Phosphoryl purine ketone compounds for treatment of cancer

Abstract

The present invention relates to compounds of formula (I) # imgabs0 # wherein R1 to R3 are as described herein, and pharmaceutically acceptable salts thereof, as well as compositions comprising the compounds and methods of using the compounds.

Description

[0001] The present invention relates to organic compounds useful for treatment and / or prevention in mammals, and in particular to agonism of TLR7 useful for the treatment of cancer. Technical Field

[0002] The present invention relates to novel phosphoryl purinone and its derivatives having Toll-like receptor agonist activity, as well as their preparation, pharmaceutical compositions containing them and their potential use as drugs for treating and / or preventing cancer.

[0003] Although immune checkpoint inhibitors have changed the treatment landscape for many tumors, durable responses are limited. Lymphocyte infiltration and IFN-γ status are key factors for effective therapy by defining a "T cell inflamed" phenotype ("hot tumors"). In contrast, "non-inflamed" or "cold tumors" are characterized by a lack of T cell infiltration of the tumor. Immunotherapy of cold tumors is a huge challenge because adaptive immune responses have not yet been established or maintained. However, cold tumors may contain a large number of myeloid cells, including macrophages, different DC subsets, and myeloid-derived suppressor cells. Agonistic approaches targeting myeloid cells aim to induce anti-tumor inflammatory responses by secreting proinflammatory cytokines, and thereby support T cell effector function and memory formation. Given that myeloid cells are also present in cold tumors, activating them in the tumor microenvironment will increase T cell infiltration and sustained activation in these tumors. This can translate into clinical benefit in inflamed and immune-excluded tumors, for which only a small number of patients have benefited from immunotherapy to date.

[0004] As part of the innate immune system, myeloid cells are able to respond to infection and abnormal cell behavior. Myeloid cells are the first to detect pathogens / abnormal cells and eliminate them through different mechanisms, including inflammatory cytokine secretion and phagocytosis. Continuous activation of T cells by providing co-stimulatory signals and antigen presentation to T cells is another key function of myeloid cells, paving the way for a lasting immune response. On the other hand, myeloid cells also play a key role in tissue repair and homeostasis by inhibiting uncontrolled immune activation. Tumors take advantage of this property of these cells by recruiting them to create an inhibitory microenvironment, thereby inhibiting T cells through direct and indirect mechanisms. By delivering the correct signals, such as TLR agonists, tumor-associated myeloid cells can still be activated in an inhibitory tumor microenvironment. After activation, they can produce inflammatory cytokines and upregulate activation markers, thereby triggering an anti-tumor immune response, ideally resulting in a lasting adaptive response.

[0005] Toll-like receptors (TLRs) detect highly conserved pathogenic danger signals, leading to a strong inflammatory response. TLR signaling stimulates innate and ultimately acquired immune responses against pathogens. Toll-like receptors (TLRs) detect a variety of conserved pathogen-associated molecular patterns (PAMPs). They play an important role in sensing invading pathogens and subsequently initiating innate immune responses. The human TLR family has 10 known members, which are type I transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic tail containing a conserved Toll / interleukin (IL)-1 receptor (TIR) ​​domain. In this family, TLR3, TLR7, TLR8, and TLR9 are localized in endosomes.

[0006] TLR7 can be activated by binding to specific small molecule ligands (i.e., TLR7 agonists) or its natural ligands (i.e., single-stranded RNA, ssRNA). After ssRNA binds to TLR7, the receptor present in its dimerized form is believed to undergo structural changes, resulting in subsequent recruitment of adapter proteins in its cytoplasmic domain, including myeloid differentiation primary response gene 88 (MyD88). After the MyD88 approach initiates the receptor signal cascade, cytoplasmic transcription factors such as interferon regulatory factor 7 (IRF-7) and nuclear factor κB (NF-κB) are activated. These transcription factors then translocate to the nucleus and initiate transcription of various genes (e.g., IFN-α and other antiviral cytokine genes). People are highly concerned about the activation of bone marrow cells in tumors, which initiate and coordinate immune responses and express TLR7 (natural ligands are single-stranded RNAs from viruses) in endosomes. The systemic administration of TLR agonists leads to strong systemic activation of different immune cells, and ultimately activates immune cells in the tumor microenvironment to achieve anti-tumor effects. In fact, several identified TLR7 agonists have been considered for therapeutic purposes. The TLR7 agonist Imiquimod (R-837) was applied topically to subcutaneous tumors in mice or human patients with skin cancer lesions. It strongly activates anti-tumor immune responses. The TLR7 / 8 dual agonist Resiquimod (R-848) is an immunomodulatory topical gel that is undergoing Phase II clinical trials at Galderma for the treatment of stage cutaneous T-cell lymphoma. MBS-8 is a micellar nanoparticle formulation of the TLR7 agonist 1V270, developed by MonTa Biosciences for intravenous immunotherapy of advanced solid tumors. BDB-001 is a TLR7 / 8 dual agonist that is undergoing Phase II clinical trials at Seven and Eight Biopharmaceuticals as an intravenous treatment for advanced or metastatic solid tumors that are refractory to anti-PD-1 / anti-PD-L1 therapy. APR-003 is an oral small molecule TLR7 agonist that is in early clinical development at Apros Therapeutics for the treatment of patients with advanced, unresectable colorectal cancer with malignant liver lesions. PRTX-007 is an oral small molecule TLR7 agonist in early clinical development at Primmune Therapeutics for the treatment of acute viral diseases (including SARS-CoV-2) and cancer. Therefore, there is still a need to identify additional compounds suitable for systemic administration and preferentially targeting tumors. There is an unmet clinical need to develop effective and safe TLR7 agonists to treat a variety of cancers. Summary of the invention

[0007] The present invention relates to novel compounds having formula (I),

[0008]

[0009] in

[0010] R 1 C 1-6 alkyl;

[0011] R 2 C 1-6 alkyl;

[0012] R 3 is pyridinyl substituted by: ((C 1-6 alkyl)2amino)C 1-6 Alkoxy, ((C 1-6 alkyl)2amino)C 1-6 Alkylamino, ((C 1-6 alkyl)2amino)C 1-6 Alkylpyrrolidyl,

[0013] (C 1-6alkyl)2-piperazinyl, (C 1-6 Alkylamino)C 1-6 Alkylamino, (C 1-6 alkylpyrrolidinyl)amino, 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl, 2,5-diazabicyclo[2.2.2]octanyl, aminopyrrolidinyl, C 1-6 Alkyl-1-oxa-4,9-diazaspiro[5.5]undecyl, C 1-6 Alkylpiperazine, C 1-6 Alkylsulfonylpiperazinyl, pyrrolidinyl C 1-6 Alkylamino or tetrahydropyranylamino;

[0014] or a pharmaceutically acceptable salt thereof.

[0015] The invention also relates to their manufacture, to medicaments based on the compounds according to the invention and their production, and to the use of the compounds of formula (I) as agonists of TLR7.

[0016] The compound of formula (I) shows good TLR7 agonist activity. In another embodiment, compared with the reference compound RO14, the compound of the present invention shows excellent kinetic solubility and thermodynamic (equilibrium) solubility. In addition, compared with the reference compound, the compound of formula (I) shows significantly improved fasting state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) dissolution results. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 X-ray crystallographic analysis of Example 15-B

[0018] Figure 2 X-ray crystallographic analysis of compound 18e-A DETAILED DESCRIPTION

[0019] definition

[0020] The term "C 1-6 "Alkyl" means a saturated, straight-chain or branched alkyl group containing 1 to 6, especially 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. In particular, "C 1-6 "Alkyl" groups are methyl, ethyl and n-propyl.

[0021] The term "C 1-6 "Alkoxy" means C 1-6 Alkyl-O-.

[0022] The terms "halogen" and "halo" are used interchangeably herein to refer to fluorine, chlorine, bromine or iodine.

[0023] The term "pharmaceutically acceptable salts" refers to salts that are not biologically or otherwise undesirable. "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts.

[0024] "Pharmaceutically acceptable acid addition salts" refer to those pharmaceutically acceptable salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the like, and organic acids, which may be selected from aliphatic, alicyclic, aromatic, aromatic aliphatic, heterocyclic, formic and sulfonic acid organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid, and the like.

[0025] The term "pharmaceutical base addition salt" refers to those pharmaceutical salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary amines, secondary amines, tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine and polyamine resins).

[0026] The term "pharmaceutically active metabolite" refers to a pharmacologically active product produced by the metabolism of a specific compound or its salt in the body. After entering the human body, most drugs are substrates for chemical reactions that may change their physical properties and biological effects. These metabolic transformations, which usually affect the polarity of the compounds of the present invention, change the way the drug is distributed in the body and excreted from the body. However, in some cases, drug metabolism is necessary for therapeutic effects.

[0027] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the invention that, when administered to a subject, (i) treats or prevents a particular disease, disorder or condition, (ii) reduces, ameliorates or eliminates one or more symptoms of a particular disease, disorder or condition, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, disorder or condition as described herein. The therapeutically effective amount depends on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending medical or veterinary doctor and other factors.

[0028] The term "pharmaceutical composition" means a mixture or solution comprising a therapeutically effective amount of an active drug ingredient together with a pharmaceutically acceptable excipient for administration to a mammal (eg, a human) in need thereof.

[0029] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," and "therapeutically inert excipient" are used interchangeably and refer to any pharmaceutical ingredient in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant used to formulate a drug product.

[0030] TLR7 agonists

[0031] The present invention relates to (i) a compound of formula (I),

[0032]

[0033] in

[0034] R 1 C 1-6 alkyl;

[0035] R 2 C 1-6 alkyl;

[0036] R 3 is pyridinyl substituted by: ((C 1-6 alkyl)2amino)C 1-6 Alkoxy, ((C 1-6 alkyl)2amino)C 1-6 Alkylamino, ((C 1-6 alkyl)2amino)C 1-6 Alkylpyrrolidyl,

[0037] (C 1-6 alkyl)2-piperazinyl, (C 1-6 Alkylamino)C 1-6 Alkylamino, (C 1-6 alkylpyrrolidinyl)amino, 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl, 2,5-diazabicyclo[2.2.2]octanyl, aminopyrrolidinyl, C 1-6 Alkyl-1-oxa-4,9-diazaspiro[5.5]undecyl, C 1-6 Alkylpiperazine, C 1-6 Alkylsulfonylpiperazinyl, pyrrolidinyl C 1-6 Alkylamino or tetrahydropyranylamino;

[0038] or a pharmaceutically acceptable salt thereof.

[0039] A further embodiment of the present invention is (ii) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to (i), wherein R 1 It is methyl or ethyl.

[0040] A further embodiment of the present invention is (iii) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to (i) or (ii), wherein R 1 It is methyl.

[0041] A further embodiment of the present invention is (iv) a compound of formula (I) according to any one of (i) to (iii), wherein R 2 It is methyl, ethyl or propyl.

[0042] A further embodiment of the present invention is (v) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of (i) to (iv), wherein R 3 for Where R 4 for ((C 1-6 alkyl)2amino)C 1-6 Alkoxy, ((C 1-6 alkyl)2amino)C 1-6 Alkylamino, ((C 1-6 alkyl)2amino)C 1-6 Alkylpyrrolidyl, (C 1-6 alkyl)2-piperazinyl, (C 1-6 Alkylamino)C 1-6 Alkylamino, (C 1-6 alkylpyrrolidinyl)amino, 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl, 2,5-diazabicyclo[2.2.2]octanyl, aminopyrrolidinyl, C 1-6 Alkyl-1-oxa-4,9-diazaspiro[5.5]undecyl, C 1-6 Alkylpiperazine, C 1-6 Alkylsulfonylpiperazinyl, pyrrolidinyl C 1-6 Alkylamino or tetrahydropyranylamino.

[0043] A further embodiment of the present invention is (vi) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of (i) to (v), wherein R 3 for Where R 4 for ((C 1-6 alkyl)2amino)C 1-6 Alkylamino or (C 1-6 Alkylamino)C 1-6 Alkylamino.

[0044] A further embodiment of the present invention is (vii) a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of (i) to (vi), wherein R 3 It is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino.

[0045] A further embodiment of the present invention is (viii) a compound of formula (I) according to any one of (i) to (vii), wherein

[0046] R 1 C 1-6 alkyl;

[0047] R 2 C 1-6 alkyl;

[0048] R 3 for Where R 4 for ((C 1-6 alkyl)2amino)C 1-6 Alkylamino or (C 1-6 Alkylamino)C 1-6 Alkylamino;

[0049] or a pharmaceutically acceptable salt thereof.

