Phosphorylpurinone compounds for the treatment of cancer

Abstract

The present invention relates to a compound of formula (I), TIFF2025522978000084.tif37170(wherein R 1 ~R 3 are as described herein), and pharmaceutically acceptable salts thereof, and also relates to compositions containing the compound and methods of using the compound.

Description

Technical Field

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

Background Art

[0002] Field of the Invention The present invention relates to novel phosphoryl purinones and their derivatives having Toll-like receptor agonism activity, their production, pharmaceutical compositions containing them, and their potential use as pharmaceuticals for the treatment and / or prophylaxis of cancer.

[0003] Immune checkpoint inhibitors have changed the treatment landscape for many tumors, but durable responses are limited. Lymphocyte infiltration and IFN-γ status are important factors for effective treatment by defining a "T cell inflammatory" phenotype ("hot tumor"). In contrast, the absence of T cells infiltrating the tumor indicates the characteristics of a "non-inflammatory" or "cold tumor". Immunological treatment of cold tumors is a major challenge because an adaptive immune response is not occurring or being maintained. However, cold tumors can contain a significant number of myeloid cells such as macrophages, different subsets of DCs, and myeloid-derived suppressor cells. An agonist approach targeting myeloid cells aims to induce an anti-tumor inflammatory response by secreting pro-inflammatory cytokines and maintain T cell effector function and memory formation. Considering that myeloid cells are also present in cold tumors, activating them in the tumor microenvironment increases T cell infiltration and sustained activation in those tumors. This could result in a clinical benefit in inflammatory tumors and immune excluded tumors, which have hitherto benefited from immunotherapy in only a portion of patients.

[0004] As part of the innate immune system, myeloid cells can respond to infection and abnormal cell behavior. Myeloid cells are the first to detect pathogens / abnormal cells and eliminate them through various mechanisms such as inflammatory cytokine secretion and phagocytosis. Another extremely important function of myeloid cells is to provide co-stimulatory signals and antigen presentation to T cells, thereby enabling the continuous activation of T cells, which opens the way to a long-lasting immune response. On the other hand, they also play an important role in tissue repair and homeostasis by suppressing uncontrolled immune activation. Tumors exploit this property of these cells by mobilizing them and having them create an inhibitory microenvironment, thereby inhibiting T cells through direct and indirect mechanisms. Tumor-associated myeloid cells can be activated even in an inhibitory tumor microenvironment, for example, by delivering the correct signal to TLR agonists. Once activated, they can produce inflammatory cytokines, upregulate activation markers, and ultimately induce an anti-tumor immune response, ideally resulting in a long-lasting adaptive response.

[0005] Toll-like receptors (TLRs) detect highly conserved pathogenic danger signals and trigger strong inflammatory responses. TLR signaling initiates innate immune responses against pathogens and ultimately adaptive immune responses. Toll-like receptors (TLRs) detect a wide range of conserved pathogen-associated molecular patterns (PAMPs). They play an important role in sensing invading pathogens and then initiating innate immune responses. There are 10 known members of the TLR family in humans, 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. Within this family, TLR3, TLR7, TLR8, and TLR9 are located within endosomes.

[0006] TLR7 can be activated by binding to specific small molecule ligands (i.e., TLR7 agonists) or its natural ligand (i.e., single-stranded RNA, ssRNA). After ssRNA binds to TLR7, the receptor in its dimeric form undergoes a structural change, and then adapter proteins including myeloid differentiation primary response gene 88 (MyD88) are thought to be recruited to its cytoplasmic domain. After the initiation of the receptor signaling cascade via the MyD88 pathway, 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 the transcription of various genes, such as IFN-α and other antiviral cytokine genes. Of great interest is the activation of intratumoral myeloid cells that express TLR7 in endosomes, which initiate and regulate the immune response (the natural ligand is viral single-stranded RNA). Systemic administration of TLR agonists results in potent systemic activation of various immune cells, ultimately activating immune cells within the tumor microenvironment to achieve an antitumor effect. In fact, several identified TLR7 agonists are being investigated for therapeutic purposes. Topical application of imiquimod (R-837) (Aldara®), a TLR7 agonist, to human patients with murine subcutaneous tumors or skin cancer lesions strongly activates the antitumor immune response. The TLR7 / 8 dual agonist Resiquimod (R-848) is an immunomodulatory topical gel for the treatment of stage cutaneous T-cell lymphoma currently in a Phase II clinical trial at Galderma. MBS-8 is a micelle nanoparticle formulation of TLR7 agonist 1V270 developed by MonTa Biosciences as an intravenous immunotherapy treatment for advanced solid tumors. BDB-001 is a TLR7 / 8 dual agonist for intravenous treatment of advanced or metastatic solid tumors refractory to anti-PD-1 / anti-PD-L1 therapy currently in a Phase II clinical trial at Seven and Eight Biopharmaceuticals. APR-003 is an oral small molecule TLR7 agonist being investigated in early clinical development by Apros Therapeutics for the treatment of patients with advanced unresectable colorectal cancer with malignant liver lesions.PRTX-007 is an orally administered small molecule TLR7 agonist for the treatment of acute viral diseases such as SARS-CoV-2 and cancer during early clinical development at Primmune Therapeutics. Accordingly, there remains a need to identify additional compounds suitable for systemic administration and preferential tumor targeting. The unmet clinical need for developing potent and safe TLR7 agonists for the treatment of a wide range of cancers is significant. SUMMARY OF THE INVENTION

[0007] SUMMARY OF THE INVENTION The present invention relates to novel compounds of formula (I), TIFF2025522978000002.tif37170(wherein, R 1 is C 1~6 alkyl, R 2 is C 1~6 alkyl, R 3 is ((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 alkylpyrrolidinyl, (C 1~6 alkyl)2piperazinyl, (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]undecanyl, C 1~6 alkylpiperazinyl, C 1~6 alkylsulfonylpiperazinyl, pyridinyl substituted with pyrrolidinylC 1~6 alkylamino or tetrahydropyranylamino) relates to the compound or its pharmaceutically acceptable salt.

[0008] The present invention also relates to their production, pharmaceuticals based on the compounds according to the invention and their production, and the use of the compounds of formula (I) thereof as agonists of TLR7.

[0009] The compounds of formula (I) exhibit good TLR7 agonism activity. In another embodiment, the compounds of the present invention showed excellent kinetic solubility and thermodynamic (equilibrium) solubility compared to the reference compound RO14. Furthermore, the compounds of formula (I) showed much improved fasting state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) dissolution results compared to the reference compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

Figure 1

Figure 2

[0011] DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS The term "C 1~6 alkyl" refers to a saturated straight-chain or branched-chain alkyl group having 1 to 6, especially 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. Specific "C 1~6 alkyl" groups are methyl, ethyl, and n-propyl.

[0012] The term "C 1~6 alkoxy" represents C 1~6 alkyl-O-.

[0013] The terms "halogen" and "halo" are used interchangeably herein and refer to fluoro, chloro, bromo, or iodo.

[0014] The term "pharmaceutically acceptable salt" means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid addition salts and base addition salts.

[0015] The term "pharmaceutically acceptable acid addition salt" refers to a pharmaceutically acceptable salt formed from an organic acid selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and 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, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.

