1-Methylbutyl] -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-2,6-diamine [0373]

<img file="PL3000813T3_D0059.tif" />

[0374] A solution of sodium methoxide in methanol (0.5 M, 9 mL, 4.5 mmol) was added to the solution of 8-bromo-N²- [(1R) -1-methylbutyl] -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-2,6-diamine (0.844 g, 2.20 mmol) in methanol (12 mL) and the resulting solution heated at reflux for 23.5 hours. Then more sodium methoxide in methanol (0.5 M, 4.5 mL) was added and refluxing was continued for another 4 hours. More sodium methoxide in methanol (0.5 M, 4.5 mL) was again added and refluxing continued for a further 16.5 hours when LCMS analysis showed the reaction was complete. The reaction mixture was cooled to room temperature, evaporated and the residue partitioned between ethyl acetate (75 ml) and water (75 ml). The aqueous phase was re-extracted with ethyl acetate (75 ml) and the combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane and purified on a 100g aminopropyl (NH2) cartridge using a gradient of 0-100% ethyl acetate in cyclohexane, followed by a gradient of 0-20% methanol (+ 1% triethylamine) for 15 min. The product-containing fractions were combined and evaporated in vacuo to give the title compound as a white foam (0.614 g).

LCMS (System D): tRET = 2.83 min; MH⁺ = 335

Intermediate 73: Trifluoroacetate A²- [(1R) -1-methylbutyl] -8- (methyloxy) -3H-purine2,6-diamine [0375]

<img file="PL3000813T3_D0060.tif" />

F [0376] Trifluoroacetic acid (1 mL, 1.48 g, 7.08 mmol) was added with stirring to a solution of N2 - [(1R) -1-methylbutyl] -8- (methyloxy) -9- (tetrahydro-2H -pyran-2-yl) -9H-purine-2,6-diamine (0.613 g, 1.833 mmol) in methanol (10 mL). The resulting mixture was stirred for 66 hours under nitrogen and then evaporated to give the title compound as an off-white solid (0.690g).

LCMS (System D): tRET = 1.89 min; MH⁺ = 251

Intermediate 74: 9- (4-Chlorobutyl) -A²- [(1R) -1-methylbutyl] -8- (methyloxy) -9H-purine-2,6-diamine [0377]

<img file="PL3000813T3_D0061.tif" />

[0378] Vv was prepared similarly to intermediate 44 from trifluoroacetate N²-1 (1R) -1-methylbutanol-8- (methyloxy) -3H-purine-2,6-diaminine and 1-bromo-4-chlorobutane.

LCMS (System D): tRET = 3.02 min; MH⁺ = 341/343

Intermediate 75: ^²- [(15) -1-Methylbutyl] -9- (tetrahydro-2 / -pyran-2-yl) -9 / -purine2,6-diamine [0379]

<img file="PL3000813T3_D0062.tif" />

[0380] Vv was prepared similarly to Intermediate 70 from 2-fluoro-9- (tetrahydro-2H-pyran-2-yl) -9H-purine-6-amino and (2S) -2-pentanamin.

LCMS (System D): tRET = 2.63 min; MH⁺ = 305

Intermediate 76: 8-Bromo²- [(15) -1-methylbutyl] -9- (tetrahydro-2 / -pyran-2-yl) 9 // - parvao-2.6-diainiaa [0381]

<img file="PL3000813T3_D0063.tif" />

[0382] Created like Intermediate 71 with N²-1 (1S) -1-methylbutol-9 (tetrahydro-2 / -pyran-2-yl) -9 / -purine-2,6-diaminyl.

LCMS (System D): tRET = 3.05 min; MH⁺ = 383/385

Intermediate 77: ^²- [(15) -1-Methylbutyl] -8- (methoxyl) -9- (tetrahydro-2 / -pyran-2-yl) -9H-purine-2,6-diamine [0383]

<img file="PL3000813T3_D0064.tif" />

[0384] A solution of sodium methoxide in methanol (0.5 M, 13 mL, 6.5 mmol) was added to the solution of 8-bromo-N²-1 (1S) -1-methylbutol-9- (tetrahydro-2 / -pyran-2-yl) -9 / -purine-2,6-diaminv (1.26 g, 3.29 mmol) in methanol (10 ml) and the solution was heated at reflux for 4 hours. Then more sodium methoxide in methanol solution (0.5 M, 12 ml, 6 mmol) was added and refluxing continued for a further 18 hours. The mixture was cooled and evaporated and the residue was partitioned between ethyl acetate (75 ml) and water (75 ml). The aqueous phase was re-extracted with ethyl acetate (75 ml) and the combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate and evaporated. The residue was dissolved in dichloromethane and purified on a 100g aminopropyl (NH2) cartridge using a gradient of 0-100% ethyl acetate in cyclohexane, followed by a gradient of 0-20% methanol (+ 1% triethylamine) for 15 min. The product-containing fractions were combined and evaporated in vacuo to give the title compound as a white foam (0.848g).

LCMS (System D): tRET = 2.83 min; MH⁺ = 335

Intermediate 78: Trifluoroacetate²- [(15) -1-methylbutyl] -8- (methyloxy) -3H-purine-2,6-diamine [0385]

<img file="PL3000813T3_D0065.tif" />

F [0386] Prepared similarly to Intermediate 73 with N²- [(1S) -1-methyl-butyl] -8- (methyloxy) -9- (tetrahydro-2H-pyran-2-yl) -9H-purine-2,6-diamine.