[0050] A further embodiment of the present invention is (ix) a compound of formula (I) according to any one of (i) to (viii), wherein

[0051] R 1 is methyl;

[0052] R 2 is methyl, ethyl or propyl;

[0053] R 3 for Where R 4 is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino;

[0054] or a pharmaceutically acceptable salt thereof.

[0055] Another embodiment of the present invention is a compound of formula (I) selected from the following compounds:

[0056] 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one;

[0057] 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one;

[0058] 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one;

[0059] 6-amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one;

[0060] 6-amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one;

[0061] 9-[[6-(2,3,3a,4,6,6a-Hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-yl)-3-pyridinyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one;

[0062] 6-amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridinyl]methyl]-7H-purin-8-one;

[0063] 6-amino-2-dimethylphosphoryl-9-[[6-(tetrahydropyran-4-ylamino)-3-pyridinyl]methyl]-7H-purin-8-one;

[0064] 6-amino-9-[[6-[3-[(dimethylamino)methyl]pyrrolidin-1-yl]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one;

[0065] 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undec-9-yl)-3-pyridinyl]methyl]-7H-purin-8-one;

[0066] 6-amino-2-dimethylphosphoryl-9-[[6-[(1-methylpyrrolidin-3-yl)amino]-3-pyridinyl]methyl]-7H-purin-8-one;

[0067] 6-amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one;

[0068] 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one;

[0069] 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one;

[0070] 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one;

[0071] 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one;

[0072] 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one;

[0073] 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one;

[0074] 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one;

[0075] 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one;

[0076] 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one;

[0077] 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one;

[0078] 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one; and

[0079] 6-amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one;

[0080] or a pharmaceutically acceptable salt thereof.

[0081] Another embodiment of the present invention relates to (xi) a method for preparing the compound according to any one of (i) to (x), the method comprising the following steps:

[0082] a) making a compound of formula (III),

[0083] With amine or alcohol HR 4 (II) reacting in a solvent-free reaction; or reacting in the presence of an inorganic base, wherein the base is sodium hydride; or reacting in the presence of an organic metal catalyst system, wherein the catalyst system is selected from Pd2(dba)3 / RuPhos / t-BuONa, Pd2(dba)3 / BrettPhos / t-BuONa and Pd-PEPPSI-IPentCl / t-BuOK; wherein R 1 , R 2 and R 4 As defined in any one of (i) to (ix).

[0084] Another embodiment of the present invention is (xii) a compound or a pharmaceutically acceptable salt according to any one of (i) to (x), which is produced according to the method of claim 11.

[0085] Another embodiment of the present invention is (xiii) a pharmaceutical composition comprising the compound according to any one of (i) to (x) and a pharmaceutically acceptable excipient.

[0086] Another embodiment of the present invention is (xiv) a compound or a pharmaceutically acceptable salt according to any one of (i) to (x) for use as a therapeutically active substance.

[0087] Another embodiment of the present invention is (xv) a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, for use in treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive-stage small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma.

[0088] Another embodiment of the present invention is (xvi) use of the compound according to any one of (i) to (x) as an agonist of TLR7.

[0089] Another embodiment of the present invention is (xvii) use of a compound according to any one of (i) to (x) for treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive-stage small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma.

[0090] Another embodiment of the present invention is (xviii) use of a compound according to any one of (i) to (x) for preparing a medicament for treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive-stage small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma.

[0091] Another embodiment of the present invention is (xix) the use of a compound according to any one of (xvii) or (xviii), wherein the cancer is selected from pancreatic ductal adenocarcinoma and colorectal cancer.

[0092] Another embodiment of the present invention is (xx) a method for treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive small cell lung cancer, non-small cell lung cancer, muscle invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma, the method comprising administering a therapeutically effective amount of a compound as defined in any one of (i) to (x).

[0093] Pharmaceutical compositions and administration

[0094] Another embodiment provides a pharmaceutical composition or medicine containing the compounds of the invention and a therapeutic inert carrier, diluent or excipient, and methods for preparing such compositions and medicines using the compounds of the invention. In one example, the compound of formula (I) can be formulated as a galenical administration form by mixing with a physiologically acceptable carrier (i.e., a carrier that is nontoxic to the recipient at the dosage and concentration used) at ambient temperature at an appropriate pH and desired purity. The pH of the formulation depends primarily on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the compound of formula (I) is formulated in an acetate buffer at pH 5. In another embodiment, the compound of formula (I) is sterile. The compound can be stored, for example, as a solid or amorphous composition, as a lyophilized formulation, or as an aqueous solution.

[0095] The composition is formulated, metered and administered in a manner consistent with good medical practice. Factors to be considered in this case include the specific illness being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the agent, the method of administration, the time schedule of administration, and other factors known to practitioners. The "effective amount" of the compound to be administered will be subject to such considerations and is the minimum amount required to activate the TLR7 receptor and cause the production of INF-α and other cytokines, which can be used for, but not limited to, the treatment of cancer. For example, the amount can be lower than the amount that is toxic to normal cells or mammals as a whole.

[0096] In one example, the pharmaceutically effective amount of the compound of the present invention administered parenterally per dose will be in the range of about 0.01 to 1000 mg / kg of patient body weight per day, alternatively about 0.01 to 1000 mg / kg of patient body weight, typically an initial range of 0.3 to 15 mg / kg / day of the compound used. In another embodiment, oral unit dosage forms such as tablets and capsules preferably contain about 1 to about 1000 mg of the compound of the present invention.

[0097] The compounds of the present invention can be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and (if necessary for local treatment) intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0098] The compounds of the present invention can be administered in any convenient administration form, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain conventional components of pharmaceutical preparations, for example, diluents, carriers, pH adjusters, sweeteners, fillers and other active agents.

[0099] Typical formulations are prepared by mixing the compounds of the invention with carriers or excipients. Suitable carriers and excipients are well known to those skilled in the art and are described in, for example, Ansel, Howard C. et al., Ansel' sPharmaceutical DosageFormsandDrugDeliverySystems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al. Remington:TheScienceandPracticeo fPharmacy .Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients.Chicago, Pharmaceutical Press, 2005. The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents, flavoring agents, diluents and other known additives to provide an aesthetically pleasing display of a drug (e.g., a compound of the present invention or a pharmaceutical composition thereof) or to facilitate the preparation of a pharmaceutical product (e.g., a drug).

[0100] An example of a suitable oral dosage form is a tablet containing about 1 to 1000 mg of a compound of the present invention and about 1 to 1000 mg of anhydrous lactose, about 1 to 1000 mg of cross-linked sodium carboxymethylcellulose, about 1 to 1000 mg of polyvinyl pyrrolidone (PVP) K30 and about 1 to 1000 mg of magnesium stearate. The powdered ingredients are first mixed together and then mixed with the PVP solution. The resulting composition can be dried, granulated, mixed with magnesium stearate and compressed into tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound of the present invention (e.g., 1 to 500 mg) in a suitable buffer solution (e.g., phosphate buffer) and adding a penetration enhancer (e.g., a salt such as sodium chloride) if necessary. The solution can be filtered, for example, using a 0.2 micron filter to remove impurities and contaminants.

[0101] Thus, embodiments include pharmaceutical compositions comprising a compound of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof. Further embodiments include a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0102] Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in treating a hyperproliferative disease.Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in treating cancer.

[0103] The following examples illustrate typical compositions of the present invention but are intended to be representative only.

[0104] Composition A

[0105] The compounds of the invention can be used as active ingredients in a manner known per se to produce tablets of the following composition:

[0106]

[0107] Composition B

[0108] The compounds of the invention can be used as active ingredients in a manner known per se to produce capsules of the following composition:

[0109]

[0110]

[0111] Indications and treatments

[0112] The compound of the present invention targets TLR7. Therefore, the compound of the present invention can be used to promote the activation of antigen presenting cells (APC), contribute to the initiation, transportation and infiltration of T cells, promote Th1 and CD8+T responses, and enhance the killing of cancer. The compound of the present invention can be used to enhance the innate immune response in the bone marrow cells expressing TLR7. Alternatively, the compound of the present invention can be used to activate T cells in tumors, and T cells are present in the tumor but are suppressed, such as by the upregulation of co-stimulatory molecules and the production of proinflammatory cytokines. More broadly, the compound can be used to treat non-inflammatory cancer types (e.g., immune desert type or immune rejection type) by enhancing antigen presentation, T cell initiation, recruitment / infiltration and tumor killing.

[0113] Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to the mammal a therapeutically effective amount of a compound of formula (I), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof. Cancer herein includes, but is not limited to, advanced solid tumors, such as pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive small cell lung cancer, non-small cell lung cancer, muscle invasive bladder cancer, nodular basal cell carcinoma, squamous cell carcinoma.

[0114] synthesis

[0115] The compounds of the present invention may be prepared by any conventional method. Suitable methods for synthesizing these compounds and their starting materials are provided in the following schemes and examples. Unless otherwise indicated, all substituents, especially R 1 To R 4 As defined above. Furthermore, unless otherwise specifically indicated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to one of ordinary skill in the art of organic chemistry.

[0116] The general synthetic route for preparing compounds of formula (I) is shown below.

[0117] Solution 1

[0118]

[0119] The compound of formula (X) is prepared by reacting the compound of formula (XI) with (4-methoxyphenyl)methylamine in the presence of an organic base such as trimethylamine or N,N-diisopropylethylamine. The compound of formula (IX) is prepared by reacting the compound of formula (X) with 1-(chloromethyl)-4-methoxybenzene in the presence of an inorganic base such as potassium carbonate, sodium hydride or cesium carbonate. The compound of formula (VII) is prepared by reacting the compound of formula (IX) with the compound of formula (VIII) in the presence of an organometallic catalyst system such as Pd(OAc)2 / XantPhos / K2CO3 or Pd2(dba)3 / XantPhos / TEA. The compound of formula (V) is prepared by deprotecting the compound of formula (VII) with an acid such as trifluoroacetic acid and then alkylating it with 2-chloro-5-(chloromethyl)pyridine in the presence of an inorganic base such as potassium carbonate, sodium hydride or cesium carbonate. The compound of formula (III) is prepared by brominating the compound of formula (V) with a brominating agent such as bromine or N-bromosuccinimide, followed by hydrolysis with aqueous sodium hydroxide. The compound of formula (Ia) is prepared by reacting the compound of formula (III) with an amine or alcohol HR 4 (Compound of formula (II)) is prepared by reacting in a solvent-free reaction, or in the presence of an inorganic base (such as sodium hydride), or in the presence of an organic metal catalyst system (such as Pd2(dba)3 / RuPhos / t-BuONa, Pd2(dba)3 / BrettPhos / t-BuONa or Pd-PEPPSI-IPentCl / t-BuOK).

[0120] The compounds of the present invention can be obtained as diastereomers or mixtures of enantiomers, which can be separated by methods well known in the art, for example, (chiral) HPLC or SFC. In another embodiment, compounds of formula (I) or (Ia) can be obtained according to the above scheme by using the corresponding chiral starting materials.

[0121] The present invention also relates to a method for preparing a compound of formula (I) or formula (Ia), the method comprising the following steps:

[0122] a) making a compound of formula (III),

[0123] With amine or alcohol HR 4 The compound (II) is reacted in a solvent-free reaction, or in the presence of an inorganic base (such as sodium hydride), or in the presence of an organometallic catalyst system (such as Pd2(dba)3 / RuPhos / t-

[0124] BuONa, Pd2(dba)3 / BrettPhos / t-BuONa or Pd-PEPPSI-IPentCl / t-

[0125] The reaction was carried out in the presence of BuOK.

[0126] A compound of formula (I), which is prepared according to the above process, is also an object of the present invention.

[0127] Examples

[0128] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention.

[0129] abbreviation

[0130] aq. water-based

[0131] BrettPhos: Dicyclohexyl-[3,6-dimethoxy-2-(2,4,6-triisopropylphenyl)phenyl]phosphine

[0132] BSA: N,O-bis(trimethylsilyl)acetamide

[0133] CDI: N,N'-Carbonyldiimidazole

[0134] DIEPA: N,N-diethylpropylamine

[0135] DMF: Dimethylformamide

[0136] DBU: 1,8-diazabicycloundec-7-ene

[0137] DPPA: diphenylphosphoryl azide

[0138] EC 50 : The molar concentration of an agonist that produces 50% of the maximal possible response for that agonist.