[0016] The term "pharmaceutically acceptable base addition salt" refers to a pharmaceutically acceptable salt formed from an organic base or an inorganic base. Examples of acceptable inorganic bases include sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, salts of substituted amines, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperidine, piperidine, N-ethylpiperidine, and polyamine resins.

[0017] The term "pharmaceutically active metabolite" refers to a pharmacologically active product produced through the metabolism in vivo of a specific compound or its salt. After entering the body, most drugs serve as substrates for chemical reactions that can change their physical properties and biological effects. These metabolic conversions usually affect the polarity of the compounds of the present invention and change the way the drugs are distributed in and excreted from the body. However, in some cases, drug metabolism is required for therapeutic effects.

[0018] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents a specific disease, condition, or disorder, (ii) attenuates, alleviates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The therapeutically effective amount can vary depending on the compound, the state of the disease 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 physician or veterinarian, and other factors.

[0019] The term "pharmaceutical composition" refers to a mixture or solution that contains a therapeutically effective amount of an active pharmaceutical ingredient together with pharmaceutically acceptable additives and is administered to a mammal, such as a human in need thereof.

[0020] The terms "pharmaceutically acceptable additive", "pharmaceutically acceptable carrier", and "therapeutically inert additive" can be used interchangeably and refer to any pharmaceutically acceptable component that has no therapeutic activity and is non-toxic to the subject being administered and is used in the formulation of a medicament, such as a disintegrant, binder, filler, solvent, buffer, isotonic agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant.

[0021] An agonist of TLR7 The present invention relates to (i) a compound of formula (I), TIFF2025522978000003.tif37170(wherein, R 1 is C 1~6 alkyl, R 2 is C 1~6 alkyl, R 3 is ((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 alkylpyrrolidinyl, (C 1~6 alkyl)2piperazinyl, (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]undecanyl, C 1~6 alkylpiperazinyl, C 1~6 alkylsulfonylpiperazinyl, pyrrolidinylC 1~6 alkylamino or pyridinyl substituted with tetrahydropyranylamino) or a pharmaceutically acceptable salt thereof.

[0022] A further embodiment of the present invention is the compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein (ii) R 1 is methyl or ethyl.

[0023] A further embodiment of the present invention is the compound of formula (I) according to (i) or (ii), or a pharmaceutically acceptable salt thereof, wherein (iii) R 1 is methyl.

[0024] A further embodiment of the present invention is (iv) R 2The compound of formula (I) according to any one of (i) to (iii), wherein it is methyl, ethyl or propyl.

[0025] A further embodiment of the present invention is (v) R 3 is TIFF2025522978000004.tif15170, and R 4 is ((C 1~6 alkyl) 2 amino) C 1~6 alkoxy, ((C 1~6 alkyl) 2 amino) C 1~6 alkylamino, ((C 1~6 alkyl) 2 amino) C 1~6 alkylpyrrolidinyl, (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]undecanyl, C 1~6 alkylpiperazinyl, C 1~6 alkylsulfonylpiperazinyl, pyrrolidinyl C 1~6 alkylamino or tetrahydropyranyl amino, the compound of formula (I) according to any one of (i) to (iv), or a pharmaceutically acceptable salt thereof.

[0026] A further embodiment of the present invention is (vi) R 3 is TIFF2025522978000005.tif15170, and R 4 is ((C 1~6 alkyl) 2 amino) C 1~6 alkylamino or (C 1~6 alkylamino) C 1~6 alkylamino, the compound of formula (I) according to any one of (i) to (v), or a pharmaceutically acceptable salt thereof.

[0027] A further embodiment of the present invention is (vii) R 3 is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino, a compound of formula (I) according to any one of (i) to (vi), or a pharmaceutically acceptable salt thereof.

[0028] A further embodiment of the present invention is (viii) R 1 is 1~6 alkyl, R 2 is 1~6 alkyl, R 3 is TIFF2025522978000006.tif15170, and R 4 is ((C 1~6 alkyl)2amino)C 1~6 alkylamino or (C 1~6 alkylamino)C 1~6 alkylamino, a compound of formula (I) according to any one of (i) to (vii), or a pharmaceutically acceptable salt thereof.

[0029] A further embodiment of the present invention is (ix) R 1 is methyl, R 2 is methyl, ethyl or propyl, R 3 is TIFF2025522978000007.tif15170, and R 4 is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino, a compound of formula (I) according to any one of (i) to (viii), or a pharmaceutically acceptable salt thereof.

[0030] Another embodiment of the present invention is the following 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one, 6-Amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one, 6-Amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridyl]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-pyridyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one, 6-Amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-dimethylphosphoryl-9-[[6-(tetrahydropyran-4-ylamino)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-9-[[6-[3-[(dimethylamino)methyl]pyrrolidin-1-yl]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one, 6-Amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-dimethylphosphoryl-9-[[6-[(1-methylpyrrolidin-3-yl)amino]-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-Amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one, 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one, and 6-Amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one, a compound of formula (I) selected therefrom, or a pharmaceutically acceptable salt thereof.

[0031] Another embodiment of the present invention is a method for preparing a compound according to any one of (i) to (x), comprising the following steps, a) a compound of formula (III), TIFF2025522978000008.tif44170 and an amine or alcohol HR 4 a solvent-free reaction or a reaction in the presence of an inorganic base (the base being sodium hydride) or a reaction in the presence of an organometallic catalyst system (the catalyst system being 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 are defined as described in any one of (i) to (ix), a method involving the reaction.

[0032] Another embodiment of the present invention is a compound according to any one of (i) to (x) or a pharmaceutically acceptable salt thereof when produced according to the method of claim 11.

[0033] Another embodiment of the present invention relates to a pharmaceutical composition comprising a compound according to any one of (i) to (x) and a pharmaceutically acceptable additive.

[0034] Another embodiment of the present invention is a compound according to any one of (i) to (x) or a pharmaceutically acceptable salt thereof for use as a therapeutic active substance.

[0035] Another embodiment of the present invention is a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, advanced small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma, and squamous cell carcinoma.

[0036] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) as an agonist of TLR7.

[0037] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) for the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, advanced small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma, and squamous cell carcinoma.

[0038] Another embodiment of the present invention is the use of a compound according to any one of (i) to (x) 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, cholangiocarcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, advanced small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma, and squamous cell carcinoma.

[0039] Another embodiment of the present invention is 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.

[0040] Another embodiment of the present invention is a method for treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, advanced small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma, and squamous cell carcinoma, and the method comprises administering a therapeutically effective amount of a compound defined in any one of (i)-(x).

[0041] Pharmaceutical Compositions and Administration Another embodiment provides a pharmaceutical composition or medicament containing a compound of the present invention and a therapeutically inert carrier, diluent, or additive, and a method of using a compound of the present invention for preparing such a composition and medicament. In one example, a compound of formula (I) can be formulated into a phytomedicine dosage form by mixing it with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used, at ambient temperature, at an appropriate pH, and at a desired degree of purity. The pH of the formulation mainly depends on the specific application and the concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate buffer at pH 5. In another embodiment, a 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.