LCMS (System D): tRET = 1.89 min; MH⁺ = 251

Intermediate 79: 9- (4-Chlorobutyl) - ^²- [(15) -1-methylbutyl] -8- (methyloxy) -9H-purine-2,6-diamine [0387]

<img file="PL3000813T3_D0066.tif" />

[0388] Prepared like Intermediate 44 from N trifluoroacetate²- [(1S) -1-methylbutyl] -8- (methyloxy) -3H-purine-2,6-diamine and 1-bromo-4-chlorobutane.

LCMS (System D): tRET = 3.02 min; MH⁺ = 341/343

Intermediate 80: 9- (3-Chloropropyl) -2 - {[(15) -1-methylbutyl] oxy} -8- (methyloxy) 9H-purin-6-amine [0389]

<img file="PL3000813T3_D0067.tif" />

[0390] Prepared like Intermediate 44 from 2 - {[(1S) -120 methylbutyl] oxy} -8- (methyloxy) -9H-purin-6-amine trifluoroacetate and 1-bromo-3-chloropropane.

LCMS (System D): tRET = 2.90 min; MH⁺ = 328/330

Intermediate 81: 9- (5-Chloropentyl) -8- (methyloxy) -2 - {[(15) -1-methylpropyl] oxy} -9H-purin-6-amine [0391]

<img file="PL3000813T3_D0068.tif" />

[0392] Prepared like Intermediate 14 from 8- (methyloxy) -2 {[(1S) -1-methylpropyl] oxy} -1H-purin-6-amine trifluoroacetate and 1-bromo-5-chloropentane.

LCMS (System A): tRET = 1.00 min; MH⁺ = 342/344

Intermediate 82: 8- (Methyloxy) -2 - {[(15) -1-methylpropyl] oksv} -9- [5- (1-piperidyl) pentyl] -9H-purin-6-amine [0393]

<img file="PL3000813T3_D0069.tif" />

[0394] Prepared like Intermediate 38 from 9- (5-chloropentyl) -8- (methyl 5 xv) -2 - {[(1S) -1-methylpropyl] oxv} -9H-purine-6-amino and pipervdvnv, but with purification on silica using a gradient of 0-25% methanol in dichloromethane.

LCMS (System A): tRET = 0.61 min; MH⁺ = 391

Intermediate 83: Salt of formic acid 2- (butyloxy) -8- (methyloxy) -9- [3- (1-piperidyl) propyl] -9H-purine-6-amino [0395]

<img file="PL3000813T3_D0070.tif" />

HCO₂h [0396] Similarly to Intermediate 20 from 2- (butyloxy) -8 (methyloxy) -1H-purine-6-amino, 1,3-dibromopropane and piperidium trifluoroacetate, but with subsequent purification by MDAP using Method A, and then Metodv D.

LCMS (System B): tRET = 1.16 min; MH⁺ = 363

Reference Example 1: 6-Amino-9- [3- (1-azetydimethyl) propyl] -2- (butyloxy) -7,9-dihydro-8H-purin-8-one [0397]

<img file="PL3000813T3_D0071.tif" />

D^N [0398] 9- [3- (1-Azetylvinyl) propyl] -2- (butyloxy) -8- (methyloxy) -9H-purine-6-amine (13 mg, 0.039 mmol) was dissolved in methanol (3 mL) and 4 M hydrogen chloride in 1,420 dioxane (0.243 mL, 0.972 mmol) was added and the mixture was stirred at room temperature for 18 hours. The solvent was removed in vacuo and the residue was dissolved in methanol and applied to an aminopropyl SPE cartridge (2 g). The cartridge was pelleted with methanol and the solvent removed to give the solid compound as a white solid (13 mg).

LCMS (System B): tRET = 1.12 min; MH⁺ = 321

Reference Example 2: 6-Amino-2- (butyloxy-9- [3- (1-pyrrolidinyl) propyl] -7,9-dihydro-8H-purin-8-one [0399]

<img file="PL3000813T3_D0072.tif" />

[0400] 2- (Butyloxy) -8- (methyloxy) -9- [3- (1-pyrrolidinyl) propyl] -9H-purin-6-amine (49 mg, 0.141 mmol) was dissolved in methanol (5 mL) and 4 M hydrogen chloride in 1,4-dioxane (0.879 ml, 3.52 mmol) was added and the mixture was stirred at room temperature for 5 hours. The solvent was removed in vacuo to give a cream-colored solid which was dissolved in methanol and applied to the aminopropyl cartridge.

SPE (2 g) and eluted with methanol. The solvent was evaporated to give the title compound as a white solid (43 mg).

LCMS (System C): tRET = 0.70 min; MH⁺ = 335

Reference Example 3: 6-Amino-2- (butyloxy) -9- [3- (hexahydro-1H-azepin-1-yl) propyl] -7,9-dihydro-8H-purin-8-one [0401]

<img file="PL3000813T3_D0073.tif" />

[0402] Prepared similar to Reference Example 1 from 2- (butyloxy) -9- [3- (hexahydro-1H-azepin-1-yl) propyl] -8- (methyloxy) -9H-purin-6-amine.

LCMS (System B): tRET = 1.33 min; MH⁺ = 363

Reference Example 4: 6-Amino-9- [4- (1-azetidinyl) butyl] -2- (butyloxy) -7,9-dihydro8H-purin-8-one [0403]

<img file="PL3000813T3_D0074.tif" />

[0404] Prepared similar to Reference Example 1 from 9- [4- (1-azetidinyl) butyl] -2- (butyloxy) -8- (methyloxy) -9H-purin-6-amine.

LCMS (System B): tRET = 1.16 min; MH⁺ = 335

Reference Example 5: 6-Amino-2- (butyloxy) -9- [4- (1-pyrrolidinyl) butyl] -7,9-dihydro-8H-purin-8-one [0405]

<img file="PL3000813T3_D0075.tif" />

[0406] Prepared similarly to Reference Example 1 from formic acid salts 25 and 2- (butyloxy) -8- (methyloxy) -9- [4- (1-pyrrolidinyl) butyl] -9H-purin-6-amine.