[0139] EDC: N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine

[0140] eq: equivalent

[0141] EtOAc or EA: Ethyl acetate

[0142] HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)

[0143] hr: hours

[0144] HPLC: High Performance Liquid Chromatography

[0145] HOBt: N-Hydroxybenzotriazole

[0146] MS(ESI): mass spectrometry (electrospray ionization)

[0147] m-CPBA: 3-Chlorobenzene

[0148] MTEB: Methyl tert-butyl ether

[0149] NMR: Nuclear Magnetic Resonance

[0150] NMP: N-Methylpyrrolidone

[0151] obsd. Observed value

[0152] Pd-PEPPSI-IPentCl: (SP-4-1)-[1,3-bis[2,6-bis(1-ethylpropyl)phenyl]-4,5-dichloro-1,3-dihydro-2H-imidazol-2-ylidene]dichloro(2-methylpyridine)palladium

[0153] PE: Petroleum ether

[0154] PMB: p-methoxybenzyl

[0155] PPA: Polyphosphoric Acid

[0156] RT or rt: room temperature

[0157] RuPhos: dicyclohexyl-[2-(2,6-diisopropoxyphenyl)phenyl]phosphine

[0158] sat. saturated

[0159] SFC: Supercritical Fluid Chromatography

[0160] TEA: Triethylamine

[0161] TFA: trifluoroacetic acid

[0162] THF: Tetrahydrofuran

[0163] TLC: Thin layer chromatography

[0164] V / V: Volume ratio

[0165] XantPhos: (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine)

[0166] General experimental conditions

[0167] Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and Quad 12 / 25 Cartridge module. ii) ISCO combi-flash chromatograph. Silica gel brand and pore size: i) KP-SIL Particle size: 40-60 μm; ii) CAS registration number: silica gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Ocean Chemical Co., Ltd., pore size: 200-300 or 300-400.

[0168] Intermediates and final compounds were purified by preparative HPLC on a reverse phase column using a Phenomenex Luna C18 (15 μm, 150×400 mm) column, a Phenomenex Luna C18 (10 μm, 100×250 mm) column, a Phenomenex Luna C18 (10 μm, 80×250 mm) column, a Shim-pack C18 (10 μm, 25×150 mm) column, a Phenomenex Synergi C18 (10 μm, 25×150 mm) column or a Waters Atlantis T3 (5 μm, 30×150 mm) column. Waters AutoP purification system (sample manager 2767, pump 2525, detector: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium bicarbonate in water; acetonitrile and 0.1% FA in water, or acetonitrile and 0.1% TFA in water). Or Gilson-281 purification system (pump 322, detector: UV 156, solvent system: acetonitrile and 0.05% ammonium bicarbonate in water; acetonitrile and 0.225% FA in water; acetonitrile and 0.05% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).

[0169] Using LC / MS (Waters TM The LC / MS spectra of the compounds were obtained using an Agilent Alliance 2795-Micromass ZQ, Shimadzu Alliance 2020-Micromass ZQ, or Agilent Alliance 6110-Micromass ZQ. The LC / MS conditions were as follows (run time 3 or 1.5 min):

[0170] Acidic conditions I: A: 0.1% TFA solution in H2O; B: 0.1% TFA solution in acetonitrile;

[0171] Acidic conditions II: A: 0.0375% TFA solution in H2O; B: 0.01875% TFA solution in acetonitrile;

[0172] Basic conditions I: A: 0.1% NH3·H2O solution in H2O; B: acetonitrile;

[0173] Basic conditions II: A: 0.025% NH3·H2O solution in H2O; B: acetonitrile;

[0174] Neutral conditions: A: H2O; B: acetonitrile.

[0175] Mass spectra (MS): Usually only ions representing the parent mass are reported, and unless otherwise stated, the mass ions quoted are positive mass ions (M+H) + .

[0176] NMR spectra were obtained using a Bruker Avance 400 MHz or a Bruker Avance 500 MHz.

[0177] All reactions involving air-sensitive reagents were performed under argon atmosphere. Unless otherwise stated, reagents were purchased from commercial suppliers as received without further purification.

[0178] Preparation Example

[0179] Example 1

[0180] 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one

[0181]

[0182] Step 1: Preparation of 2-chloro-N-[(4-methoxyphenyl)methyl]-9H-purin-6-amine (Compound 1a)

[0183]

[0184] To a solution of 2,6-dichloro-9H-purine (50.0 g, 264.55 mmol, 1 eq) and TEA (73.63 mL, 529.7 mmol, 2 eq) in DMF (400 mL) was added (4-methoxyphenyl)methylamine (36.29 mL, 277.78 mmol, 1.05 eq). The mixture was stirred at 100 ° C for 16 hours. The reaction mixture was cooled to 15 ° C and then filtered. The filter cake was washed with EA (3×150 mL) and H2O (3×150 mL). The residue was dried under reduced pressure to give 2-chloro-N-[(4-methoxyphenyl)methyl]-9H-purine-6-amine (60 g, compound 1a) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δppm 8.55 (br s, 1H), 8.13 (s, 1H), 7.29 (d, J = 8.8Hz, 2H), 6.89 (d, J = 8.4Hz, 2H), 4.56 (s, 2H), 3.71 (s, 3H).

[0185] Step 2: Preparation of 2-chloro-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 1b)

[0186]

[0187] To a solution of 2-chloro-N-[(4-methoxyphenyl)methyl]-9H-purine-6-amine (180.0 g, compound 1a, 621.29 mmol, 1 eq) in DMF (1.8 L) was added K2CO3 (89.0 g, 683.42 mmol, 1.1 eq) and 1-(chloromethyl)-4-methoxy-benzene (84.24 mL, 621.29 mmol, 1 eq). The mixture was stirred at 15 °C for 16 hours and then diluted with H2O (2.0 L). After filtration, the filter cake was purified by grinding (PE / EA=5 / 1, 2×600 mL) and vacuum dried to provide 2-chloro-N,9-bis[(4-methoxyphenyl)methyl]purine-6-amine (180 g, compound 1b) as a pink solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:410.1. 1 H NMR(400MHz,DMSO-d6)δppm 8.78-8.75(m,1H),8.23(s,1H),7.27(d,J=8.4Hz,4H),6.91-6.85(m,4H),5.25(s,2H),5.06-4.54(m,2H),3.74-3.69(m,6H).

[0188] Step 3: Preparation of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 1c)

[0189]

[0190] To a solution of 2-chloro-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine solution (35 g, compound 1b, 85.4 mmol, 1 eq) and methylphosphonomethane (10.0 g, 128.09 mmol, 1.5 eq) in DMF (500 mL) was added K2CO3 (23.6 g, 170.79 mmol, 2 eq), XantPhos (4.9 g, 8.54 mmol, 0.1 eq) and Pd(OAc)2 (1.9 g, 8.54 mmol, 0.1 eq). Under N2 atmosphere, the mixture was stirred at 150°C for 2 hours. The mixture was filtered through celite, and the filtrate was concentrated in vacuo to provide 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (76 g, compound 1c, crude) as a yellow oil, which was used in the next step without further purification. MS observations (ESI + )[(M+H) + ]:452.2.

[0191] Step 4: Preparation of 2-dimethylphosphoryl-9H-purin-6-amine (Compound 1d)

[0192]

[0193] A solution of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (76.0 g, compound 1c, crude) in TFA (760.0 mL) was stirred at 60°C for 16 hours. The mixture was concentrated in vacuo, and the residue was triturated with MeOH (3×400 mL) and EA (3×60 mL) to provide 2-dimethylphosphoryl-9H-purin-6-amine (30 g, compound 1d, crude) as a light yellow solid and used in the next step without further purification. MS observed value (ESI + )[(M+H) + ]:211.0.

[0194] Step 5: Preparation of 9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1e)

[0195]

[0196] To a solution of 2-dimethylphosphoryl-9H-purine-6-amine (30.0 g, compound 1d, 142.07 mmol, 1 eq) in DMF (300 mL) was added K2CO3 (37.0 g, 284.14 mmol, 2 eq) and 2-chloro-5-(chloromethyl)pyridine (23.0 g, 142.07 mmol, 1 eq). The mixture was stirred at 40 ° C for 16 hours. The mixture was filtered. The filtrate was concentrated in vacuo, and the residue was purified by preparative HPLC to provide 9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purine-6-amine (22 g, compound 1e) as a light yellow solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:337.0. 1 H NMR(400MHz,DMSO-d6)δppm 8.55(d,J=2.0Hz,1H),8.45(s,1H),7.90(dd,J=8.8,2.8Hz,1H),7.72(brs,2H),7.52(d,J=8.4Hz,1H),5.46(s,2H),1.72(d,J=13.6Hz,6H).

[0197] Step 6: Preparation of 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1f)

[0198]

[0199] To a solution of 9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purine-6-amine (22.0 g, compound 1e, 65.34 mmol, 1 eq), NaOAc (11.8 g, 143.74 mmol, 2.2 eq) and AcOH (3.9 g, 65.34 mmol, 1 eq) in water (220 mL) was added Br2 (20.9 g, 130.67 mmol, 2 eq) dropwise at 15°C. The mixture was then stirred at 15°C for 16 hours. The mixture was extracted with DCM / MeOH (v / v=10 / 1, 3×200 mL). The organic layers were combined and dried over Na2SO4. After filtration, the filtrate was concentrated in vacuo and the residue was purified by preparative HPLC to provide 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-purin-6-amine (11 g, compound 1f) as a red solid. MS observed (ESI + )[{ 35 Cl& 79 Br}(M+H) + ]:416.9.1 HNMR (400MHz, DMSO-d6) δppm 8.48 (s, 1H), 7.87-7.68 (m, 3H), 7.53 (d, J = 8.0Hz, 1H), 5.44 (s 2H), 1.71 (d, J = 13.6Hz, 6H).

[0200] Step 7: Preparation of 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g)

[0201]

[0202] A solution of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purine-6-amine (11.5 g, compound 1f, 27.67 mmol, 1 eq) and NaOH (8.8 g, 221.36 mmol, 8 eq) in water (80 mL) and tert-butanol (80 mL) was stirred at 80 ° C for 16 hours. The mixture was concentrated in vacuo and then diluted with H2O (80 mL). HCl aq. (6 M) was added to pH = 8. After filtration, the filter cake was washed with H2O (3×60 mL) and dried in vacuo. The residue was purified by trituration (EA / MeOH=10 / 1, 3×20 mL) to provide 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (8.5 g, compound 1 g) as a gray solid, which was used in the next step without further purification. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:353.0. 1 HNMR(400MHz,DMSO-d6)δppm 8.45(d,J=2.0Hz,1H),7.82(dd,J=8.0,2.4Hz 1H),7.50(d,J=8.0Hz,1H),6.74(br s, 2H), 4.99 (s, 2H), 1.67 (d, J = 13.6Hz, 6H).

[0203] Step 8: Preparation of 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (Example 1)

[0204]

[0205] A mixture of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purine-8-one (7.5 g, compound 1 g, 21.26 mmol, 1 eq) in N', N'-dimethylethane-1,2-diamine (90.0 mL, 212.63 mmol, 10 eq) was stirred at 170 ° C for 72 hours in a sealed tube. The reaction mixture was concentrated in vacuo, and the residue was purified by preparative HPLC. The crude product was dissolved in H2O / MeOH=1 / 1 (60 mL) and adjusted to pH=8 by adding NaHCO3 solid, and then concentrated in vacuo. The residue was dissolved in DCM / MeOH=5 / 1 (100 mL), and the mixture was filtered, and the filtrate was then concentrated in vacuo. The residue was dissolved in MeCN / MeOH=20 / 1 (70 mL) and stirred at 85 ° C for 2 hours, then cooled to 15 ° C. After filtration, the filter cake was dried in vacuo to provide 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (2.2 g, Example 1) as a white solid. MS observed (ESI + )[(M+H) + ]:405.1. 1 H NMR(500MHz,DMSO-d6)δppm 10.49(br d,J=2.0Hz,1H),8.01(d,J=2.1Hz,1H),7.39(dd,J=8.6,2.4Hz,1H),6.70(br s,2H),6.42(d,J=8.7Hz,1H),6.36(t,J=5.4Hz,1H),4.75(s,2H),3.27(q, J=6.5Hz,2H),2.35(t,J=6.6Hz,2H),2.14(s,6H),1.68(d,J=13.6Hz,6H).