[0042] The compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to medical practitioners. The "effective amount" of the compound to be administered is determined by such considerations and is the minimum amount necessary to activate the TLR7 receptor and produce INF-α and other cytokines, which can be used, but are not limited to, the treatment of cancer. For example, such an amount can be less than an amount that is toxic to normal cells or to the entire mammal.

[0043] In one example, the pharmaceutically effective amount of the compound of the present invention administered parenterally per single dose ranges from about 0.01 to 1000 mg / kg of the patient's body weight per day, or from about 0.01 to 1000 mg / kg of the patient's body weight per day, and a typical initial range of the compound used is 0.3 to 15 mg / kg / day. In another embodiment, oral unit dosage forms such as tablets and capsules preferably contain from about 1 to about 1000 mg of the compound of the present invention.

[0044] 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, intralung, intradermal, intrathecal and epidural, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0045] The compounds of the present invention can be administered in any convenient dosage form, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions can contain ingredients conventional in pharmaceutical preparations, for example, diluents, carriers, pH adjusters, sweeteners, bulking agents and additional active agents.

[0046] Typical formulations are prepared by mixing the compounds of the present invention with carriers or additives. Suitable carriers and additives are well known to those skilled in the art and are described in detail, for example, in Ansel, Howard C., et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004, Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000, and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also contain one or more buffers, stabilizers, surfactants, wetting agents, smoothing agents, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, flow promoters, processing aids, colorants, sweeteners, flavors, fragrances, diluents and other known additives for providing an aesthetically pleasing presentation of the drug (i.e., the compound of the present invention or its pharmaceutical composition) or for assisting in the manufacture of a pharmaceutical product (i.e., a pharmaceutical).

[0047] Examples of suitable oral dosage forms are tablets containing from about 1 to 1000 mg of anhydrous lactose, from about 1 to 1000 mg of croscarmellose sodium, from about 1 to 1000 mg of polyvinylpyrrolidone (PVP) K30, and from about 1 to 1000 mg of magnesium stearate, and from about 1 to 1000 mg of the compound of the present invention. The powdered components are first mixed together and then mixed with a solution of PVP. The resulting composition can be dried, granulated, mixed with magnesium stearate, and compressed into tablet form using conventional equipment. Examples of aerosol formulations can be prepared, for example, by dissolving 1 to 500 mg of the compound of the present invention in a suitable buffer solution, such as phosphate buffer, and adding a salt, such as sodium chloride, as an isotonic agent if desired. The solution can be filtered, for example, through a 0.2 micron filter to remove impurities and contaminants.

[0048] Accordingly, one embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof. In a further embodiment, it includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or additive.

[0049] Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of a proliferative disorder. Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of cancer.

[0050] The following embodiments illustrate typical compositions of the present invention, but merely serve as representative examples thereof.

[0051] Composition A The compound of the present invention can be used in a manner known per se as an active ingredient to produce tablets of the following composition. Per tablet Active ingredient 200 mg Microcrystalline cellulose 155 mg Corn starch 25 mg Talc 25 mg Hydroxypropylmethylcellulose 20 mg 425 mg

[0052] Composition B The compound of the present invention can be used in a method known per se for producing a capsule preparation having the following composition as an active ingredient. Per capsule Active ingredient 100.0 mg Corn starch 20.0 mg Lactose 95.0 mg Talc 4.5 mg Magnesium stearate 0.5 mg 220.0 mg

[0053] Indications and treatment methods The compound of the present invention targets TLR7. Therefore, the compound of the present invention is useful for promoting the activation of antigen-presenting cells (APCs), contributing to T cell priming, trafficking and infiltration, promoting Th1 and CD8+ T responses, and promoting cancer cell death. The compound of the present invention is useful for enhancing the innate immune response in myeloid cells expressing TLR7. Alternatively, the compound of the present invention is useful for activating T cells in tumors where T cells are present but are suppressed, for example, by upregulation of costimulatory molecules and production of pro-inflammatory cytokines. More generally, the present compound can be used for the treatment of cancer types that are not inflamed, such as immune desert or immune excluded states, by enhancing antigen presentation, T cell priming, mobilization / infiltration, and tumor cell death.

[0054] Another embodiment includes a method of treating or preventing cancer in a mammal in need of treatment or prevention, the method comprising administering to the mammal a therapeutically effective amount of a compound of formula (I), a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof. Cancers herein include, but are not limited to, progressive solid tumors such as pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, head and neck squamous cell carcinoma, adenoid cystic carcinoma, advanced small cell lung cancer, non-small cell lung cancer, muscle-invasive bladder cancer, nodular basal cell carcinoma, squamous cell carcinoma, etc.

[0055] Synthesis The compounds of the present invention can be prepared by any conventional means. Suitable methods for synthesizing these compounds and their starting materials are shown in the following schemes and examples. All substituents, especially R 1 ~R 4 are as defined above unless otherwise specified. Further, unless otherwise explicitly stated, all reactions, reaction conditions, abbreviations, and symbols have meanings well known to those skilled in the art of organic chemistry.

[0056] A general synthetic route for preparing the compound of formula (I) is shown below.

[0057] Scheme 1 TIFF2025522978000009.tif117170

[0058] The compound of formula (X) is prepared by the reaction of the compound of formula (XI) with (4-methoxyphenyl)methanamine in the presence of an organic base such as trimethylamine or N,N-diisopropylethylamine. The compound of formula (IX) is prepared by the reaction of the compound of formula (X) with 1-(chloromethyl)-4-methoxy-benzene in the presence of an inorganic base such as potassium carbonate, sodium hydride or cesium carbonate. The compound of formula (VII) is prepared by the reaction of 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, followed by alkylation 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 an aqueous sodium hydroxide solution. The compound of formula (Ia) is prepared by the reaction of the compound of formula (III) with an amine or alcohol HR 4 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-BuONa, Pd2(dba)3 / BrettPhos / t-BuONa, or Pd-PEPPSI-IPentCl / t-BuOK with the compound of formula (II).

[0059] The compounds of the present invention can be obtained as mixtures of diastereomers or enantiomers, which can be separated by methods well known in the art, such as (chiral) HPLC or SFC. In another embodiment, the compounds of formula (I) or (Ia) can be obtained according to the above scheme by using the corresponding chiral starting materials.

[0060] The present invention also relates to a method for preparing a compound of formula (I) or formula (Ia), comprising the following steps a) A compound of formula (III), TIFF2025522978000010.tif44170 and an amine or alcohol HR 4 a reaction in the absence of a solvent, 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-BuONa, Pd2(dba)3 / BrettPhos / t-BuONa, or Pd-PEPPSI-IPentCl / t-BuOK between (II),

[0061] A compound of formula (I) when produced by the above method is also an object of the present invention.

Examples

[0062] The present invention will be more fully understood by referring to the following examples. However, these examples should not be construed as limiting the scope of the present invention.