LCMS (System B): tRET = 1.23 min; MH⁺ = 349

Reference Example 6: 6-Amino-2- (butyloxy) -9- [4- (1-piperidinyl) butyl 1-7.9-dihydro-8H-purin-8-one [0407]

<img file="PL3000813T3_D0076.tif" />

[0408] Prepared similar to Reference Example 1 from formic acid salts and 2- (butyloxy) -8- (methyloxy) -9- [4- (1-piperidinyl) butyl 1-9H-purin-6-amine.

LCMS (System B): tRET = 1.29min; MH⁺ = 363

Reference Example 7: 6-Amino-2- (butyloxy) -9- [4- (hexahydro-1H-azepin-1-yl) butyl] -7,9-dihydro-8H-purin-8-one [0409]

<img file="PL3000813T3_D0077.tif" />

[0410] Prepared similar to Reference Example 1 from 2- (butyloxy) -9- [4- (hexahydro-1H-azepin-1-yl) butyl 1-8- (methyloxy) -9H-purin-6-amine.

LCMS (System B): tRET = 1.37 min; MH⁺ = 377

Reference Example 8: 6-Amino-9- [5- (1-azetidinyl) pentyl-1-2- (butyloxy) -7,9-dihydro-8H-purin-8-one [0411]

<img file="PL3000813T3_D0078.tif" />

[0412] Prepared similar to Reference Example 1 from 9- [5- (1-azetidinyl) pentyl-1-2- (butyloxy) -8- (methyloxy) -9H-purin-6-amine.

LCMS (System B): tRET = 1.25 min; MH⁺ = 349

Reference Example 9: 6-Amino-2- (butyloxy) -9- [5- (1-pyrrolidinyl) pentyl1-7,9-di20 hydro-8H-purin-8-one [0413]

<img file="PL3000813T3_D0079.tif" />

[0414] Prepared similar to Reference Example 1 from 2- (butyloxy) -8- (methyloxy) -9- [5- (1-pyrrolidinyl) pentyl] -9H-purin-6-amine.

LCMS (System B): tRET = 1.28 min; MH⁺ = 363

Reference Example 10: 6-Amino-2- (butyloxy) -9- [5- (1-piperidinyl) pentylol-7,9-di5 hydro-8H-purin-8-one [0415]

<img file="PL3000813T3_D0080.tif" />

[0416] Prepared similar to Reference Example 1 from 2- (butyloxy) -8 (methyloxy) -9- [5- (1-piperidinyl) pentylol-9H-purin-6-amine.

LCMS (System B): tRET = 1.35 min; MH⁺ = 377

Reference Example 11: 6-Amino-2- (butyloxy) -9- [5- (hexahydro-1H-azepin-1-yl) pentylol-7,9-dihydro-8H-purin-8-one [0417]

<img file="PL3000813T3_D0081.tif" />

Method A [0418] Prepared similar to Reference Example 2 of 2- (butyloxy) -9- [515 (hexahydro-1H-azepin-1-yl) pentylol-8- (methyloxy) -9H-purin-6-amine.

LCMS (System B): tRET = 1.55 min; MH⁺ = 391 Method B [0419] Prepared similar to Reference Example 19 from 2 (butyloxy) -8- (methyloxy) -1H-purin-6-amine trifluoroacetate, 1H-purin-6-amine, 1-bromo-5-chloropentane and 1H-azepine hexahydro20.

LCMS (System B): tRET = 1.54 min; MH⁺ = 391

Reference Example 12: 6-Amino-2- (butyloxy) -9- [5- (hexahydro-1 (2H) -azocinyl) pentylol-7,9-dihydro-8H-purin-8-one [0420]

<img file="PL3000813T3_D0082.tif" />

[0421] Prepared similar to Reference Example 1 from 2- (butyloxy) -9- [5- (hexahydro-1 (2H) -azocinyl) pentylol-8- (methyloxy) -9H-purin-6-amine.

LCMS (System D): tRET = 3.17 min; MH⁺ = 405

Reference Example 13: 6-Amino-2- (butyloxy) -9- [6- (1-pyrrolidinyl) hexyl] -7,9-dihydro-8 / -purin-8-one [0422]

<img file="PL3000813T3_D0083.tif" />

[0423] Prepared as in Reference Example 1 of 2- (butyloxy) -8- (methyloxy) -9- [6- (1-pyrrolidyl) hexyl] -9H-purine-6-amino.

LCMS (System D): tRET = 2.47 min; MH⁺ = 377

Reference Example 14: 6-Amino-2- (butyloxy) -9- [6- (1-pipervidinyl) hexyl] -7,9-dihydro-8 / pipin-8-one [0424]

<img file="PL3000813T3_D0084.tif" />

[0425] Prepared as in Reference Example 1 of 2- (butyloxy) -8- (methyloxy) -9- [6- (1-piperidyl) hexyl] -9H-purine-6-amino.

LCMS (System D): tRET = 2.68 min; MH⁺ = 391

Reference Example 15: 6-Amino-2- (butyloxy) -9- [6- (hexahydro-1 / -azepin-1-yl) hexyl] -7,9-dihydro- 8H-purvn-8-one [0426]

<img file="PL3000813T3_D0085.tif" />

[0427] Created as in Reference Example 1 of 2- (butyloxy) -9- [6 (hexahvdro-1 / -azepin-1-yl) hexyl] -8- (methyl) -9 / -purine-6-amino .