[0206] Example 2

[0207] 6-Amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one

[0208]

[0209] To a solution of 2-(dimethylamino)ethanol (126.36 mg, 1.42 mmol, 10 eq) in THF (3 mL) was added NaH (56.7 mg, 60 wt.%, 1.42 mmol, 10 eq) at 0 ° C. The mixture was stirred at 0 ° C for 1 hour. The mixture was then concentrated in vacuo. The residue was dissolved in DMF (2 mL) and 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purine-8-one (50.0 mg, compound 1 g, 0.140 mmol, 1 eq) was added. The mixture was stirred at 130 ° C for 15 hours. H2O (10 mL) was added dropwise at 0 ° C, and FA was added to make pH = 6. The mixture was then concentrated in vacuo and the residue was purified by preparative HPLC to provide 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (12.6 mg, Example 2) as a light yellow solid. MS observed (ESI + )[(M+H) + ]:406.1. 1 H NMR(400MHz,CD3OD)δppm 8.26(d,J=2.4Hz,1H),7.81(dd,J=8.8,2.8Hz,1H),6.77(d,J=8.4Hz,1H),5.02(s,2H ), 4.38 (t, J = 5.6Hz, 2H), 2.75 (t, J = 5.6Hz, 2H), 2.31 (s, 6H), 1.85 (d, J = 13.6Hz, 6H).

[0210] Example 3

[0211] 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one

[0212]

[0213] Step 1: Preparation of tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]pyrrolidin-3-yl]carbamate (Compound 3a)

[0214]

[0215] To a mixture of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (100.0 mg, compound 1 g, 0.280 mmol, 1 eq), tert-butyl N-pyrrolidin-3-ylcarbamate (79.2 mg, 0.430 mmol, 1.5 eq) and t-BuONa (54.4 mg, 0.570 mmol, 2 eq) in NMP (2 mL) was added RuPhos (13.2 mg, 0.030 mmol, 0.1 eq) and Pd2(dba)3 (25.9 mg, 0.030 mmol, 0.1 eq). The mixture was stirred at 120° C. for 16 h under N2 atmosphere. The mixture was filtered. The filtrate was concentrated in vacuo and purified by preparative HPLC to provide tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]pyrrolidin-3-yl]carbamate (15 mg, compound 3a) as a light yellow solid. MS observed (ESI + )[(M+H) + ]:503.2.

[0216] Step 2: Preparation of 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (Example 3)

[0217]

[0218] A solution of tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (15.0 mg, compound 3a, 0.030 mmol, 1 eq) in HCl / dioxane (1.0 mL, 4 M) was stirred at 15 ° C for 1 hour. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC to provide 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one (8.7 mg, Example 3) as a light yellow solid. MS observed value (ESI + )[(M+H) + ]:402.9. 1H NMR(400MHz,CD3OD)δppm 8.17(d,J=2.0Hz,1H),7.69-7.65(m,1H),6.46(d,J=8.4Hz,1H),4.99(s,2H),3.68-3.63(m,3H),3 .57-3.48(m,1H),3.31-3.30(m,1H),2.27-2.22(m,1H),1.91-1.89(m,1H),1.84(d,J=13.6Hz,6H).

[0219] Example 4

[0220] 6-Amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one

[0221]

[0222] Step 1: Preparation of 4-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]-2,2-dimethyl-piperazine-1-carboxylic acid tert-butyl ester (Compound 4a)

[0223]

[0224] The title compound was prepared by using tert-butyl 2,2-dimethylpiperazine-1-carboxylate instead of tert-butyl N-pyrrolidin-3-ylcarbamate in a similar manner to Example 3, step 1. A light yellow oily 4-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridinyl]-tert-butyl 2,2-dimethylpiperazine-1-carboxylate (80 mg, compound 4a) was obtained. MS observed value (ESI + )[(M+H) + ]:531.0.

[0225] Step 2: Preparation of 6-amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 4)

[0226]

[0227] The title compound was prepared similarly to Example 3, step 2 by using 4-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridinyl]-2,2-dimethyl-piperazine-1-carboxylic acid tert-butyl ester (compound 4a) instead of N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridinyl]pyrrolidin-3-yl]carbamic acid tert-butyl ester (compound 3a). 6-amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazine-1-yl)-3-pyridinyl]methyl]-7H-purine-8-one (6.1 mg, Example 4) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:431.2. 1 H NMR(400MHz,CD3OD)δppm 8.30(d,J=2.0Hz,1H),7.71(dd,J=8.8,2.4Hz,1H),6.88(d,J=8.8Hz,1H),5.00(s,2H),3. 77(t,J=5.6Hz,2H),3.60(s,2H),3.32-3.30(m,2H),1.88(d,J=13.6Hz,6H),1.40(s,6H).

[0228] Example 5

[0229] 6-Amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one

[0230]

[0231] Step 1: Preparation of tert-butyl 5-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (Compound 5a)

[0232]

[0233] The title compound was prepared by using tert-butyl 2,5-diazabicyclo [2.2.2] octane-2-carboxylate instead of tert-butyl N-pyrrolidin-3-ylcarbamate similar to Example 3, step 1. 5-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridyl]-tert-butyl 2,5-diazabicyclo [2.2.2] octane-2-carboxylate (35 mg, compound 5a) was obtained as a brown solid. MS observed value (ESI +)[(M+H) + ]:529.1.

[0234] Step 2: Preparation of 6-amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (Example 5)

[0235]

[0236] The title compound was prepared by using 5-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridyl]-2,5-diazabicyclo[2.2.2]octane-2-carboxylic acid tert-butyl ester (compound 5a) instead of N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamic acid tert-butyl ester (compound 3a) in a similar manner to Example 3, step 2. 6-amino-9-[[6-(2,5-diazabicyclo[2.2.2]octane-2-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one (9.3 mg, Example 5) was obtained as a green solid. MS observed value (ESI + )[(M+H) + ]:428.9. 1 H NMR(400MHz,CD3OD)δppm 8.26(s,1H),7.75(d,J=7.6Hz,1H),6.60(d,J=8.8Hz,1H),4.98(s,2H),3.82 -3.77(m,2H),3.44-3.43(m,4H),2.14-2.04(m,4H),1.88(d,J=13.6Hz,6H).

[0237] Example 6

[0238] 9-[[6-(2,3,3a,4,6,6a-Hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-yl)-3-pyridinyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one

[0239]

[0240] Step 1: Preparation of tert-butyl 2-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]-1,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5-carboxylate (Compound 6a)

[0241]

[0242] The title compound was prepared by using 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrole-5-carboxylic acid tert-butyl ester instead of N-pyrrolidin-3-ylcarbamic acid tert-butyl ester in analogy to Example 3, step 1. A light yellow oily 2-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purine-9-yl)methyl]-2-pyridyl]-1,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5-carboxylic acid tert-butyl ester (80 mg, compound 6a) was obtained. MS observed value (ESI + )[(M+H) + ]:529.0.

[0243] Step 2: Preparation of 9-[[6-(2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-yl)-3-pyridinyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one (Example 6)

[0244]

[0245]

[0246] The title compound was prepared in analogy to Example 3, step 2, by using 2-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]-1,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5-carboxylic acid tert-butyl ester (compound 6a) instead of N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridinyl]pyrrolidin-3-yl]carbamic acid tert-butyl ester (compound 3a). 9-[[6-(2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-yl)-3-pyridinyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one (28 mg, Example 6) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:429.0. 1 H NMR(400MHz,CD3OD)δppm 8.25(d,J=2.0Hz,1H),7.75(dd,J=8.8,2.4Hz,1H),6.60(d,J=8.8Hz,1H), 5.04(s,2H),3.59-3.55(m,6H),3.33-3.29(m,4H),1.84(d,J=13.6Hz,6H).

[0247] Example 7

[0248] 6-Amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridinyl]methyl]-7H-purin-8-one

[0249]

[0250] The title compound was prepared by using 2-pyrrolidin-1-ylethylamine instead of N',N'-dimethylethane-1,2-diamine in analogy to Example 1, step 8. 6-amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridinyl]methyl]-7H-purin-8-one (13.3 mg, Example 7) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:431.1. 1 H NMR(400MHz,CD3OD)δppm 8.08(d,J=2.0Hz,1H),7.57(dd,J=8.8,2.4Hz,1H),6.46(d,J=8.4Hz,1H),4.92(s,2H),3.41(t,J =6.8Hz,2H),2.69(t,J=6.8Hz,2H),2.62-2.58(m,4H),1.84(d,J=13.6Hz,6H),1.83-1.77(m,4H).

[0251] Example 8

[0252] 6-Amino-2-dimethylphosphoryl-9-[[6-(tetrahydropyran-4-ylamino)-3-pyridinyl]methyl]-7H-purin-8-one

[0253]

[0254] The title compound was prepared by using tetrahydropyran-4-amine instead of N',N'-dimethylethane-1,2-diamine in analogy to Example 1, step 8. 6-amino-2-dimethylphosphoryl-9-[[6-(tetrahydropyran-4-ylamino)-3-pyridinyl]methyl]-7H-purin-8-one (18.4 mg, Example 8) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:418.1. 1HNMR(400MHz,CD3OD)δppm 8.06(d,J=2.0Hz,1H),7.55(dd,J=8.8,2.4Hz,1H),6.46(d,J=8.4Hz,1H),4.91(s,2H),3.96-3.91(m,2 H),3.86-3.83(m,1H),3.55-3.49(m,2H),1.95-1.91(m,2H),1.84(d,J=13.6Hz,6H),1.50-1.46(m,2H).

[0255] Example 9

[0256] 6-amino-9-[[6-[3-[(dimethylamino)methyl]pyrrolidin-1-yl]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one

[0257]

[0258] The title compound was prepared by using N,N-dimethyl-1-pyrrolidin-3-yl-methylamine instead of tert-butyl N-pyrrolidin-3-ylcarbamate in analogy to Example 3, step 1. 6-amino-9-[[6-[3-[(dimethylamino)methyl]pyrrolidin-1-yl]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one (12.1 mg, Example 9) was obtained as a light yellow oil. MS observed value (ESI + )[(M+H) + ]:445.0. 1 HNMR(400MHz,CD3OD)δppm 8.19(d,J=2.0Hz,1H),7.71(dd,J=8.4,2.0Hz,1H),6.51(d,J=8.8Hz,1H),4.96(s,2H),3.75-3.72(m,1H),3.61-3.55(m,1H),3.4 6-3.39(m,2H),3.29-3.27(m,2H),3.18-3.13(m,1H),2.94(s,6H),2.83-2.87(m,1H),2.31-2.27(m,1H),1.85(d,J=13.6Hz,6H).

[0259] Example 10

[0260] 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undec-9-yl)-3-pyridinyl]methyl]-7H-purin-8-one

[0261]

[0262] Step 1: Preparation of 4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane (Compound 10a)

[0263]

[0264] To a solution of tert-butyl 1-oxa-4,9-diazaspiro[5.5]undecane-4-carboxylate (500.0 mg, 1.95 mmol, 1 eq) in THF (10 mL) was added LiAlH4 (148.05 mg, 3.9 mmol, 2 eq) at 0 °C. The reaction mixture was stirred at 80 °C for 2 hours. MeOH (2 mL) and H2O (1 mL) were added at 0 °C. The reaction mixture was dried over Na2SO4. After filtration, the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC to provide 4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane (200 mg, compound 10a) as a colorless oil. MS observed values ​​(ESI + )[(M+H) + ]:171.1.

[0265] Step 2: Preparation of 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undec-9-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 10)

[0266]

[0267] The title compound was prepared by using 4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane instead of tert-butyl N-pyrrolidin-3-ylcarbamate similar to Example 3, step 1. 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane-9-yl)-3-pyridyl]methyl]-7H-purine-8-one (4.7 mg, Example 10) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:487.2. 1H NMR (400MHz, CD3OD) δ (ppm) 8.21 (d, J = 2.0Hz, 1H), 7.67 (dd, J = 8.8, 2.4Hz, 1H), 6.78 (d, J = 8.8Hz, 1H), 4.96 (s, 2H), 3.88-3.81 (m, 2H), 3.74 (t, J =4.8Hz,2H),3.28-3.20(m,2H),2.37(t,J=4.8Hz,2H),2.25(s,2H),2.2 1(s,3H),1.99-1.92(m,2H),1.84(d,J=13.6Hz,6H),1.61-1.50(m,2H).