[0063] Abbreviations aq. Aqueous solution BrettPhos: dicyclohexyl-[3,6-dimethoxy-2-(2,4,6-triisopropylphenyl)phenyl]phosphane BSA: N,O-bis(trimethylsilyl)acetamide CDI: N,N’-carbonyldiimidazole DIEPA: N,N-diethylpropylamine DMF: Dimethylformamide DBU: 1,8-diazabicycloundec-7-ene DPPA: Diphenylphosphoryl azide EC 50 : The molar concentration of an agonist that produces 50% of the maximum possible response of the agonist. EDC: N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine eq: Equivalent EtOAc or EA: Ethyl acetate HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) hr(s): hour(s) HPLC: High Performance Liquid Chromatography HOBt: N-Hydroxybenzotriazole MS(ESI): Mass Spectrometry (Electron Spray Ionization) m-CPBA: 3-Chloroperbenzoic acid MTEB: Methyl tert-butyl ether NMR: Nuclear Magnetic Resonance NMP: N-Methylpyrrolidone obsd.: Observed value 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 PE: Petroleum ether PMB: p-Methoxybenzyl PPA: Polyphosphoric acid RT or rt: Room temperature RuPhos: Dicyclohexyl-[2-(2,6-diisopropoxyphenyl)phenyl]phosphane sat.: Saturated SFC: Supercritical Fluid Chromatography TEA: Triethylamine TFA: Trifluoroacetic acid THF: Tetrahydrofuran TLC: Thin Layer Chromatography V / V: Volume ratio XantPhos: (9,9-Dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine)

[0064] General experimental conditions The intermediate and final compound were purified by flash chromatography using one of the following equipment: i) Biotage SP1 system and Quad 12 / 25 cartridge module, ii) ISCO combi-flash chromatography equipment, silica gel brand and pore size: i) KP-SIL 60Å, particle size: 40 - 60μm, ii) CAS registration number: silica gel: 63231 - 67-4, particle size: 47 - 60 micron silica gel, iii) ZCX manufactured by Qingdao Haiyang Chemical Co., Ltd., pore: 200 - 300 or 300 - 400.

[0065] The intermediate and final compound were purified by preparative HPLC on a reverse-phase column using a Phenomenex Luna C18 (15μm, 150×400mm) column, a Phenomenex Luna C18 (10μm, 100×250mm) column, a Phenomenex Luna C18 (10μm, 80×250mm) column, a Shim-pack C18 (10μm, 25×150mm) column, a Phenomenex Synergi C18 (10μm, 25×150mm) column or a Waters Atlantis T3 (5μm, 30×150mm) column. Using a Waters AutoP purification system (sample manager 2767, pump 2525, detectors: 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 a 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), purified by Prep-HPLC on a reverse-phase column.

[0066] The LC / MS spectrum of the compound was obtained using LC / MS (Waters™ Alliance 2795 - Micromass ZQ, Shimadzu Alliance 2020 - Micromass ZQ, or Agilent Alliance 6110 - Micromass ZQ), and the LC / MS conditions were as follows (run time 3 minutes or 1.5 minutes). Acidic condition I: A: 0.1% TFA in H2O, B: 0.1% TFA in acetonitrile, Acidic condition II: A: 0.0375% TFA in H2O, B: 0.01875% TFA in acetonitrile, Basic condition I: A: 0.1% NH3·H2O in H2O, B: acetonitrile, Basic condition II: A: 0.025% NH3·H2O in H2O, B: acetonitrile, Neutral condition: A: H2O, B: acetonitrile.

[0067] Mass spectrum (MS): Generally, only the ion showing the parent mass is reported, and unless otherwise specified, the mass ion cited is the positive mass ion (M + H) + is used.

[0068] The NMR spectrum was obtained using a Bruker Avance 400 MHz or a Bruker Avance 500 MHz.

[0069] All reactions involving air - sensitive reagents were carried out under an argon atmosphere. The reagents were used as received from commercial suppliers without further purification, unless otherwise specified.

[0070] Preparation Examples Example 1 6 - Amino - 9 - [[6 - [2 - (dimethylamino)ethylamino]-3 - pyridyl]methyl]-2 - dimethylphosphoryl - 7H - purin - 8 - one TIFF2025522978000011.tif30170

[0071] Project 1: Preparation of 2-chloro-N-[(4-methoxyphenyl)methyl]-9H-purin-6-amine (Compound 1a) TIFF2025522978000012.tif43170To a solution of 2,6-dichloro-9H-purine (50.0 g, 264.55 mmol, 1 equiv) and TEA (73.63 mL, 529.7 mmol, 2 equiv) in DMF (400 mL), (4-methoxyphenyl)methanamine (36.29 mL, 277.78 mmol, 1.05 equiv) was added. The mixture was stirred at 100 °C for 16 h. The 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 obtain 2-chloro-N-[(4-methoxyphenyl)methyl]-9H-purin-6-amine (60 g, Compound 1a) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.55 (brs, 1H), 8.13 (s, 1H), 7.29 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 4.56 (s, 2H), 3.71 (s, 3H).

[0072] Project 2: Preparation of 2-chloro-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 1b) TIFF2025522978000013.tif53170To a solution of 2-chloro-N-[(4-methoxyphenyl)methyl]-9H-purin-6-amine (180.0 g, Compound 1a, 621.29 mmol, 1 equiv) in DMF (1.8 L), K2CO3 (89.0 g, 683.42 mmol, 1.1 equiv) and 1-(chloromethyl)-4-methoxy-benzene (84.24 mL, 621.29 mmol, 1 equiv) were added. The mixture was stirred at 15 °C for 16 h and then diluted with H2O (2.0 L). After filtration, the filter cake was triturated (PE / EA = 5 / 1, 2 × 600 mL) for purification and dried in vacuo to obtain 2-chloro-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (180 g, Compound 1b) as a pink solid. MS found (ESI + )[{ 35Cl}(M+H) + :410.1. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 8.78 - 8.75 (m, 1H), 8.23 (s, 1H), 7.27 (d, J = 8.4 Hz, 4H), 6.91 - 6.85 (m, 4H), 5.25 (s, 2H), 5.06 - 4.54 (m, 2H), 3.74 - 3.69 (m, 6H).

[0073] Step 3: Preparation of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 1c) TIFF2025522978000014.tif52170To a solution of 2-chloro-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (35 g, Compound 1b, 85.4 mmol, 1 equiv) and methylphosphonomethane (10.0 g, 128.09 mmol, 1.5 equiv) in DMF (500 mL), K2CO3 (23.6 g, 170.79 mmol, 2 equiv), XantPhos (4.9 g, 8.54 mmol, 0.1 equiv) and Pd(OAc)2 (1.9 g, 8.54 mmol, 0.1 equiv) were added. The mixture was stirred at 150 °C for 2 h under a N2 atmosphere. The mixture was filtered through celite, and the filtrate was concentrated in vacuo to give 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (76 g, Compound 1c, crude product) as a yellow oil, which was used in the next step without further purification. MS found (ESI + )[(M+H) + :452.2.

[0074] Step 4: Preparation of 2-dimethylphosphoryl-9H-purin-6-amine (Compound 1d) A solution of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (76.0 g, Compound 1c, crude product) in TIFF2025522978000015.tif 24170 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 give 2-dimethylphosphoryl-9H-purin-6-amine (30 g, Compound 1d, crude product) as a pale yellow solid, which was used in the next step without further purification. MS found (ESI + )[(M+H) + : 211.0.