LCMS (System D): tRET = 2.76 min; MH⁺ = 405

Reference Example 16: 6-Amino-2- (butylamino) -9- [3- (1-pipervidinyl) propyl] -7,9-dihydro-8 / pipin-8-one [0428]

<img file="PL3000813T3_D0086.tif" />

[0429] Mixture N²-butyl-9- (3-chloropropyl) -8- (methyloxy) -9H-purine-2,6-diamine (250 mg, 0.8 mmol), piperidine (340 mg, 4 mmol) and sodium iodide (360 mg , 2.4 mmol) in THF (8 mL) was heated to reflux for 48 hours. The solvent was evaporated and the residue was purified by preparative method

TLC and then dissolved in methanol (5 mL). Hydrogen chloride in methanol (0.5 ml) was added and the mixture was stirred at room temperature for 16 hours. The solvent was then evaporated and the pH of the residue was adjusted to 7-8 by the addition of sodium bicarbonate solution. The product was extracted into ethyl acetate and the extract was evaporated and the residue was purified by preparative HPLC to give the title compound (16 mg).

LCMS (System A): tRET = 0.55 Min; MH⁺ = 348

Reference Example 17: 6-Amino-2- (butylamino) -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8H-purin-8-one [0430]

<img file="PL3000813T3_D0087.tif" />

[0431] Prepared similar to Reference Example 1 of N²-butyl-8- (methyloxy) -9- [4- (1-piperidinyl) butyl] -9H-purine-2,6-diamine.

LCMS (System B): tRET = 0.96 min; MH⁺ = 362

Reference Example 18: 6-Amino-2- (butylamino) -9- [4- (hexahydro-1H-azepin-1-yl) butyl] -7,9-dihydro-8H-purin-8-one [0432]

<img file="PL3000813T3_D0088.tif" />

[0433] Prepared similar to Reference Example 1 with N²-butyl-9- [4- (hexahydro-1H-azepin-1-yl) butyl] -8- (methyloxy) -9H-purine-2,6-diamine.

LCMS (System B): tRET = 1.12 min; MH⁺ = 376

Reference Example 19: 6-Amino-2- (butylamino) -9- [5- (hexahydro-1H-azepin-1-yl) pentyl] -7,9-dihydro-8H-purin-8-one [0434]

<img file="PL3000813T3_D0089.tif" />

[0435] Trifluoroacetate A^;-biityl-8- (inoxy) -3 // - pyridyo-2.6-diamino (192 mg, 0.547 mmol) and potassium carbonate (189 mg, 1.368 mmol) were suspended

DMF (3 mL) and heated to 60 ° C for 1 hour. The reaction mixture was cooled to room temperature and 1-bromo-5-chloropentane (0.072 mL, 0.547 mmol) was added and the reaction mixture was stirred for another 18 hours. Hexahydro-1H-azepine (54.2 mg, 0.547 mmol) and triethylamine (0.076 mL, 0.547 mmol) were added and the reaction mixture was heated to 70 ° C for 24 hours. LCMS analysis showed the main peak at MH⁺ 404 corresponding to the creation of N²-butvlo-9- [5- (hexahvdro-1 / -azepin-1-yl) pentyl] -8- (methyloxy) -9 / purple-2,6-diaminine. The solvent was removed in vacuo and the residue was partitioned between DCM (2 mL) and water (2 mL). The aqueous layer was re-extracted with DCM (2 mL) and the combined organic extracts concentrated and the residue dissolved in 1: 1 MeOH: DMSO (2 mL) and purified by MDAP (Method C). In the evaporation of the fractions containing the product, a residue of TFA salt was obtained, whose LCMS analysis showed that the 8-methoxyl group was hydrolyzed, presumably during concentration in the presence of TFA. This crude substance was once again dissolved in 1: 1

MeOH: DMSO (2 ml) and again purified by MDAP (Method A). The product-containing fractions were evaporated under a stream of nitrogen to give the solid compound as a white solid (39 mg).

LCMS (System B): tRET = 1.18 min; MH⁺ = 390

Example 1: 6-Amino-2 - {[(15) -1-methylbutvlo] oksvł-9- [4- (1-pipervdvnvlo) -butvlo] -7,9dihvdro-8 / -purvn-8-on [0436]

<img file="PL3000813T3_D0090.tif" />

[0437] Created as in Reference Example 19 from trifluoroacetate 2 {[(1S) -1-methylbutyl] oksv} -8- (methyll) -1 / purple-6-amino, 1-bromo-4-chlorobutane and pipervdvnv .

LCMS (System B): tRET = 1.38 min; MH⁺ = 377

Example_2: _6-Amino-9- [4- (hexahvdro-1 / -azepin-1-yl) butyl] -2 - {[(15) -1methylbutyl] oksv} -7,9-dihvdro-8 / -purvn- 8-on [0438]

<img file="PL3000813T3_D0091.tif" />

[0439] Created as in Reference Example 1 of 9-14- (hexahvdro-1 / azepin-1-yl) butvolol-2- {1 (1S) - 1-methylbutoxyl} -8- (methyll) -9 / - purin-6-amine.

LCMS (System B): tRET = 1.48 min; MH⁺ = 391

Example 3: 6-Amino-2 - {[(15) -1-methylbutvlo] oksv} -9- [5- (1-pipervdvnvlo) pentvlo] -7,95 dihvdro-8 // - piirvn-8-on [ 0440]

<img file="PL3000813T3_D0092.tif" />

[0441] Created as in Reference Example 1 of 2- {1 (1 S) -1-methylbutoxyl} -8- (methylox) -9-15- (1-piperidyl) pentvlol-9H-purine-6-amino in as follows:

A solution of hydrogen chloride in dioxane (4 M, 0.71 mL) was added to a solution of 2- {1 (1S) -1-methyl butoxyl} -8- (methylox) -9-15- (1-piperidol) pentvolol-9 / -purine-6 -aminv (0.046 g, 0.126 mmol) in methanol (3 mL). The resulting mixture was allowed to stand overnight at room temperature and then purged under nitrogen. The residue was dissolved in methanol and applied to a 2 g cartridge of aminopropyl SPE (previously methanol condensed), methanol and the resulting solution was purged under a nitrogen atmosphere, whereby the solid compound was obtained in the form of a yellow solid (40.97 mg).