[0268] Example 11

[0269] 6-amino-2-dimethylphosphoryl-9-[[6-[(1-methylpyrrolidin-3-yl)amino]-3-pyridinyl]methyl]-7H-purin-8-one

[0270]

[0271] The title compound was prepared analogously to Example 3, step 1, by using 1-methylpyrrolidin-3-amine instead of tert-butyl N-pyrrolidin-3-ylcarbamate and BrettPhos instead of RuPhos. 6-amino-2-dimethylphosphoryl-9-[[6-[(1-methylpyrrolidin-3-yl)amino]-3-pyridinyl]methyl]-7H-purin-8-one (23.92 mg, Example 11) was obtained as a white solid. MS observed (ESI + )[(M+H) + ]:417.1. 1 H NMR(400MHz,CD3OD)δppm 8.17(d,J=2.0Hz,1H),7.62(dd,J=8.4,2.0Hz,1H),6.53(d,J=8.8Hz,1H),4.94(s,2H),4.47-4.44(m,1H),3.68-3.67(m ,1H),3.47-3.44(m,2H),3.26-3.23(m,1H),2.93(s,3H),2.52-2.48(m,1H),2.15-2.08(m,1H),1.85(d,J=13.6Hz,6H).

[0272] Example 12

[0273] 6-amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one

[0274]

[0275] Step 1: Preparation of N'-ethyl-N'-methyl-ethane-1,2-diamine (Compound 12a)

[0276]

[0277] 2-(2-bromoethyl)isoindoline-1,3-dione (20.0 g, 78.72 mmol, 1.0 eq) was added to a solution of N-methylethylamine (14.2 mL, 165.3 mmol, 2.1 eq) in m-xylene (45 mL) at 20°C. The reaction mixture was stirred at 150°C for 12 hours. The reaction mixture was cooled to room temperature and then basified with Na2CO3 (aq. 2M) until pH = 9. The mixture was extracted with EtOAc (3×70 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo to provide a residue. Water (5 mL) and HCl (30.0 mL, 12 M, 360.0 mmol, 4.57 eq) were then added to the residue at 0°C. The mixture was heated to 130°C and stirred for 6 hours while a precipitate was formed. The precipitate was filtered and washed with cold water (50 mL). The filtrate was concentrated in vacuo to provide a residue. The residue was dissolved in MeOH (200 mL), basified with solid NaOH to pH = 11 and filtered. The filtrate was concentrated in vacuo and the crude product was purified by distillation at 100 °C (760 mmHg) to provide N'-ethyl-N'-methyl-ethane-1,2-diamine (5.0 g, compound 12a) as a colorless liquid. 1 HNMR (400MHz, DMSO-d6) δppm: 2.55 (t, J = 6.4Hz, 2H), 2.30 (q, J = 7, 2Hz, 2H), 2.26 (t, J = 6.4Hz, 2H), 2.09 (s, 3H), 0.95 (t, J = 7.2Hz, 3H).

[0278] Step 2: Preparation of 6-amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (Example 12)

[0279]

[0280] The title compound was prepared by using N'-ethyl-N'-methyl-ethane-1,2-diamine instead of N',N'-dimethylethane-1,2-diamine in analogy to Example 1, step 8. 6-amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purine-8-one (8.28 mg, Example 12) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:419.1. 1 H NMR (400MHz, CD3OD) δppm: 8.08 (d, J = 2.0Hz, 1H), 7.57 (dd, J = 8.8, 2.0Hz, 1H), 6.46 (d, J = 8.4Hz, 1H), 4.92 (s, 2H), 3.38 (t ,J=6.8Hz,2H),2.59(t,J=6.8Hz,2H),2.50(q,J=7,2Hz,2H),2.27(s,3H),1.83(d,J=13.6Hz,6H),1.07(t,J=7.2Hz,3H).

[0281] Example 13-A and Example 13-B 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 13-A) and 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 13-B)

[0282]

[0283] Step 1: Preparation of 1-methylphosphonooxyethane (Compound 13a)

[0284]

[0285] To diethoxy(methyl)phosphine (60.0 g, 440.76 mmol, 1 eq) was added H2O (7.94 g, 440.76 mmol, 1 eq) at 0° C. The mixture was then stirred at 25° C. for 12 hours. 1 H NMR showed that the reaction was complete and the desired product was obtained, which contained 50 mol% ethanol. The reaction solution was used directly in the next step. 1H NMR (400MHz, CDCl3) δppm 7.90-7.88 (m, 0.5H), 6.55-6.54 (m, 0.5H), 4.21-4.06 (m, 2H), 1.56 (dd, J = 14.8, 2.0Hz, 3H), 1.37 (t, J = 3.2Hz, 3H).

[0286] Step 2: Preparation of 1-methylphosphonoethane (Compound 13b)

[0287]

[0288] At 0 ° C under N2, ethylmagnesium bromide (440mL, 3M, 1321mmol, 3eq) was added dropwise to a solution of 1-methylphosphonooxyethane (47.6g, compound 13a, 440.41mmol, 1eq) in THF (900mL) over 30 minutes. The mixture was then warmed to 25 ° C and stirred for 12 hours. The reaction mixture was cooled to 0 ° C. A solution of potassium carbonate (182.6g, 1321mmol, 3eq) in water (200mL) was added dropwise to the reaction mixture. The mixture was extracted with DCM (3×800mL). The organic layers were then combined and dried over Na2SO4. After filtration, the filtrate was concentrated in vacuo to provide 1-methylphosphonoethane (47g, compound 13b, crude product) as a light yellow liquid. 1 H NMR (400MHz, CDCl3) δppm 7.53-7.50 (m, 0.5H), 6.40-6.37 (m, 0.5H), 1.85-1.82 (m, 2H), 1.57 (dd, J = 14.8, 2.0Hz, 3H), 1.24-1.15 (m, 3H).

[0289] Step 3: Preparation of 2-[ethyl(methyl)phosphoryl]-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 13c)

[0290]

[0291] The title compound was prepared by using 1-methylphosphonoethane (Compound 13b) instead of methylphosphonomethane in analogy to Example 1, Step 3. 2-[Ethyl(methyl)phosphoryl]-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (78 g, Compound 13c, crude product) was obtained as a black oil and used in the next step without further purification. MS observed value (ESI + )[(M+H) + ]:466.2.

[0292] Step 4: Preparation of 2-[ethyl(methyl)phosphoryl]-9H-purin-6-amine (Compound 13d)

[0293]

[0294] The title compound was prepared by using 2-[ethyl(methyl)phosphoryl]-N,9-bis[(4-methoxyphenyl)methyl]purine-6-amine (Compound 13c) instead of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purine-6-amine (Compound 1c) in analogy to Example 1, Step 4. Black oily 2-[ethyl(methyl)phosphoryl]-9H-purine-6-amine (37 g, Compound 13d) was obtained and used in the next step without further purification. MS observed value (ESI + )[(M+H) + ]:226.0.

[0295] Step 5: Preparation of 9-[(6-chloro-3-pyridyl)methyl]-2-[ethyl(methyl)phosphoryl]purin-6-amine (Compound 13e)

[0296]

[0297] The title compound was prepared by using 2-[ethyl(methyl)phosphoryl]-9H-purine-6-amine (Compound 13d) instead of 2-dimethylphosphoryl-9H-purine-6-amine (Compound 1d) in analogy to Example 1, Step 5. 9-[(6-chloro-3-pyridyl)methyl]-2-[ethyl(methyl)phosphoryl]purine-6-amine (30 g, Compound 13e) was obtained as a light yellow solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:351.0. 1 H NMR(400MHz,DMSO-d6)δppm 8.54(d,J=2.4Hz,1H),8.48(s,1H),7.88(dd,J=8.0,2.4Hz,1H),7.50(d,J=8.0Hz,1H) ,5.46(s,2H),2.06-1.97(m,2H),1.69(d,J=13.2Hz,3H),1.01(dt,J=17.6,7.6Hz,3H).

[0298] Step 6: Preparation of 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]purin-6-amine (Compound 13f-A) and 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]purin-6-amine (Compound 13f-B)

[0299]

[0300] The title compound was prepared by using 9-[(6-chloro-3-pyridinyl)methyl]-2-[ethyl(methyl)phosphoryl]purine-6-amine (Compound 13e) instead of 9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-purine-6-amine (Compound 1e) in analogy to Example 1, Step 6. After preparative HPLC purification, the racemic product was separated by chiral SFC to provide 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]purine-6-amine as a brown solid (faster elution, 15 g, Compound 13f-A) and 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]purine-6-amine as a brown solid (slower elution, 15.7 g, Compound 13f-B). Chiral SFC conditions: DAICELCHIRALPAK AD (10 μm, 50×250 mm) column; mobile phase: phase A is CO 2 , and phase B is EtOH (0.05% DEA); gradient elution: EtOH (0.05% DEA), in CO 2 , 5% to 40%, flow rate: 3 mL / min; detector: PDA.

[0301] Compound 13f-A: MS observed value (ESI + )[{ 35 Cl& 79 Br}(M+H) + ]:430.9. 1 HNMR(400MHz,DMSO-d6)δppm 8.47(d,J=2.4Hz,1H),7.77(br s,2H),7.73(dd,J=8.0,2.4Hz,1H),7.52(d,J=8.0Hz,1H),5.44(s,2H),2.04-1.93(m,2H),1.65(d,J=13.2Hz,3H),1.01(dt,J=17.6,7.6Hz,3H).

[0302] Compound 13f-B: MS observed value (ESI + )[{ 35 Cl& 79 Br}(M+H)+ ]:430.9. 1 HNMR(400MHz,DMSO-d6)δppm 8.47(d,J=2.4Hz,1H),7.77(br s,2H),7.73(dd,J=8.0,2.4Hz,1H),7.51(d,J=8.4Hz,1H),5.44(s,2H),2.04-1.93(m,2H),1.64(d,J=13.2Hz,3H),1.02(dt,J=17.6,7.6Hz,3H).

[0303] Step 7: Preparation of 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A) and 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B)

[0304]

[0305] Compound 13g-A was prepared by using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]purin-6-amine (compound 13f-A) instead of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (compound 1f) in analogy to Example 1, Step 7. 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (11 g, compound 13g-A) was obtained as a gray solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:367.0. 1 H NMR (400MHz, DMSO-d6) δppm 8.43(d,J=2.0Hz,1H),7.77(dd,J=8.4,2.4Hz,1H),7.46(d,J=8.4Hz,1H),7.09(br s,2H),4.99(s,2H),2.00-1.88(m,2H),1.59(d,J=13.2Hz,3H),1.00(dt,J=17.6,7.6Hz,3H).

[0306] Compound 13g-B was prepared similarly to compound 13g-A by using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]purin-6-amine (compound 13f-B) instead of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]purin-6-amine (compound 13f-A). 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (11.5 g, compound 13g-B) was obtained as a gray solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:367.0. 1 H NMR (400MHz, DMSO-d6) δppm 8.42 (d, J = 2.4Hz, 1H), 7.77 (dd, J = 8.4, 2.4Hz 1H), 7.46 (d, J = 8.4Hz, 1H), 6.98 (br s,2H),4.98(s,2H),1.99-1.88(m,2H),1.58(d,J=13.2Hz,3H),0.99(dt,J=17.6,7.6Hz,3H).

[0307] Step 8: Preparation of 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 13-A) and 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 13-B)

[0308]

[0309] A solution of 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (300.0 mg, compound 13g-A, 0.820 mmol, 1 eq) in 1-methylpiperazine (8.0 mL, 40.9 mmol, 50 eq) was stirred at 170° C. for 6 hours. The solution was purified by preparative HPLC. The residue was purified by SFC (DAICELCHIRALCEL OD (10 μm, 30×250 mm) column; mobile phase: [0.1% NH 3 H 2 O EtOH]; B%: 40%-40%, 3.2 min; 25 min) to provide 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (108.3 mg, Example 13-A) as a white solid. MS observed value (ESI + )[(M+H) + ]:431.2. 1 H NMR (400MHz, DMSO-d6) δppm 10.41 (s, 1H), 8.15 (d, J = 2.4Hz, 1H), 7.54 (dd, J = 8.8, 2.4Hz, 1H), 6.78 (d, J = 8.8Hz, 1H), 6.67 (br s,2H),4.81(s,2H),3.43(t,J=4.8Hz,4H),2.37(t,J=4.8Hz,4H),2.21(s,3 H), 2.05-1.98 (m, 2H), 1.63 (d, J = 13.2Hz, 3H), 1.04 (dt, J = 17.6, 7.6Hz, 3H).

[0310] Similarly to Example 13-A, Example 13-B was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A). 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (119.3 mg, Example 13-B) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:431.2. 1H NMR (400MHz, DMSO-d6) δppm 10.41 (s, 1H), 8.14 (d, J = 2.4Hz, 1H), 7.53 (dd, J = 8.8, 2.4Hz, 1H), 6.77 (d, J = 8.8Hz, 1H), 6.67 (br s,2H),4.80(s,2H),3.42(t,J=4.8Hz,4H),2.34(t,J=4.8Hz,4H),2.18(s,3 H), 2.05-1.90 (m, 2H), 1.62 (d, J = 12.8Hz, 3H), 1.03 (dt, J = 17.6, 7.6Hz, 3H).