[0075] Step 5: Preparation of 9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1e) To a solution of 2-dimethylphosphoryl-9H-purin-6-amine (30.0 g, Compound 1d, 142.07 mmol, 1 equiv) in TIFF2025522978000016.tif 29170 DMF (300 mL) were added K2CO3 (37.0 g, 284.14 mmol, 2 equiv) and 2-chloro-5-(chloromethyl)pyridine (23.0 g, 142.07 mmol, 1 equiv). 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 give 9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (22 g, Compound 1e) as a pale yellow solid. MS found (ESI + )[{ 35 Cl}(M+H) + : 337.0. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 8.55 (d, J = 2.0 Hz, 1H), 8.45 (s, 1H), 7.90 (dd, J = 8.8, 2.8 Hz, 1H), 7.72 (brs, 2H), 7.52 (d, J = 8.4 Hz, 1H), 5.46 (s, 2H), 1.72 (d, J = 13.6 Hz, 6H).

[0076] Step 6: Preparation of 8-Bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1f) To a solution of 9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (22.0 g, Compound 1e, 65.34 mmol, 1 equiv), NaOAc (11.8 g, 143.74 mmol, 2.2 equiv) and AcOH (3.9 g, 65.34 mmol, 1 equiv) in water (220 mL), Br2 (20.9 g, 130.67 mmol, 2 equiv) was added dropwise at 15 °C. Then the mixture was stirred at 15 °C for 16 h. 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 give 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (11 g, Compound 1f) as a red solid. MS found (ESI + )[{ 35 Cl& 79 Br}(M + H) + : 416.9. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.48 (s, 1H), 7.87 - 7.68 (m, 3H), 7.53 (d, J = 8.0 Hz, 1H), 5.44 (s 2H), 1.71 (d, J = 13.6 Hz, 6H).

[0077] Step 7: Preparation of 6-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g) TIFF2025522978000018.tif29170Water (80 mL) and tert-butanol (80 mL) were charged with a solution of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (11.5 g, Compound 1f, 27.67 mmol, 1 equiv) and NaOH (8.8 g, 221.36 mmol, 8 equiv), and the mixture was stirred at 80 °C for 16 h. The mixture was concentrated in vacuo and then diluted with H2O (80 mL). Aqueous HCl solution (6 M) was added to adjust the pH to 8. After filtration, the filter cake was washed with H2O (3 × 60 mL) and dried in vacuo. The residue was triturated (EA / MeOH = 10 / 1, 3 × 20 mL) for purification to give 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (8.5 g, Compound 1g) as a gray solid, which was used in the next step without further purification. MS found (ESI + )[{ 35 Cl}(M+H) + : 353.0. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.45 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 8.0, 2.4 Hz 1H), 7.50 (d, J = 8.0 Hz, 1H), 6.74 (brs, 2H), 4.99 (s, 2H), 1.67 (d, J = 13.6 Hz, 6H).

[0078] Step 8: Preparation of 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (Example 1) TIFF2025522978000019.tif30170N’,N’-Dimethylethane-1,2-diamine (90.0 mL, 212.63 mmol, 10 eq) was added to 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (7.5 g, Compound 1 g, 21.26 mmol, 1 eq), and the mixture was stirred in a sealed tube at 170 °C for 72 h. 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), adjusted to pH = 8 by adding solid NaHCO3, and then concentrated in vacuo. The residue was dissolved in DCM / MeOH = 5 / 1 (100 mL), the mixture was filtered, and then the filtrate was concentrated in vacuo. The residue was dissolved in MeCN / MeOH = 20 / 1 (70 mL), stirred at 85 °C for 2 h, and then cooled to 15 °C. After filtration, the filter cake was dried in vacuo to obtain 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (2.2 g, Example 1) as a white solid. MS found (ESI + )[(M + H) + : 405.1. 1 H NMR (500 MHz, DMSO-d6) δ ppm 10.49 (brd, J = 2.0 Hz, 1H), 8.01 (d, J = 2.1 Hz, 1H), 7.39 (dd, J = 8.6, 2.4 Hz, 1H), 6.70 (brs, 2H), 6.42 (d, J = 8.7 Hz, 1H), 6.36 (t, J = 5.4 Hz, 1H), 4.75 (s, 2H), 3.27 (q, J = 6.5 Hz, 2H), 2.35 (t, J = 6.6 Hz, 2H), 2.14 (s, 6H), 1.68 (d, J = 13.6 Hz, 6H).

[0079] Example 2 6-Amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one TIFF2025522978000020.tif A solution of 2-(dimethylamino)ethanol (126.36 mg, 1.42 mmol, 10 equiv) in 29170 THF (3 mL) was added with NaH (56.7 mg, 60 wt%, 1.42 mmol, 10 equiv) at 0 °C. The mixture was stirred at 0 °C for 1 h. Then the mixture was concentrated in vacuo. The residue was dissolved in DMF (2 mL), and 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (50.0 mg, Compound 1g, 0.140 mmol, 1 equiv) was added. The mixture was stirred at 130 °C for 15 h. H2O (10 mL) was added dropwise at 0 °C, and then FA was added to adjust the pH to 6. Then the mixture was concentrated in vacuo, and the residue was purified by preparative HPLC to obtain 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (12.6 mg, Example 2) as a pale yellow solid. MS found (ESI + )[(M+H) + :406.1. 1 H NMR (400 MHz, CD3OD) δ ppm 8.26 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 8.8, 2.8 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 5.02 (s, 2H), 4.38 (t, J = 5.6 Hz, 2H), 2.75 (t, J = 5.6 Hz, 2H), 2.31 (s, 6H), 1.85 (d, J = 13.6 Hz, 6H).

[0080] Example 3 6-Amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one TIFF2025522978000021.tif32170

[0081] Step 1: Preparation of tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (Compound 3a) To a mixture of TIFF2025522978000022.tif 32170 NMP (2 mL), 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (100.0 mg, Compound 1g, 0.280 mmol, 1 equiv), tert-butyl N-pyrrolidin-3-ylcarbamate (79.2 mg, 0.430 mmol, 1.5 equiv) and t-BuONa (54.4 mg, 0.570 mmol, 2 equiv), RuPhos (13.2 mg, 0.030 mmol, 0.1 equiv) and Pd2(dba)3 (25.9 mg, 0.030 mmol, 0.1 equiv) were added. The mixture was stirred at 120 °C for 16 h under a N2 atmosphere. The mixture was filtered. The filtrate was concentrated in vacuo and purified by preparative HPLC to give tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (15 mg, Compound 3a) as a pale yellow solid. MS found (ESI + )[(M+H) + :503.2.

[0082] Step 2: Preparation of 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (Example 3) A solution of tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (15.0 mg, Compound 3a, 0.030 mmol, 1 equiv) in TIFF2025522978000023.tif 32170 HCl / dioxane (1.0 mL, 4 M) was stirred at 15 °C for 1 h. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC to give 6-amino-9-[[6-(3-aminopyrrolidin-1-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (8.7 mg, Example 3) as a pale yellow solid. MS found (ESI + )[(M+H) + :402.9. 11H NMR (400 MHz, CD3OD) δ ppm 8.17 (d, J = 2.0 Hz, 1H), 7.69 - 7.65 (m, 1H), 6.46 (d, J = 8.4 Hz, 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 = 13.6 Hz, 6H).