LCMS (System D): tRET = 2.70 min; MH + = 391 [0442] A similarly prepared sample (1.7 g) was recrystallized from ethyl acetate (approximately 50 ml). The crystals were collected, washed with ice-cold ethyl acetate (15 ml) and dried under vacuum at 50 ° C for 3 hours, whereby the resulting compound was obtained in the form of a cream-colored solid (1.33 g). Onset melting point (DSC): 207.4 ° C (see Fig. 2)

XRPD: (see Fig. 1 and Table 1)

Example_4: _6-Amino-9- [5- (hexahvdro- 1 / -azepin-1-yl) pentvlo] -2 - {[(15) -1methylbutvlo] oksv} -7,9-dihvdro-8 / -purvn- 8-on [0443]

<img file="PL3000813T3_D0093.tif" />

[0444] Prepared as in Reference Example 19 from trifluoroacetate 2 {[(1S) -1-methylbutyl] oxv} -8- (methyll) -1 / -purine-6-amino, 1-bromo-5-chloropentane and hexahvdro -1 / -azepinv.

LCMS (System B): tRET = 1.54 min; MH⁺ = 405

Reference Example 20: 6-Amino-2 - {[(15) -1-metvlopropvlo] oksv} -9- [4- (1-piperidyl) butyl] -7,9-dihydro-8 / -purin-8-one [0445]

<img file="PL3000813T3_D0094.tif" />

[0446] Created as in Reference Example 25 of 9- (4-chlorobutyl) -8 (methyloxy) -2 - {[(1S) -1-methylpropyl] oksv} -9 / -purine-6-amino and pipervdvnv.

LCMS (System D): tRET = 2.27 min; MH⁺ = 363

A sample of the intermediate 8-methoxyl derivative 8- (methoxyl) -2- {[(1S) -1-methylpropyl] oxv} -9- [4- (1-piperidyl) butyl] -9H-purine-6-amino is also isolated.

LCMS (System D): tRET = 2.56 min; MH⁺ = 377

Reference Example 21: 6-Amino-2- {[(15) -1-methylpentyl] oxy} -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8 / -purin-8-one [0447]

<img file="PL3000813T3_D0095.tif" />

[0448] Created as in Reference Example 25 of 9- (4-chlorobutyl) -8 (methyloxy) -2 - {[(15) -1-methylpentyl] oksv} -9 / -purine-6-amino and pipervdvnv.

LCMS (System D): tRET = 2.72 min; MH⁺ = 391

A sample of an intermediate 8-methoxyl derivative was also isolated

8- (methyloxy) -2 - {[(15) -1-methylpentyl] oksv} -9- [4- (1-piperidyl) butyl] -9 / -purine-6-amino.

LCMS (System D): tRET = 3.01 min; MH⁺ = 405

Reference Example 22: 6-Amino-2 - [(1-methylethyl) oxv] -9- [5- (1-pipervidyl) pentyl] -7,9-dihydro-8 / -purin-8-one [0449]

<img file="PL3000813T3_D0096.tif" />

[0450] Prepared as in Reference Example 25 from 9- (5-chloropentyl) -2 [(1-methylethyl) oxy] -8- (methyloxy) -9H-purin-6-amine and piperidine.

LCMS (System D): tRET = 2.18 min; MH⁺ = 363

A sample of the intermediate 8-methoxy derivative was also isolated

2 - [(1-methylethyl) oxy] -8- (methyloxy) -9- [5- (1-piperidinyl) pentyl] -9H-purin-6-amine.

LCMS (System D): tRET = 2.43 min; MH⁺ = 377

Reference Example 23: 6-Amino-2- (cyclobutyloxy) -9- [4- (1-piperidinyl) butyl] -7,910 dihydro-8H-purin-8-one [0451]

<img file="PL3000813T3_D0097.tif" />

[0452] Prepared similar to Reference Example 25 from 9- (4-chlorobutyl) -2 (cyclobutyloxy) -8- (methyloxy) -9H-purin-6-amine and piperidine.

LCMS (System D): tRET = 2.24 min; MH⁺ = 361

A sample of the intermediate 8-methoxy derivative was also isolated

2- (cyclobutyloxy) -8- (methyloxy) -9- [4- (1-piperidinyl) butyl] -9H-purin-6-amine.

LCMS (System D): tRET = 2.49min; MH⁺ = 375

Reference Example 24: 6-Amino-2- (cyclopentyloxy) -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8H-purin-8-one [0453]

<img file="PL3000813T3_D0098.tif" />

[0454] Prepared similar to Reference Example 25 from 9- (4-chlorobutyl) -2 (cyclopentyloxy) -8- (methyloxy) -9H-purin-6-amine and piperidine.

LCMS (System D): tRET = 2.38 min; MH⁺ = 375

A sample of intermediate 8-methoxy derivative 2- (cyclopentyloxy) -8- (methyloxy) -9- [4- (1-piperidinyl) butyl] -9H-purin-6-amine was also isolated.