[0311] Example 14-A and Example 14-B 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 14-A) and 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one (Example 14-B)

[0312]

[0313] A solution of 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purine-8-one (250.0 mg, compound 13g-A, 0.680 mmol, 1 eq) and 1-methylsulfonylpiperazine (1.1 g, 6.82 mmol, 10 eq) in THF (2 mL) was heated to 170 ° C. The solvent was evaporated and the residue was stirred at 170 ° C for 8 hours. The mixture was purified by reverse flash chromatography and preparative HPLC to provide 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazine-1-yl)-3-pyridinyl]methyl]-7H-purine-8-one (19.8 mg, Example 14-A) as a white solid. MS observed value (ESI + )[(M+H) + ]:495.4. 1H NMR (400MHz, DMSO-d6) δppm 10.63 (br s, 1H), 8.19 (t, J = 2.4Hz, 1H), 7.60 (dd, J = 8.8, 2.4Hz, 1H), 6.86 (d, J = 8.8Hz, 1H), 6.71 (br s,2H),4.83(s,2H),3.59(t,J=4.8Hz,4H),3.15(t,J=4.8Hz,4H),2.88(s,3 H), 2.06-1.88 (m, 2H), 1.65 (d, J = 13.2Hz, 3H), 1.04 (dt, J = 17.2, 7.6Hz, 3H).

[0314] Similarly to Example 14-A, Example 14-B was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A). 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (46.5 mg, Example 14-B) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:495.4. 1 H NMR (400MHz, DMSO-d6) δppm 10.63 (br s, 1H), 8.18 (t, J = 2.4Hz, 1H), 7.60 (dd, J = 8.8, 2.4Hz, 1H), 6.86 (d, J = 8.8Hz, 1H), 6.62 (br s,2H),4.81(s,2H),3.59(t,J=4.8Hz,4H),3.15(t,J=4.8Hz,4H),2.87(s,3 H), 2.03-1.85 (m, 2H), 1.64 (d, J = 13.2Hz, 3H), 1.04 (dt, J = 17.2, 7.6Hz, 3H).

[0315] Example 15-A and Example 15-B 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Example 15-A) and 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Example 15-B)

[0316]

[0317] Analogously to Example 1, Step 8, Example 15-A was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g). 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (1.1 g, Example 15-A) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:419.2. 1 H NMR (400MHz, DMSO-d6) δppm 10.46 (br s, 1H), 8.01 (d, J = 2.0Hz, 1H), 7.39 (dd, J = 8.8, 2.4Hz, 1H), 6.69 (br s,2H),6.41(d,J=8.8Hz,1H),6.36(t,J=5.6Hz,1H),4.74(s,2H),3.27(q,J=6.4Hz,2H),2.35(t,J =6.4Hz,2H),2.14(s,6H),2.08-1.92(m,2H),1.65(d,J=13.2Hz,3H),1.03(dt,J=17.6,7.6Hz,3H).

[0318] Similarly to Example 15-A, Example 15-B was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A). 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (2.2 g, Example 15-B) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:419.2. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.46 (br s, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 8.8, 2.4 Hz, 1H), 7.02 (br s, 2H), 6.39 (d, J = 8.8 Hz, 1H), 6.26 (t, J = 5.6 Hz, 1H), 4.75 (s, 2H), 3.25 (q, J = 6.4 Hz, 2H), 2.34 (t, J = 6.4 Hz, 2H), 2.13 (s, 6H), 2.12-1.95 (m, 2H), 1.62 (d, J = 13.2 Hz, 3H), 1.03 (dt, J = 17.6, 7.6 Hz, 3H). The stereocenter configuration of Example 15-B was confirmed by X-ray crystallography.

[0319] Example 16-A and Example 16-B 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Example 16-A) and 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Example 16-B)

[0320]

[0321] Similarly to Example 2, Example 16-A was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g). 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (6.1 mg, Example 16-A) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:418.1. 1H NMR(400MHz,CD3OD)δppm 8.25(d,J=2.4Hz,1H),7.79(dd,J=8.8,2.4Hz,1H),6.76(d,J=8.4Hz,1H),5.02(s,2H),4.39(t,J=5.6Hz,2H) ,2.74(t,J=5.6Hz,2H),2.31(s,6H),2.20-2.11(m,2H),1.80(d,J=13.2Hz,3H),1.16(dt,J=18.0,7.6Hz,3H).

[0322] Similarly to Example 16-A, Example 16-B was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A). 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (7.8 mg, Example 16-B) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:418.1. 1 H NMR(400MHz,DMSO-d6)δppm10.45(br s,1H),8.18(d,J=2.4Hz,1H),7.68(dd,J=8.4,2.4Hz,1H),6.76(d,J=8.4Hz,1H),6.70(s,2H),4.89(s,2H),4.30(t,J=5 .6Hz,2H),2.57(t,J=5.6Hz,2H),2.17(s,6H),2.05-1.86(m,2H),1.62(d,J=13.2Hz,3H),1.02(dt,J=18.0,7.6Hz,3H).

[0323] Example 17

[0324] 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one

[0325]

[0326]

[0327] Step 1: Preparation of N-[2-[[5-[[6-amino-2-[(R)-ethyl(methyl)phosphoryl]-8-oxo-7H-purin-9-yl]methyl]-2-pyridinyl]amino]ethyl]-N-methyl-carbamic acid tert-butyl ester (Compound 17a)

[0328]

[0329] The title compound was prepared similarly to Example 1, step 8, by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purine-8-one (compound 13g-A) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purine-8-one (compound 1g) and using N-(2-aminoethyl)-N-methyl-tert-butyl carbamate instead of N',N'-dimethylethane-1,2-diamine. N-[2-[[5-[[6-amino-2-[(R)-ethyl(methyl)phosphoryl]-8-oxo-7H-purine-9-yl]methyl]-2-pyridyl]amino]ethyl]-N-methyl-tert-butyl carbamate (50 mg, compound 17a) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:505.2.

[0330] Step 2: Preparation of 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one (Example 17)

[0331]

[0332] To a solution of tert-butyl N-[2-[[5-[[6-amino-2-[(R)-ethyl(methyl)phosphoryl]-8-oxo-7H-purin-9-yl]methyl]-2-pyridinyl]amino]ethyl]-N-methyl-carbamate (30.0 mg, compound 17a, 0.060 mmol, 1 eq) in DCM (2 mL) was added TFA (0.5 mL). The reaction mixture was stirred at 20 ° C for 2 hours, then concentrated in vacuo, and the residue was purified by preparative HPLC to provide 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one (7.2 mg, Example 17) as a white solid. MS observed value (ESI + )[(M+H) + ]:405.2. 1H NMR(400MHz,CD3OD)δppm 8.26(d,J=2.0Hz,1H),7.73(dd,J=8.4,2.0Hz,1H),6.67(d,J=8.8Hz,1H),4.97(s,2H),3.81(t,J=6.0Hz,2H) ,3.16(t,J=6.0Hz,2H),3.04(s,3H),2.21-2.12(m,2H),1.81(d,J=13.6Hz,3H),1.18(dt,J=18.0,7.6Hz,3H).

[0333] Example 18-A and Example 18-B 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-A) and 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-B)

[0334]

[0335] Step 1: Preparation of 1-methylphosphonopropane (Compound 18a)

[0336]

[0337] The title compound was prepared by using n-propylmagnesium bromide instead of ethylmagnesium bromide in analogy to Example 13, Step 2. 1-Methylphosphonopropane (crude, 16 g, compound 18a) was obtained as a pale yellow liquid. 1 H NMR (400MHz, CDCl3) δppm: 7.63-7.54 (m, 0.5H), 6.48-6.41 (m, 0.5H), 1.89-1.64 (m, 4H), 1.56 (dd, J = 13.6, 4.0Hz, 3H), 1.07 (t, J = 6.8Hz, 3H).

[0338] Step 2: Preparation of N,9-bis[(4-methoxyphenyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (Compound 18b)

[0339]

[0340] The title compound was prepared by using 1-methylphosphonopropane (Compound 18a) instead of methylphosphonomethane in analogy to Example 1, Step 3. Black oily N,9-bis[(4-methoxyphenyl)methyl]-2-[methyl(propyl)phosphoryl]purine-6-amine (crude, 35 g, Compound 18b) was obtained and used in the next step without further purification. MS observed value (ESI + )[(M+H) + ]:480.1.

[0341] Step 3: Preparation of 2-[methyl(propyl)phosphoryl]-9H-purin-6-amine (Compound 18c)

[0342]

[0343] The title compound was prepared by using N,9-bis[(4-methoxyphenyl)methyl]-2-[methyl(propyl)phosphoryl]purine-6-amine (compound 18b) instead of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purine-6-amine (compound 1c) in analogy to Example 1, step 4. A red oily 2-[methyl(propyl)phosphoryl]-9H-purine-6-amine (crude, 16 g, compound 18c) was obtained and used in the next step without further purification. MS observed value (ESI + )[(M+H) + ]:240.0.

[0344] Step 4: Preparation of 9-[(6-chloro-3-pyridyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (Compound 18d)

[0345]

[0346] The title compound was prepared by using 2-[methyl(propyl)phosphoryl]-9H-purine-6-amine (Compound 18c) instead of 2-dimethylphosphoryl-9H-purine-6-amine (Compound 1d) in analogy to Example 1, Step 5. 9-[(6-chloro-3-pyridyl)methyl]-2-[methyl(propyl)phosphoryl]purine-6-amine (15 g, Compound 18d) was obtained as a light yellow solid. MS observed value (ESI + )[(M+H) + ]:365.1. 1H NMR(400MHz,DMSO-d6)δppm:8.53-8.52(m,2H),7.87(dd,J=8.4,2.4Hz,1H),7.50(d,J=8.4Hz,1H) ,5.49(s,2H),2.06-1.93(m,2H),1.71(d,J=13.2Hz,3H),1.51-1.40(m,2H),0.92(t,J=7.6Hz,3H).

[0347] Step 5: Preparation of 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-A) and 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-B)

[0348]

[0349] The title compound was prepared by using 9-[(6-chloro-3-pyridinyl)methyl]-2-[methyl(propyl)phosphoryl]purine-6-amine (Compound 18d) instead of 9-[(6-chloro-3-pyridinyl)methyl]-2-dimethylphosphoryl-purine-6-amine (Compound 1e) in analogy to Example 1, Step 6. After preparative HPLC purification, the racemic product was separated by chiral SFC to provide 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purine-6-amine as a brown solid (faster elution, 6.0 g, Compound 18e-A) and 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]purine-6-amine as a brown solid (slower elution, 6.0 g, Compound 18e-B). Chiral SFC conditions: DAICELCHIRALPAK AD (10 μm, 50×250 mm) column; mobile phase: phase A is CO2, phase B is iPrOH (0.1% NH3H2O); gradient elution: iPrOH (0.1% NH3H2O) in CO2, 40%-40%; flow rate: 3 mL / min; detector: PDA.

[0350] Compound 18e-A: MS observed value (ESI + )[{ 35 Cl& 79 Br}(M+H) + ]:444.9. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.46 (s, 1H), 7.81-7.74 (m, 2H), 7.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 5.44 (s, 2H), 2.10-1.85 (m, 2H), 1.65 (d, J = 13.2 Hz, 3H), 1.54-1.42 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). The stereocenter configuration of compound 18e-A was confirmed by X-ray crystallography.

[0351] Compound 18e-B: MS observed value (ESI + )[{ 35 Cl& 79 Br}(M+H) + ]:444.9. 1 HNMR(400MHz,DMSO-d6)δppm 8.46(s,1H),7.81-7.74(m,2H),7.72(dd,J=8.4,2.4Hz,1H),7.51(d,J=8.4Hz,1H),5.44(s ,2H),2.10-1.85(m,2H),1.65(d,J=13.2Hz,3H),1.54-1.42(m,2H),0.92(t,J=7.2Hz,3H).