[0083] Example 4 6-Amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one TIFF2025522978000024.tif32170

[0084] Step 1: Preparation of tert-butyl 4-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-2,2-dimethyl-piperazine-1-carboxylate (Compound 4a) TIFF2025522978000025.tif34170The title compound was prepared in the same manner as in Step 1 of Example 3 by using tert-butyl 2,2-dimethylpiperazine-1-carboxylate instead of tert-butyl N-pyrrolidin-3-ylcarbamate. tert-Butyl 4-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-2,2-dimethyl-piperazine-1-carboxylate (80 mg, Compound 4a) was obtained as a pale yellow oil. MS found (ESI + ) [(M + H) + : 531.0.

[0085] Step 2: Preparation of 6-amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 4) By using tert-butyl 4-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-2,2-dimethyl-piperazine-1-carboxylate (Compound 4a) instead of tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (Compound 3a), the title compound was prepared in the same manner as in Step 2 of Example 3. 6-Amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (6.1 mg, Example 4) was obtained as a white solid. MS found (ESI + )[(M+H) + : 431.2. 1 H NMR (400 MHz, CD3OD) δ ppm 8.30 (d, J = 2.0 Hz, 1H), 7.71 (dd, J = 8.8, 2.4 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 5.00 (s, 2H), 3.77 (t, J = 5.6 Hz, 2H), 3.60 (s, 2H), 3.32 - 3.30 (m, 2H), 1.88 (d, J = 13.6 Hz, 6H), 1.40 (s, 6H).

[0086] Example 5 6-Amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one TIFF2025522978000027.tif32170

[0087] Step 1: Preparation of tert-butyl 5-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (Compound 5a) TIFF2025522978000028.tif34170The title compound was prepared in the same manner as in Step 1 of Example 3 by using tert-butyl 2,5-diazabicyclo[2.2.2]octane-2-carboxylate instead of tert-butyl N-pyrrolidin-3-ylcarbamate. tert-Butyl 5-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (35 mg, Compound 5a) was obtained as a brown solid. MS found (ESI + )[(M+H) + :529.1.

[0088] Step 2: Preparation of 6-amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (Example 5) TIFF2025522978000029.tif32170The title compound was prepared in the same manner as in Step 2 of Example 3 by using tert-butyl 5-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-2,5-diazabicyclo[2.2.2]octane-2-carboxylate (Compound 5a) instead of tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (Compound 3a). 6-Amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (9.3 mg, Example 5) was obtained as a green solid. MS found (ESI + )[(M+H) + :428.9. 11H NMR (400 MHz, CD3OD) δ ppm 8.26 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 8.8 Hz, 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 = 13.6 Hz, 6H).

[0089] Example 6 9 - [[6-(2,3,3a,4,6,6a - Hexahydro - 1H - pyrrolo[3,4 - c]pyrrol - 5 - yl)-3 - pyridyl]methyl]-6 - amino - 2 - dimethylphosphoryl - 7H - purin - 8 - one TIFF2025522978000030.tif33170

[0090] Step 1: Preparation of tert - butyl 2 - [5 - [(6 - amino - 2 - dimethylphosphoryl - 8 - oxo - 7H - purin - 9 - yl)methyl]-2 - pyridyl]-1,3,3a,4,6,6a - hexahydropyrrolo[3,4 - c]pyrrole - 5 - carboxylate (Compound 6a) TIFF2025522978000031.tif37170The title compound was prepared in the same manner as in Step 1 of Example 3 by using tert - butyl 2,3,3a,4,6,6a - hexahydro - 1H - pyrrolo[3,4 - c]pyrrole - 5 - carboxylate instead of tert - butyl N - pyrrolidin - 3 - ylcarbamate. tert - butyl 2 - [5 - [(6 - amino - 2 - dimethylphosphoryl - 8 - oxo - 7H - purin - 9 - yl)methyl]-2 - pyridyl]-1,3,3a,4,6,6a - hexahydropyrrolo[3,4 - c]pyrrole - 5 - carboxylate (80 mg, Compound 6a) was obtained as a pale yellow oil. MS found (ESI + )[(M + H) + : 529.0.

[0091] Step 2: Preparation of 9 - [[6-(2,3,3a,4,6,6a - hexahydro - 1H - pyrrolo[3,4 - c]pyrrol - 5 - yl)-3 - pyridyl]methyl]-6 - amino - 2 - dimethylphosphoryl - 7H - purin - 8 - one (Example 6) Instead of TIFF2025522978000032.tif33170 tert-butyl N-[1-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]pyrrolidin-3-yl]carbamate (Compound 3a), the title compound was prepared in the same manner as in Step 2 of Example 3 by using tert-butyl 2-[5-[(6-amino-2-dimethylphosphoryl-8-oxo-7H-purin-9-yl)methyl]-2-pyridyl]-1,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5-carboxylate (Compound 6a). 9-[[6-(2,3,3a,4,6,6a-Hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-yl)-3-pyridyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purin-8-one (28 mg, Example 6) was obtained as a white solid. MS found (ESI + )[(M+H) + : 429.0. 1 H NMR (400 MHz, CD3OD) δ ppm 8.25 (d, J = 2.0 Hz, 1H), 7.75 (dd, J = 8.8, 2.4 Hz, 1H), 6.60 (d, J = 8.8 Hz, 1H), 5.04 (s, 2H), 3.59 - 3.55 (m, 6H), 3.33 - 3.29 (m, 4H), 1.84 (d, J = 13.6 Hz, 6H).

[0092] Example 7 6-Amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridyl]methyl]-7H-purin-8-one Instead of N’,N’-dimethylethane-1,2-diamine, the title compound was prepared in the same manner as in Step 8 of Example 1 by using 2-pyrrolidin-1-ylethylamine. 6-Amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidin-1-ylethylamino)-3-pyridyl]methyl]-7H-purin-8-one (13.3 mg, Example 7) was obtained as a white solid. MS found (ESI + )[(M+H) + : 431.1.1 1H NMR (400 MHz, CD3OD) δ ppm 8.08 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.8, 2.4 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 4.92 (s, 2H), 3.41 (t, J = 6.8 Hz, 2H), 2.69 (t, J = 6.8 Hz, 2H), 2.62 - 2.58 (m, 4H), 1.84 (d, J = 13.6 Hz, 6H), 1.83 - 1.77 (m, 4H).

[0093] Example 8 6 - Amino - 2 - dimethylphosphoryl - 9 - [[6 - (tetrahydropyran - 4 - ylamino) - 3 - pyridyl]methyl] - 7H - purin - 8 - one TIFF2025522978000034.tif30170The title compound was prepared in the same manner as in Step 8 of Example 1 by using tetrahydropyran - 4 - amine instead of N,N′ - dimethylethane - 1,2 - diamine. 6 - Amino - 2 - dimethylphosphoryl - 9 - [[6 - (tetrahydropyran - 4 - ylamino) - 3 - pyridyl]methyl] - 7H - purin - 8 - one (18.4 mg, Example 8) was obtained as a white solid. MS found (ESI + ) [(M + H) + : 418.1. 1 1H NMR (400 MHz, CD3OD) δ ppm 8.06 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 8.8, 2.4 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 4.91 (s, 2H), 3.96 - 3.91 (m, 2H), 3.86 - 3.83 (m, 1H), 3.55 - 3.49 (m, 2H), 1.95 - 1.91 (m, 2H), 1.84 (d, J = 13.6 Hz, 6H), 1.50 - 1.46 (m, 2H).