LCMS (System D): tRET = 2.64 min; MH⁺ = 389

Reference Example 25: 6-Amino-2- (cyclohexyloxy) -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8H-purin-8-one [0455]

<img file="PL3000813T3_D0099.tif" />

[0456] Sodium iodide (0.006 g, 0.04 mmol) was added with stirring to a mixture of 9- (4-chlorobutyl) -2- (cyclohexyloxy) -8- (methyloxy) -9H-purin-6-amine (0.103 g, 0.303 mmol), N, N-diisopropylethylamine (0.105 M, 0.079 g, 0.609 mmol) and piperidine (0.120 mL, 0.103 g, 1.215 mmol) in DMF (1.5 mL). The resulting mixture was heated at 80 ° C for 20 hours, when LCMS analysis showed the formation of two products, one corresponding to the chloride exchange for the piperidine moiety and the other corresponding to the hydrolyzing 8-methoxy moiety. The reaction mixture was partitioned between dichloromethane (6 ml) and water (6 ml) and the phases separated using a hydrophobic frit. The solvent was removed from the organic phase under a stream of nitrogen in a blowdown device and the residue was dissolved in 1: 1 MeOH: DMSO (2 mL) and separated using a mass spectrometer-coupled autopreparative method (Method A) to give the title compound in white form solid (16.6 mg).

LCMS (System D): tRET = 2.53 min; MH⁺ = 389

Intermediate 2- (cyclohexyloxy) -8- (methyloxy) -9- [4- (1-piperidinyl) butyl 1-9H-purin-6-amine was also isolated as a colorless solid (55.2 mg).

LCMS (System D): tRET = 2.80 min; MH⁺ = 403

Reference Example 26: 6-Amino-2 - {[(1ffi-1-methylbutyl1amino} -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8H-purin-8-one [0457]

<img file="PL3000813T3_D0100.tif" />

[0458] Prepared similar to Reference Example 25 from 9- (4-chlorobutyl) -N²[(1R) -1-methylbutyl 1-8- ( methyloxy) -9H-purine-2,6-diamine and piperidine.

LCMS (System D): tRET = 2.47 min; MH⁺ = 376

A sample of the intermediate 8-methoxy derivative was also isolated

N²- [(1R) -1-metylobutylo1-8- (methyloxy) -9- [4- (1-piperidinyl) butyl1-9-purine-2,6-diamine.

LCMS (System D): tRET = 2.76 min; MH⁺ = 390

Reference Example 27: 6-Amino-2 - {[(15) -1-methylbutyl] amino} -9- [4- (1-piperidinyl) butyl] -7,9-dihydro-8H-purin-8-one [ 0459]

<img file="PL3000813T3_D0101.tif" />

[0460] Prepared similar to Reference Example 25 from 9- (4-chlorobutyl) -N²[(1S) -1-methylbutyl] -8- (methyloxy) -9H-purine-2,6-diamine and piperidine.

LCMS (System D): tRET = 2.47 min; MH⁺ = 376

A sample of the intermediate 8 N-methoxy derivative 5 N was also isolated²- [(1S) -1-methylbutyl] -8- (methyloxy) -9- [4- (1-piperidinyl) butyl] -9H-purine-2,6-diamine.

LCMS (System D): tRET = 2.76 min; MH⁺ = 390

Example 5: 6-Amino-2- {[(15) -1-methylbutyl] oxy} -9- [3- (1-piperidinyl) propyl] -7,9-dihydro-8H-purin-8-one [0461]

<img file="PL3000813T3_D0102.tif" />

[0462] Prepared similar to Reference Example 25 from 9- (3-chloropropyl) -2 {[(15) -1-methylbutyl] oxy} -8- (methyloxy) -9H-purin-6-amine and piperidine.

LCMS (System D): tRET = 2.52 min; MH⁺ = 363

A sample of the intermediate 8-methoxy derivative 2 - {[(15) -1-methylbutyl] oxy} -8- (methyloxy) -9- [3- (1-piperidinyl) propyl] -9H-purine-6-amine was also isolated. .

LCMS (System D): tRET = 2.87 min; MH⁺ = 377

Reference Example 28: 6-Amino-2 - {[(15) -1-methylpropyl] oxy} -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purin-8-one [ 0463]

<img file="PL3000813T3_D0103.tif" />

[0464] Prepared similar to Reference Example 1 of 8- (methyloxy) -2 - {[(15) 1-methylpropyl] oxy} -9- [5- (1-piperidinyl) pentyl] -9H-purin-6- amine.

LCMS (System D): tRET = 2.39min; MH⁺ = 377

Reference Example 29: 6-Amino-2- (butyloxy) -9- [3- (1-piperidinyl) propyl] -7,9-di25 hydro-8H-purin-8-one [0465]

<img file="PL3000813T3_D0104.tif" />

[0466] Prepared similar to Reference Example 1 from 2- (butyloxy) -8 (methyloxy) -9- [3- (1-piperidinyl) propylol-9H-purin-6-amine.

LCMS (System B): tRET = 1.23 min; MH⁺ = 349

Polymorphism [0467] X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) were performed on 6-amino-2 - {[(1S) -1-methylbutyloloxy} -9- [5- (1-piperidinyl) pentylol-7, 9-dihydro-8H-purin-8-ones according to the following methods.

XRPD [0468] XRPD data was recorded on a PANalytical X'Pert Pro powder diffractometer equipped with an X'Celerator detector. Data collection conditions were: radiation: Cu Ka, generator voltage: 40 kV, generator current: 45 mA, initial angle: 2.0 ° 2θ, final angle: 40.0 ° 2θ, step size: 0.0167 ° 2θ. The time per step was 31.750 s. A sample was prepared by mounting a few milligrams of sample on a Si wafer plate (zero background), which gave a thin layer of powder.

[0469] The characteristic peak positions and calculated intervals d are summarized in Table 1. They were calculated from primary data using Highscore software. The experimental error at the peak positions was approximately ± 0.1 ° 2θ. Relative peak intensities will vary depending on the preferred orientation.