[0352] Step 6: Preparation of 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Compound 18f-A) and 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Compound 18f-B)

[0353]

[0354] Compound 18f-A was prepared by using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purine-6-amine (compound 18e-A) instead of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purine-6-amine (compound 1f) in analogy to Example 1, Step 7. 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purine-8-one (3.6 g, compound 18f-A) was obtained as a brown solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:381.1.1 H NMR (400MHz, DMSO-d6) δppm 10.70 (br s, 1H), 8.43 (d, J = 2.4Hz, 1H), 7.78 (dd, J = 8.4, 2.4Hz, 1H), 7.48 (d, J = 8.4Hz, 1H), 6.80 (br s,2H),4.99(s,2H),2.00-1.83(m,2H),1.60(d,J=13.2Hz,3H),1.52-1.38(m,2H),0.92(t,J=7.2Hz,3H).

[0355] Compound 18f-B was prepared similarly to compound 18f-A by using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]purine-6-amine (compound 18e-B) instead of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purine-6-amine (compound 18e-A). 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purine-8-one (5.0 g, compound 18f-B) was obtained as a brown solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:381.1. 1 H NMR(400MHz,DMSO-d6)δppm 10.51(br s,1H),8.43(d,J=2.4Hz,1H),7.78(dd,J=8.4,2.4Hz,1H),7.48(d,J=8.0Hz,1H),6.73(brs,2H), 4.99(s,2H),2.03-1.80(m,2H),1.60(d,J=13.2Hz,3H),1.53-1.35(m,2H),0.92(t,J=7.2Hz,3H).

[0356] Step 7: Preparation of 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-A) and 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-B)

[0357]

[0358] A mixture of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (2.5 g, compound 18f-A, 6.57 mmol, 1.0 eq) in N',N'-dimethylethane-1,2-diamine (30.0 mL, 32.83 mmol, 5.0 eq) was stirred in a sealed tube at 170 ° C for 72 hours. The reaction mixture was concentrated in vacuo, and the residue was purified by preparative HPLC and SFC (DAICEL CHIRALPAK IC (10 μm, 30×250 mm) column; mobile phase: [0.1% NH 3 H 2 O i PrOH]; B%: 55%-55%, 6.6 minutes) to provide 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one as a white solid (1.0 g, Example 18-A). MS observed value (ESI + )[(M+H) + ]:433.2. 1 H NMR (400MHz, DMSO-d6) δppm8.01 (d, J=2.0Hz, 1H), 7.38 (dd, J=8.8, 2.4Hz, 1H), 6.90 (br s,2H),6.40(d,J=8.6Hz,1H),6.32(t,J=5.4Hz,1H),4.74(s,2H),3.26(q,J=6.4Hz,2H),2.34(t,J=6.8H z,2H),2.13(s,6H),2.04-1.87(m,2H),1.63(d,J=13.2Hz,3H),1.56-1.42(m,2H),0.96(t,J=7.2Hz,3H).

[0359] Similarly to Example 18-A, Example 18-B was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Compound 18f-B) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Compound 18f-A). 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (1.0 g, Example 18-B) was obtained as a white solid. MS observed value (ESI + )[(M+H) + ]:433.2. 1H NMR (400MHz, DMSO-d6) δppm8.01 (d, J=2.0Hz, 1H), 7.37 (dd, J=8.8, 2.4Hz, 1H), 6.68 (br s,2H),6.41(d,J=8.6Hz,1H),6.36(t,J=5.4Hz,1H),4.74(s,2H),3.28(q,J=6.4Hz,2H),2.33(t,J=6.8H z,2H),2.13(s,6H),2.04-1.87(m,2H),1.63(d,J=13.2Hz,3H),1.56-1.42(m,2H),0.96(t,J=7.2Hz,3H).

[0360] Example 19

[0361] 6-amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one

[0362]

[0363] Step 1: Preparation of 1-ethylphosphonoethane (Compound 19a)

[0364]

[0365] At 0 ° C under N2, ethylmagnesium bromide (144.8mL, 3M solution in THF, 434.47mmol, 3eq) was added dropwise to a solution of diethyl phosphite (20.0g, 144.8mmol, 1eq) in THF (120mL) over 30 minutes. The mixture was then warmed to 25 ° C and stirred for 12 hours. After the reaction mixture was cooled to 0 ° C, a solution of potassium carbonate (60.05g, 434.47mmol, 3eq) in water (40mL) was added dropwise to the stirred solution. The mixture was diluted with DCM (3×50mL) and filtered. The filtrate was collected, dried over Na2SO4, and concentrated under reduced pressure to provide a light yellow liquid 1-ethylphosphonoethane (crude, 15.0g, compound 19a). 1 HNMR (400MHz, CDCl3) δppm 7.43-7.41 (m, 0.5H), 6.29-6.27 (m, 0.5H), 1.93-1.88 (m, 4H), 1.23-1.15 (m, 6H).

[0366] Step 2: Preparation of 2-diethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 19b)

[0367]

[0368] The title compound was prepared by using 1-ethylphosphonopropane (Compound 19a) instead of methylphosphonomethane in analogy to Example 1, Step 3. 2-Diethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (23 g, Compound 19b) was obtained as a red oil. MS observed value (ESI + )[(M+H) + ]:480.2.

[0369] Step 3: Preparation of 2-diethylphosphoryl-9H-purin-6-amine (Compound 19c)

[0370]

[0371] The title compound was prepared by using 2-diethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purine-6-amine (Compound 19b) instead of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purine-6-amine (Compound 1c) in analogy to Example 1, Step 4. A red oily 2-diethylphosphoryl-9H-purine-6-amine (crude, 20 g, Compound 19c) was obtained. MS observed value (ESI + )[(M+H) + ]:240.0.

[0372] Step 4: Preparation of 9-[(6-chloro-3-pyridinyl)methyl]-2-diethylphosphoryl-purin-6-amine (Compound 19d)

[0373]

[0374] The title compound was prepared by using 2-diethylphosphoryl-9H-purine-6-amine (Compound 19c) instead of 2-dimethylphosphoryl-9H-purine-6-amine (Compound 1d) in analogy to Example 1, Step 5. 9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-purine-6-amine (20 g, Compound 19d) was obtained as a light yellow oil. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:365.0.

[0375] Step 5: Preparation of 8-bromo-9-[(6-chloro-3-pyridinyl)methyl]-2-diethylphosphoryl-purin-6-amine (Compound 19e)

[0376]

[0377] The title compound was prepared by using 9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-purine-6-amine (Compound 19d) instead of 9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purine-6-amine (Compound 1e) in analogy to Example 1, Step 6. 8-Bromo-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-purine-6-amine (14 g, Compound 19e) was obtained as a light yellow solid. MS observed value (ESI + )[{ 35 Cl& 79 Br}(M+H) + ]:444.9. 1 H NMR (400MHz, DMSO-d6) δppm 8.46 (d, J = 2.4Hz, 1H), 7.76 (br s,2H),7.72(dd,J=8.4,2.4Hz,1H),7.52(d,J=8.0Hz,1H),5.43(s,2H),2.02-1.92(m,4H),1.05-0.97(m,6H).

[0378] Step 6: Preparation of 6-amino-9-[(6-chloro-3-pyridinyl)methyl]-2-diethylphosphoryl-7H-purin-8-one (Compound 19f)

[0379]

[0380] The title compound was prepared by using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-purine-6-amine (Compound 19e) instead of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purine-6-amine (Compound 1f) in analogy to Example 1, Step 7. 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-7H-purine-8-one (11 g, Compound 19f) was obtained as a purple solid. MS observed value (ESI + )[{ 35 Cl}(M+H) + ]:381.1. 1 H NMR (400MHz, DMSO-d6) δppm 8.42 (d, J = 2.4Hz, 1H), 7.79 (dd, J = 8.0, 2.4Hz, 1H), 7.48 (d, J = 8.4Hz, 1H), 6.83 (br s,2H),4.98(s,2H),1.97-1.87(m,4H),1.02-0.94(m,6H).

[0381] Step 7: Preparation of 6-amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one (Example 19)

[0382]

[0383] Analogously to Example 1, step 8, the title compound was prepared by using 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-7H-purine-8-one (Compound 19f) instead of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purine-8-one (Compound 1g). 6-amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-7H-purine-8-one (3.2 g, Example 19) was obtained as a pink solid. MS observed value (ESI + )[(M+H) + ]:433.2. 1 H NMR (400MHz, DMSO-d6) δppm 8.00 (d, J=2.4Hz, 1H), 7.38 (dd, J=8.4, 2.4Hz, 1H), 6.72 (br s,2H),6.41(d,J=8.4Hz,1H),6.36(t,J=5.6Hz,1H),4.74(s,2H),3.27(q,J=6.4Hz,2H),2.35( t,J=6.4Hz,2H),2.14(s,6H),2.14-1.95(m,4H),1.05(t,J=7.6Hz,3H),1.01(t,J=7.6Hz,3H).

[0384] Reference compounds

[0385] Compound RO14 of Example 14 disclosed in prior art WO 2016180695 was selected as a reference compound for comparison.

[0386]

[0387] Example 20

[0388] Activity of compounds of the invention in HEK293-h TLR7 assay

[0389] HEK293-Blue-h TLR7 cell assay:

[0390] The stable HEK293-Blue-h TLR7 cell line was purchased from InvivoGen (Catalog No.: hkb-htlr7, San Diego, California, USA). These cells were designed to study human TLR7 stimulation by monitoring NF-κB activation. The SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. NF-κB and AP-1 were activated to induce SEAP by stimulating HEK-Blue hTLR7 cells with TLR7 ligands. Therefore, reporter expression was regulated by NF-κB promoters for 20 hours with human TLR7 stimulation. Using QUANTI-Blue TM The kit (Cat.#: rep-qb1, Invivogen, San Diego, Ca, USA) measures the activity of the SEAP reporter gene in the cell culture supernatant at a wavelength of 640 nm. In the presence of alkaline phosphatase, the detection medium turns purple or blue.

[0391] HEK293-Blue-hTLR7 cells were incubated at a density of 250,000 to 450,000 cells / mL in a 96-well plate containing 4.5 g / L glucose, 50 U / mL penicillin, 50 mg / mL streptomycin, 100 mg / mL Normocin, 2 mM L-glutamine, 10% (V / V) heat-inactivated fetal bovine serum in a volume of 180 μL. HEK293-Blue-hTLR7 cells were then added with 20 μL of test compounds in a serial dilution in the presence of 1% in the final DMSO, and incubated at 37°C in a CO2 incubator for 20 hours. The 20 μL supernatant in each well was then incubated with 180 μL Quanti-blue substrate solution at 37°C for 2 hours, and the absorbance was read at 620 to 655 nm using a spectrophotometer. It is widely accepted that TLR7 activation leads to the signaling pathway of downstream NF-κB activation, and thus similar reporter assays are also widely used to evaluate TLR7 agonists (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Vol. 29, No. 7, July 2008, p. 329.sci; Hiroaki Hemmi et al., Nature Immunology 3, 196-200 (2002)).

[0392] The compounds of the invention were tested for TLR7 agonist activity in the HEK293-hTLR7 assay, as described herein, and the results are listed in Table 1. The EC of Example 1 was found to be 50The EC of a particular compound of the invention was found to be about 0.001 μM to about 0.08 μM. 50 It is about 0.001 μM, which is much better than the reference compound RO14.

[0393] Table 1. EC of compounds of the present invention 50

[0394]

[0395]

[0396] Example 21

[0397] Solubility: Lyophilization Solubility Assay (LYSA) and Thermodynamic Solubility Assay (THESA)

[0398] Aqueous solubility is a key physicochemical property of any potential drug candidate and is routinely measured in both drug discovery and development. Poor solubility is detrimental to absorption after oral administration and may mask compound activity in various ways in bioassays, including underestimated activity, reduced hit rates in high throughput screening (HTS), changes in data output, inaccurate structure-activity relationships (SAR), and inaccurate results of in vitro absorption, distribution, metabolism, excretion, and toxicity (ADMET) tests. In early drug profiling in drug development, the importance of solubility data for predicting oral absorption has also been widely discussed. Therefore, it is best to identify solubility limitations as early as possible before performing functional assays. Therefore, it is crucial to evaluate solubility as early as possible in the drug discovery process.

[0399] The solubility of a substance can be broadly defined as the maximum amount of that substance that dissolves in a specified volume of solvent. However, it is important to understand that the solubility of a compound can vary greatly depending on solvent conditions (such as temperature and pH) and the physicochemical properties of the compound (such as ionization and crystallinity). These key factors need to be considered during solubility determination in order to generate high-quality solubility data that will help compounds progress through the discovery and development stages. The kinetic solubility of a compound is the maximum solubility of the fastest precipitating substance of the compound; this is usually measured using a stock solution of the compound dissolved in an organic solvent (usually dimethyl sulfoxide (DMSO)) as the starting material. Kinetic solubility values ​​are strongly dependent on time and method, and are therefore expected to be non-reproducible between different laboratories using different protocols. The precipitate formed is rarely determined during the assessment, and may be any combination of the various possible solid states of the compound. Taking into account the level of supersaturation that may occur when diluting an organic solvent in water, kinetic solubility values ​​are usually higher than the corresponding thermodynamic (equilibrium) solubility values. Solubility assays during early discovery usually determine kinetic solubility. The lyophilization solubility assay (LYSA) is a high-throughput kinetic solubility assay used in the early drug discovery process.