[0094] Example 9 6 - Amino - 9 - [[6 - [3 - [(dimethylamino)methyl]pyrrolidin - 1 - yl] - 3 - pyridyl]methyl] - 2 - dimethylphosphoryl - 7H - purin - 8 - one TIFF2025522978000035.tif32170The title compound was prepared in the same manner as in Step 1 of Example 3 by using N,N-dimethyl-1-pyrrolidin-3-yl-methanamine instead of tert-butyl N-pyrrolidin-3-ylcarbamate. 6-Amino-9-[[6-[3-[(dimethylamino)methyl]pyrrolidin-1-yl]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purin-8-one (12.1 mg, Example 9) was obtained as a pale yellow oil. MS found (ESI + )[(M+H) + :445.0. 1 H NMR(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),3.46-3.39(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).

[0095] Example 10 6-Amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)-3-pyridyl]methyl]-7H-purin-8-one TIFF2025522978000036.tif33170

[0096] Step 1: Preparation of 4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane (Compound 10a) A solution of 17170 THF (10 mL) in tert-butyl 1-oxa-4,9-diazaspiro[5.5]undecane-4-carboxylate (500.0 mg, 1.95 mmol, 1 equiv) was added with LiAlH4 (148.05 mg, 3.9 mmol, 2 equiv) at 0 °C. The reaction mixture was stirred at 80 °C for 2 h. 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 give 4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane (200 mg, Compound 10a) as a colorless oil. MS found (ESI + )[(M+H) + : 171.1.

[0097] Step 2: Preparation of 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 10) By using 4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane instead of tert-butyl N-pyrrolidin-3-ylcarbamate, the title compound was prepared in the same manner as in Step 1 of Example 3. 6-Amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)-3-pyridyl]methyl]-7H-purin-8-one (4.7 mg, Example 10) was obtained as a white solid. MS found (ESI + )[(M+H) + : 487.2. 11H NMR (400 MHz, CD3OD) δ (ppm) 8.21 (d, J = 2.0 Hz, 1H), 7.67 (dd, J = 8.8, 2.4 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 4.96 (s, 2H), 3.88 - 3.81 (m, 2H), 3.74 (t, J = 4.8 Hz, 2H), 3.28 - 3.20 (m, 2H), 2.37 (t, J = 4.8 Hz, 2H), 2.25 (s, 2H), 2.21 (s, 3H), 1.99 - 1.92 (m, 2H), 1.84 (d, J = 13.6 Hz, 6H), 1.61 - 1.50 (m, 2H).

[0098] Example 11 6 - Amino - 2 - dimethylphosphoryl - 9 - [[6 - [(1 - methylpyrrolidin - 3 - yl)amino]-3 - pyridyl]methyl]-7H - purin - 8 - one The title compound was prepared in the same manner as in Step 1 of Example 3 by using 1 - methylpyrrolidin - 3 - amine instead of tert - butyl N - pyrrolidin - 3 - ylcarbamate and using BrettPhos instead of RuPhos. 6 - Amino - 2 - dimethylphosphoryl - 9 - [[6 - [(1 - methylpyrrolidin - 3 - yl)amino]-3 - pyridyl]methyl]-7H - purin - 8 - one (23.92 mg, Example 11) was obtained as a white solid. MS found (ESI + )[(M + H) + : 417.1. 1 1H NMR (400 MHz, CD3OD) δ ppm 8.17 (d, J = 2.0 Hz, 1H), 7.62 (dd, J = 8.4, 2.0 Hz, 1H), 6.53 (d, J = 8.8 Hz, 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 = 13.6 Hz, 6H).

[0099] Example 12 6-Amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one TIFF2025522978000040.tif30170

[0100] Step 1: Preparation of N’-ethyl-N’-methyl-ethane-1,2-diamine (Compound 12a) TIFF2025522978000041.tif12170To a solution of N-methylethylamine (14.2 mL, 165.3 mmol, 2.1 equiv) dissolved in m-xylene (45 mL), 2-(2-bromoethyl)isoindoline-1,3-dione (20.0 g, 78.72 mmol, 1.0 equiv) was added at 20 °C. The reaction mixture was stirred at 150 °C for 12 h. The reaction mixture was cooled to room temperature and then basified with Na2CO3 (2 M aqueous solution) 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 give a residue. Then, water (5 mL) and HCl (30.0 mL, 12 M, 360.0 mmol, 4.57 equiv) were added to the residue at 0 °C. The mixture was heated to 130 °C, stirred for 6 h, and a precipitate formed. The precipitate was filtered and washed with cold water (50 mL). The filtrate was concentrated in vacuo to give a residue. The residue was dissolved in MeOH (200 mL), basified to pH = 11 with solid NaOH, and filtered. The filtrate was concentrated in vacuo and the crude product was purified by distillation at 100 °C (760 mmHg) to give N’-ethyl-N’-methyl-ethane-1,2-diamine (5.0 g, Compound 12a) as a colorless liquid. 1 H NMR (400 MHz, DMSO-d6) δ ppm: 2.55 (t, J = 6.4 Hz, 2H), 2.30 (q, J = 7.2 Hz, 2H), 2.26 (t, J = 6.4 Hz, 2H), 2.09 (s, 3H), 0.95 (t, J = 7.2 Hz 3H).

[0101] Procedure 2: Preparation of 6-Amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (Example 12) TIFF2025522978000042.tif29170The title compound was prepared in the same manner as in Step 8 of Example 1 by using N’-ethyl-N’-methyl-ethane-1,2-diamine instead of N’,N’-dimethylethane-1,2-diamine. 6-Amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (8.28 mg, Example 12) was obtained as a white solid. MS found (ESI + )[(M+H) + :419.1. 1 H NMR (400 MHz, CD3OD) δ ppm: 8.08 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.8, 2.0 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 4.92 (s, 2H), 3.38 (t, J = 6.8 Hz, 2H), 2.59 (t, J = 6.8 Hz, 2H), 2.50 (q, J = 7,2 Hz, 2H), 2.27 (s, 3H), 1.83 (d, J = 13.6 Hz, 6H), 1.07 (t, J = 7.2 Hz, 3H).

[0102] Example 13-A and Example 13-B 6-Amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 13-A) and 6-Amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 13-B) TIFF2025522978000043.tif68170

[0103] Procedure 1: Preparation of 1-methylphosphonoyloxyethane (Compound 13a) To 2-ethoxy(methyl)phosphane (60.0 g, 440.76 mmol, 1 equiv), H2O (7.94 g, 440.76 mmol, 1 equiv) was added at 0 °C. The mixture was then stirred at 25 °C for 12 h. 1 1H NMR indicated that the reaction was complete and the desired product containing 50 mol% ethanol was obtained. The reaction solution was used directly in the next step. 1 1H NMR (400 MHz, CDCl3) δ ppm 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, 2.0 Hz, 3H), 1.37 (t, J = 3.2 Hz, 3H).