Table 1

<td colspan="2">Characteristic positions of the XRPD peaks for solid Form 1 6-amino-2 - {[(1S) -1-</td>

<td colspan="2">metylobutyloloksy} -9- [5- (1-piperidinyl) -pentyl-7,9-dihydro-8H-purin-8-one</td>

<td colspan="2">Character 1</td>

<td>2θ / °</td><td>space d / A</td>

<td>5.0</td><td>17.6</td>

<td>10.0</td><td>8.8</td>

<td>12.7</td><td>7.0</td>

<td>13.5</td><td>6.5</td>

<td>13.8</td><td>6.4</td>

<td>16.6</td><td>5.3</td>

<td>18.9</td><td>4.7</td>

<td>20.0</td><td>4.4</td>

<td>22.2</td><td>4.0</td>

<td>23.3</td><td>3.8</td>

<td>24.2</td><td>3.7</td>

<td>26.1</td><td>3.4</td>

[0470] A representative XRPD pattern of 6-amino-2 - {[(1S) -1-methylbutyloloxy} -9 [5- (1-piperidinyl) pentylol-7,9-dihydro-8H-purin-8-one is shown in Fig. 1.

DSC [0471] A DSC thermogram was obtained using a TA Instruments calorimeter. The sample was weighed into an aluminum crucible, the crucible lid was placed on top and lightly crimped without sealing the crucible. The experiment was carried out using a heating rate of 10 ° C_min^-1.

[0472] A representative DSC thermogram of 6-amino-2 - {[(1S) -1-methylbutylloxy} -9- [5- (1-piperidinyl) pentylol-7,9-dihydro-8H-purin-8-one is shown in Fig. 2.

Biological data [0473] The compounds of the disclosure were tested for in vitro biological activity according to the following tests, or similar tests:

Interferon-α induction test using low-temperature preserved peripheral blood human mononuclear cells (PBMCs)

Preparation of the compound [0474] The compounds were dissolved in DMSO. Servial 2-fold dilutions with DMSO and 0.25 μl were dispensed into 384-well transparent Greiner polypropylene plates.

Preparation of PBMC [0475] Blood samples up to 200 ml were obtained from healthy human donors. Whole blood in 25 ml volumes was applied to 15 ml Ficoll gradients in Leucosep tubes, and centrifuged at 1000 g for 20 min. Cells at the plasma / Histopaque interface were carefully removed and washed twice with PBS (centrifuged at 400 g for 5 min to collect). The final pellet was resuspended in freezing medium (90% temperature inactivated serum, 10% DMSO) to a concentration of 4x10 cells⁷ cells / ml. The re-suspended cells were then preserved at low temperature (frozen) using a controlled freezing rate freezer and stored at -140 ° C for up to 4 months.

Incubation and interferon-α assay [0476] Just before the test, vials with low temperature preserved (frozen) PBMCs were thawed rapidly in a 37 ° C water bath. A 1:10 dilution of trypan blue cells was prepared and counted. PBMC was then diluted in growth medium [RPMI 1640 containing 10% fetal calf serum (Invitrogen), penicillin + streptavidin (Gibco cat. No. 25030-024, 1:50), 2 mM Lglutamine, and 1000 units / ml recombinant human IFN-gamma (Preprotech Catalog No. 300-02)] up to a density of 1 x 10⁶ cells / ml, and 50 μl / well were dosed into 384-well transparent Greiner polypropylene plates containing 0.25 μl DMSO or test compound in 0.25 μl DMSO. The upper final compound concentration was typically 50 μΜ or 5 μΜ (to obtain curve fitting for highly active compounds). Plates were incubated for 24 hours at 37 ° C in 5% CO2.

[0477] An immunoassay for many isoforms was used to count IFN-α in PBMC supernatants. Polyclonal rabbit anti-human IFN-α antibody (catalog number 31101, Stratech Scientific) was diluted 1: 10,000 in assay buffer (RPMI 1640 containing 10% fetal calf serum, Invitrogen) and 20 μl was added to each well of MSD (Meso-Scale Discovery) single 384-well plate with small GAR spots (coated with goat anti-rabbit antibody). The plate was incubated for 1 hour at room temperature with vigorous shaking. After three washes with PBS, 20 ml cell supernatant was added to each well of the plate. The plate was then incubated for 1 hour at room temperature with vigorous shaking. A pair of monoclonal antibodies to IFN-α (catalog numbers 21100 and 21112, Stratech Scientific) were labeled with sulfo-TAG (MSD), diluted 1: 1000 in assay buffer and 20 μl added to each well of the plate. The plate was then incubated for 1 hour at room temperature with vigorous shaking. After three washes with PBS, 30 μl x2 T buffer (MSD) was added to each well and the plate was read on an MSD Sector 6000 plate reader.

[0478] Data were normalized for internal plate controls with 1 μM resiquimod (n = 16) and DMSO (n = 16). The pEC50 values ​​were obtained by fitting a 4-parameter curve with IRLS in ActivityBase, from 11-point, 2-fold serial dilutions of test compounds.

Results [0479] The compounds of reference examples 1 to 30 and examples 1 to 4 showed average pEC50&gt; 5.5.

Interferon-α and TNF-α induction test using fresh human peripheral blood mononuclear cells (PBMCs)

Compound Preparation [0480] The compounds were dissolved and serially diluted in DMSO to give 100x the desired concentration range using Biomek 2000. 1 Pl of test compound was transferred to 96 well tissue culture plates using Biomek FX. Each compound was tested twice for each donor. Each plate contained a dilution series of TLR7 / 8 agonist resiquimod as standard, and Column 11 contained 1 μl 200 μΜ resiquimod (resulting in a final concentration of 2 μΜ, used to define the approximate maximum response to resiquimod).