[0400] Lyophilization Solubility Assay (LYSA) Method

[0401] Samples were prepared in duplicate from a 10 mM DMSO stock solution. After evaporating the DMSO with a centrifugal vacuum evaporator, the compound was dissolved in 0.05 M phosphate buffer (pH 6.5), stirred for one hour and shaken for two hours. After one night, the solution was filtered using a microtiter filter plate. The filtrate and its 1 / 10 dilution were then analyzed by HPLC-UV. In addition, a four-point calibration curve was prepared from the 10 mM stock solution and used for solubility determination of the compound. The results are in μg / mL. If the maximum value of the percentage of the sample measured in the solution after evaporation divided by the calculated sample amount is greater than 80%, the solubility is reported as greater than this value. It should be noted that a metastable solid phase may be formed during the evaporation step, so this method may produce higher solubility values ​​than assays using stable polymorphs as starting materials.

[0402] Thermodynamic (equilibrium) solubility is the saturated solubility of a compound at the end of the dissolution process, where the dissolved compound is in equilibrium with excess undissolved material. Thermodynamic solubility is determined by partitioning a solid compound into a solvent. This is generally considered the true solubility of a compound and is key information in formulation development. Thermodynamic solubility assay (THESA) is a widely used tool for measuring equilibrium solubility in late-stage drug discovery processes and is considered the gold standard solubility assay.

[0403] Thermodynamic Solubility Assay (THESA) Method

[0404] About 1 to 2 mg of each compound was added to 300 μL 50 mM phosphate buffer (pH 6.5) at room temperature (22 ± 2 ° C). Each sample was placed in a microanalysis tube, sonicated for 1 hour and vibrated for 2 hours. All suspensions were placed overnight (about 16 hours). The next day, the sample was filtered into a V-bottom plate with a micron filter plate to separate the solid matter from the solution. Then, all solutions were diluted into a 96-well V-bottom microplate and analyzed by UPLC-UV. Using 5 different dilutions of the compound in a DMSO stock solution (concentration of about 1 mg / mL) prepared in a micron tube, a calibration line was established by UPLC. Dilution was performed with DMSO. The solubility of the compound can be determined from the regression equation. In the case where the drug is completely dissolved in the buffer, it is assumed that the equilibrium solubility value is higher than the value determined by UPLC and reported accordingly.

[0405] The results clearly show that both the kinetic solubility (LYSA) and the thermodynamic (equilibrium) solubility (THESA) of the compounds of the present invention are greatly improved compared to the reference compound RO14.

[0406] Table 2. LYSA and THESA of the compounds of the present invention

[0407]

[0408]

[0409] Example 22

[0410] Dissolution testing: Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF)

[0411] The dissolution of a drug in the physiological environment of the gastrointestinal tract is the first step in the oral absorption process of a drug dosage form. Since only dissolved drug can penetrate the mucosa of the gastrointestinal absorption site, the solubility of the drug and its dissolution rate are crucial to its in vivo behavior. It is essential to perform dissolution testing under conditions that closely resemble the key parameters of human gastrointestinal physiology. In addition to selecting adequate equipment and appropriate instrument parameters, it is also very important to use physiologically relevant dissolution media.

[0412] Biorelevant gastrointestinal media simulating fasted and fed states have been developed to mimic in vivo conditions as closely as possible. These media have been used to examine the solubility and dissolution properties of several classes of drugs to help predict in vivo absorption behavior. Biorelevant in vitro dissolution testing can be used to qualitatively predict the effects of formulation and food on the dissolution and availability of orally administered drugs. It has been observed that biorelevant media provide more accurate simulations of pharmacokinetic profiles than simulated gastric or simulated intestinal fluids. The formulations and preparation instructions for biorelevant media are detailed below.

[0413] Fasting state simulated intestinal fluid (FaSSIF)

[0414] Step 1: Preparation of blank FaSSIF

[0415] Dissolve 0.420 g NaOH (pellets), 3.954 g NaH2PO4 (monohydrate) and 6.186 g NaCl in about 0.9 L purified water. Adjust pH to 6.5 with 1 N NaOH or 1 N HCl. Make up the total volume of the mixture to 1.0 L with purified water at room temperature.

[0416] Step 2: Preparation of FaSSIF

[0417] 0.2240 g of SIF raw powder (Biorelevant.com Ltd, product code: FFF03, UK) was added to about 50 mL of blank FaSSIF buffer and the mixture was stirred until the powder was completely dissolved. The total volume of the mixture was made up to 100 mL with blank FaSSIF buffer at room temperature. Fed State Simulated Intestinal Fluid (FeSSIF)

[0418] Step 1: Preparation of blank FeSSIF

[0419] Dissolve 4.040 g NaOH (pellets), 8.650 g glacial acetic acid and 11.874 g NaCl in about 0.9 L purified water. Adjust the pH to 5 with 1 N NaOH or 1 N HCl. Make up the total volume of the mixture to 1.0 L with purified water at room temperature.

[0420] Step 2: Preparation of FeSSIF

[0421] 1.120 g of SIF raw powder (Biorelevant.com Ltd, product code: FFF03, UK) was added to about 50 mL of blank FeSSIF buffer and the mixture was stirred until the powder was completely dissolved. The total volume of the mixture was made up to 100 mL with blank FeSSIF buffer at room temperature.

[0422] Dissolution Test Method

[0423] Approximately 1 to 2 mg of each compound was added excessively to 300 μL buffer at room temperature. Each sample was placed in a microanalytical glass tube, ultrasonically treated for 1 hour and stirred for 2 hours. All suspensions were placed overnight (approximately 16 hours). The next day, all pH values ​​were measured with a pH meter, and the samples were filtered into a V-bottom plate with a micron-grade filter plate to separate solid matter from the solution. Then, all solutions were diluted into a 96-well V-bottom microplate and analyzed by UPLC-UV.

[0424] A calibration line was established by UPLC using 5 different dilutions of the compound in a DMSO stock solution (concentration of approximately 1 mg / mL) prepared in micrometer tubes. Dilutions were performed with DMSO (in special cases, with acetonitrile or 80% HCl 0.1 N / 20% acetonitrile). From this regression equation the solubility of the compound can be determined. In cases where the compound is completely dissolved in the buffer, the equilibrium solubility value is assumed to be higher than the value determined by UPLC and reported accordingly.

[0425] The results clearly show that the dissolution of the compounds of the present invention in both fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) is greatly improved compared to the reference compound RO14. Table 3. FaSSIF and FeSSIF of the compounds of the present invention.

[0426] Instance Number FaSSIF (μg / mL) FeSSIF (μg / mL) RO14 14 21 Example 1 2527 3077 Example 2 3519 4801 Example 13-A 2368 4672 Example 15-A 3074 3987 Example 15-B >3733 >4267 Example 16-A 1799 2371 Example 16-B 784 1464 Example 18-A 3436 4536 Example 18-B >3767 >5000

Claims

1. A compound of formula (I), in R 1 C 1-6 alkyl; R 2 C 1-6 alkyl; R 3 is pyridinyl substituted by: ((C 1-6 alkyl)2amino)C 1-6 Alkoxy, ((C 1-6 alkyl)2amino)C 1-6 Alkylamino, ((C 1-6 alkyl)2amino)C 1-6 Alkylpyrrolidyl, (C 1-6 alkyl)2-piperazinyl, (C 1-6 Alkylamino)C 1-6 Alkylamino, (C 1-6 alkylpyrrolidinyl)amino, 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl, 2,5-diazabicyclo[2.2.2]octanyl, aminopyrrolidinyl, C 1-6 Alkyl-1-oxa-4,9-diazaspiro[5.5]undecyl, C 1-6 Alkylpiperazine, C 1-6 Alkylsulfonylpiperazinyl, pyrrolidinyl C 1-6 Alkylamino or tetrahydropyranylamino; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein R 1 It is methyl or ethyl.

3. The compound according to claim 1 or 2, wherein R 1 It is methyl.

4. A compound according to any one of claims 1 to 3, wherein R 2 It is methyl, ethyl or propyl.

5. A compound according to any one of claims 1 to 4, wherein R 3 for Where R 4 for ((C 1-6 alkyl)2amino)C 1-6 Alkoxy, ((C 1-6 alkyl)2amino)C 1-6 Alkylamino, ((C 1-6 alkyl)2amino)C 1-6 Alkylpyrrolidyl, (C 1-6 alkyl)2-piperazinyl, (C 1-6 Alkylamino)C 1-6 Alkylamino, (C 1-6 alkylpyrrolidinyl)amino, 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl, 2,5-diazabicyclo[2.2.2]octanyl, aminopyrrolidinyl, C 1-6 Alkyl-1-oxa-4,9-diazaspiro[5.5]undecyl, C 1-6 Alkylpiperazine, C 1-6 Alkylsulfonylpiperazinyl, pyrrolidinyl C 1-6 Alkylamino or tetrahydropyranylamino.

6. A compound according to any one of claims 1 to 5, wherein R 3 for Where R 4 for ((C 1-6 alkyl)2amino)C 1-6 Alkylamino or (C 1-6 Alkylamino)C 1-6 Alkylamino.

7. A compound according to any one of claims 1 to 6, wherein R 3 It is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino.

8. A compound according to any one of claims 1 to 7, wherein R 1 C 1-6 alkyl; R 2 C 1-6 alkyl; R 3 for Where R 4 for ((C 1-6 alkyl)2amino)C 1-6 Alkylamino or (C 1-6 Alkylamino)C 1-6 Alkylamino; or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 8, wherein R 1 is methyl; R 2 is methyl, ethyl or propyl; R 3 for Where R 4 is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino; or a pharmaceutically acceptable salt thereof.

10. A compound selected from: 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one; 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one; 6-amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one; 9-[[6-(2,3,3a,4,6,6a-Hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-yl)-3-pyridinyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one; 6-amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-dimethylphosphoryl-9-[[6-(tetrahydropyran-4-ylamino)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-9-[[6-[3-[(dimethylamino)methyl]pyrrolidin-1-yl]-3-pyridinyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one; 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undec-9-yl)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-dimethylphosphoryl-9-[[6-[(1-methylpyrrolidin-3-yl)amino]-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridinyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one; 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one; 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one; and 6-amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridinyl]methyl]-7H-purin-8-one; or a pharmaceutically acceptable salt thereof.

11. A method for preparing a compound according to any one of claims 1 to 10, comprising the following steps: a) making a compound of formula (III), With amine or alcohol HR 4 (II) reacting in a solvent-free reaction; or reacting in the presence of an inorganic base, wherein the base is sodium hydride; or reacting in the presence of an organic metal catalyst system, wherein the catalyst system is selected from Pd2(dba)3 / RuPhos / t-BuONa, Pd2(dba)3 / BrettPhos / t-BuONa and Pd-PEPPSI-IPentCl / t-BuOK; wherein R 1 , R 2 and R 4 As defined in any one of claims 1 to 9.

12. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, which is prepared by the process according to claim 11.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

14. A compound or a pharmaceutically acceptable salt according to any one of claims 1 to 10 for use as a therapeutically active substance.

15. A compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, for use in treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive small cell lung cancer, non-small cell lung cancer, muscle invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma.

16. Use of a compound according to any one of claims 1 to 10 as an agonist of TLR7.

17. Use of a compound according to any one of claims 1 to 10 for the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive small cell lung cancer, non-small cell lung cancer, muscle invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma.

18. The use of a compound according to any one of claims 1 to 10 for the preparation of a medicament for the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive small cell lung cancer, non-small cell lung cancer, muscle invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma.

19. Use according to claim 17 or 18, wherein the cancer is selected from pancreatic ductal adenocarcinoma and colorectal cancer.

20. A method of treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, bile duct carcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, extensive small cell lung cancer, non-small cell lung cancer, muscle invasive bladder cancer, nodular basal cell carcinoma and squamous cell carcinoma, the method comprising administering a therapeutically effective amount of a compound as defined in any one of claims 1 to 10.

21. The invention as herein described.

Images