Preparation of PBMC [0481] Blood samples from two human donors were collected into sodium heparin (10 U / ml). 25 ml volumes of whole blood were applied to 15 ml Histopaque in Leucosep tubes, which were centrifuged at 800 g for 20 min and the band at the plasma / Histopaque interface was carefully removed. Harvested cells were centrifuged at 2500 rpm for 10 min and the pellet was resuspended in 10 ml medium (RPMI 1640 (low endotoxin) supplemented with 10% v / v. fetal calf serum (FCS, low endotoxin) 100 U / ml penicillin G, 100 gg / ml streptomycin, 10 mM L-glutamine and 1 x endogenous amino acids). A 1:20 dilution of cells was made using trypan blue and cells were counted using a hemocytometer. PBMC was diluted to give a final concentration of 2x10⁶/ ml and 100 g of this cell suspension were added to wells containing 1 g of diluted test compound.

Incubation and tests for interferon-α and TNF-α [0482] Cell preparations were incubated for 24 h (37 ° C, 95% air, 5% CO2), after which the supernatant sample was removed using Biomek FX and tested for IFN-α and TNF -α using the MSD (Mesoscale Discovery) electrochemiluminescence test platform. The IFN-α test was carried out similarly as described above. The TNF-α test was carried out according to the instructions in the kit (catalog number K111BHB).

[0483] Cytokine release is expressed as a percentage of control 2 μΜ resiquimod (column 11). These percentages were plotted as a function of compound concentration and pEC50 for the response was determined by curve fitting by a non-linear least squares method. A 4-parameter logistic model was generally chosen for the IFN-α response. For TNF responses where a clear maximal response was obtained (i.e. a well-defined plateau was observed in response) a 4-parameter model was generally used. If the upper curve asymptote was not well defined, the curve fit was generally narrowed to a maximum response of 100% (i.e. to response to 2 μΜ of resiquimod) or to response to the highest test concentration if greater than the response of resiquimod. Certain curves were bell shape for one or both cytokines and cytokine data on the falling edge of the bell shape response (i.e. concentrations above maximal response) were generally excluded from matching, usually with the exception of concentrations just above the peak response. Curve fitting therefore focused on the increasing slope of the dose response curve.

Results [0484] Compounds of reference examples 5 and 9 showed average pEC50s for induction of IFNα and TNF-α&gt; 7.5 and &lt;5.5, respectively. Compounds of reference examples 6, 7, 10 to 12, 14 and 18 showed average pEC50s for induction of IFN-α and TNF-α&gt; 8 and &lt;6, respectively. The compounds of reference examples 13 and 15 and examples 1 to 4 showed average pEC50s for the induction of IFN-α and TNF-α&gt; 9 and &lt;6, respectively.

Allergen-based cytokine test using fresh human peripheral blood mononuclear cells (PBMCs) from atopic volunteers [0485] A test based on co-culture of atopic human donor peripheral blood mononuclear cells (PBMCs) with the allergen and test compounds was developed. After 5-6 days of culture, cell supernatants were tested for a number of cytokines.

Compound preparation [0486] The compounds were dissolved in DMSO, then serially diluted in growth medium (RPMI 1640 medium supplemented with 100 U / ml penicillin G, 100 gg / ml streptomycin, 10 mM L-glutamine) to give 4x the desired concentration range in the presence of 0, 04% DMSO. Each compound was tested in triplicate at all concentrations. Preparation of PBMC [0487] Defibrinated human blood from volunteers with timothy allergy was centrifuged at 2500 rpm for 15 minutes. The upper serum layer was collected and heat inactivated at 56 ° C for 30 minutes (HI-autologous serum). The lower cell layer was resuspended in 50 ml PBS (+ Ca + Mg), 25 ml diluted blood was applied to 20 ml Lymphoprep in 50 ml tubes, then centrifuged at 2500 rpm for 20 minutes at RT The band at the serum / Lymphoprep interface was carefully removed. Harvested cells were washed with PBS and resuspended at 4x10⁶ per ml in growth medium with Hl-autologous serum. PBMCs were seeded at 0.4x10⁶ cells per well in flat-bottomed 96-well plates in the presence of 10 ug / ml Timothy meadow antigen (Alk Abello) and test compounds at appropriate concentrations in a total volume of 200 g.

Incubation and cytokine tests [0488] Plates were incubated at 37 ° C in 5% CO2 for up to 6 days. Cell medium from each well was harvested and stored at -20 ° C prior to analysis. Cytokines and chemokines in the supernatants were detected using Meso Scale Discovery 10-spot human TH1 / Th2 cytokines.

[0489] In the above assay, data from separate PBMC studies from three allergic donors showed that the compound of Example 3 reduces the production of Th2 IL-5 and IL-13 Th2 cytokines in a dose-dependent manner with a reduction&gt; 50% observed at 0.04 μΜ compared with the control allergen.

[0490] The compound of Examples 2 and 3 of the disclosure was also tested for in vivo biological activity in the following model:

Interferon-α induction test after nasal dosing in mice.

[0491] The compounds were dissolved in 0.2% Tween 80 in physiological saline and administered intranasally (5 μl total between nostrils) to female BALB / c mice (n = 6) under general anesthesia. Animals were sacrificed 2 hours post-dose and a post-mortem blood sample was taken, and serum levels of interferon-α were measured using an ELISA.

[0492] In this model, the compound of Example 2 showed average serum levels of interferon-α 20326 pg / ml, and the compound of Example 3 showed average levels in serum of interferon-α 21029 pg / ml. No interferon-α was detected in vehicle-treated control samples.