[0104] Step 2: Preparation of 1-methylphosphonoylethane (Compound 13b) Ethylmagnesium bromide (440 mL, 3 M, 1321 mmol, 3 equiv) was added dropwise to a solution of 1-methylphosphonoyloxyethane (47.6 g, Compound 13a, 440.41 mmol, 1 equiv) dissolved in THF (900 mL) at 0 °C over 30 min under N2. The mixture was then warmed to 25 °C and stirred for 12 h. The reaction mixture was cooled to 0 °C. A solution of potassium carbonate (182.6 g, 1321 mmol, 3 equiv) dissolved in water (200 mL) was added dropwise to the reaction mixture. The mixture was extracted with DCM (3 × 800 mL). The organic layers were combined and dried over Na2SO4. After filtration, the filtrate was concentrated in vacuo to give 1-methylphosphonoylethane (47 g, Compound 13b, crude product) as a pale yellow liquid. 1 1H NMR (400 MHz, CDCl3) δ ppm 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, 2.0 Hz, 3H), 1.24 - 1.15 (m, 3H).

[0105] Step 3: Preparation of 2-[ethyl(methyl)phosphoryl]-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 13c) TIFF2025522978000046.tif52170By using 1-methylphosphonoylethane (Compound 13b) instead of methylphosphonoylmethane, the title compound was prepared in the same manner as in Step 3 of Example 1. 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 found (ESI + )[(M+H) + :466.2.

[0106] Step 4: Preparation of 2-[Ethyl(methyl)phosphoryl]-9H-purin-6-amine (Compound 13d) TIFF2025522978000047.tif24170By using 2-[Ethyl(methyl)phosphoryl]-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 13c) instead of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 1c), the title compound was prepared in the same manner as in Step 4 of Example 1. 2-[Ethyl(methyl)phosphoryl]-9H-purin-6-amine (37 g, Compound 13d) was obtained as a black oil and used in the next step without further purification. MS found (ESI + )[(M+H) + :226.0.

[0107] Step 5: Preparation of 9-[(6-chloro-3-pyridyl)methyl]-2-[Ethyl(methyl)phosphoryl]purin-6-amine (Compound 13e) The title compound was prepared in the same manner as in Step 5 of Example 1 by using 2-[ethyl(methyl)phosphoryl]-9H-purin-6-amine (Compound 13d) instead of TIFF2025522978000048.tif301702-dimethylphosphoryl-9H-purin-6-amine (Compound 1d). 9-[(6-Chloro-3-pyridyl)methyl]-2-[ethyl(methyl)phosphoryl]purin-6-amine (30 g, Compound 13e) was obtained as a pale yellow solid. MS found (ESI + )[{ 35 Cl}(M+H) + : 351.0. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.54 (d, J = 2.4 Hz, 1H), 8.48 (s, 1H), 7.88 (dd, J = 8.0, 2.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 5.46 (s, 2H), 2.06 - 1.97 (m, 2H), 1.69 (d, J = 13.2 Hz, 3H), 1.01 (dt, J = 17.6, 7.6 Hz, 3H).

[0108] Step 6: Preparation of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]purin-6-amine (Compound 13f-A) and 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]purin-6-amine (Compound 13f-B) TIFF2025522978000049.tif601709 - Instead of [(6 - chloro - 3 - pyridyl)methyl]-2 - dimethylphosphoryl - purin - 6 - amine (Compound 1e), 9 - [(6 - chloro - 3 - pyridyl)methyl]-2 - [ethyl(methyl)phosphoryl]purin - 6 - amine (Compound 13e) was used, and the title compound was prepared in the same manner as in Step 6 of Example 1. After preparative HPLC purification, the racemic product was separated by chiral SFC to obtain 8 - bromo - 9 - [(6 - chloro - 3 - pyridyl)methyl]-2 - [(R)-ethyl(methyl)phosphoryl]purin - 6 - amine (faster elution, 15 g, Compound 13f - A) as a brown solid, and 8 - bromo - 9 - [(6 - chloro - 3 - pyridyl)methyl]-2 - [(S)-ethyl(methyl)phosphoryl]purin - 6 - amine (slower elution, 15.7 g, Compound 13f - B) as a brown solid. Chiral SFC conditions: DAICEL CHIRALPAK AD (10μm, 50×250mm) column, mobile phase: Phase A for CO2, Phase B for EtOH (0.05% DEA), gradient elution: 5% - 40% EtOH (0.05% DEA) in CO2, flow rate: 3 mL / min, detector: PDA.

[0109] Compound 13f - A: MS found (ESI + )[{ 35 Cl& 79 Br}(M + H) + : 430.9. 1 1H NMR (400 MHz, DMSO - d6) δ ppm 8.47 (d, J = 2.4 Hz, 1H), 7.77 (brs, 2H), 7.73 (dd, J = 8.0, 2.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 5.44 (s, 2H), 2.04 - 1.93 (m, 2H), 1.65 (d, J = 13.2 Hz, 3H), 1.01 (dt, J = 17.6, 7.6 Hz, 3H).

[0110] Compound 13f - B: MS found (ESI + )[{ 35 Cl& 79 Br}(M + H) + : 430.9. 1HNMR (400 MHz, DMSO-d6) δ ppm 8.47 (d, J = 2.4 Hz, 1H), 7.77 (brs, 2H), 7.73 (dd, J = 8.0, 2.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 5.44 (s, 2H), 2.04 - 1.93 (m, 2H), 1.64 (d, J = 13.2 Hz, 3H), 1.02 (dt, J = 17.6, 7.6 Hz, 3H).

[0111] Step 7: Preparation of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A) and 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B) TIFF2025522978000050.tif60170

[0112] Compound 13g-A was prepared in the same manner as in Step 7 of Example 1 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). 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 found (ESI + )[{ 35 Cl}(M + H) + : 367.0. 1 HNMR (400 MHz, DMSO-d6) δ ppm 8.43 (d, J = 2.0 Hz, 1H), 7.77 (dd, J = 8.4, 2.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.09 (brs, 2H), 4.99 (s, 2H), 2.00 - 1.88 (m, 2H), 1.59 (d, J = 13.2 Hz, 3H), 1.00 (dt, J = 17.6, 7.6 Hz, 3H).

[0113] Compound 13g-B was prepared in the same manner as 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 found (ESI + )[{ 35 Cl}(M+H) + : 367.0. 1 HNMR (400 MHz, DMSO-d6) δ ppm 8.42 (d, J = 2.4 Hz, 1H), 7.77 (dd, J = 8.4, 2.4 Hz 1H), 7.46 (d, J = 8.4 Hz, 1H), 6.98 (brs, 2H), 4.98 (s, 2H), 1.99 - 1.88 (m, 2H), 1.58 (d, J = 13.2 Hz, 3H), 0.99 (dt, J = 17.6, 7.6 Hz, 3H).

[0114] Step 8: Preparation of 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 13-A) and 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 13-B) TIFF2025522978000051.tif681701 - 6 - amino - 9 - [(6 - chloro - 3 - pyridyl)methyl] - 2 - [(R) - ethyl(methyl)phosphoryl] - 7H - purin - 8 - one(300.0 mg, Compound 13g - A, 0.820 mmol, 1 eq) was dissolved in 1 - methylpiperazine(8.0 mL, 40.9 mmol, 50 eq), and the solution was stirred at 170 °C for 6 h. The solution was purified by preparative HPLC. The residue was purified by SFC(DAICEL CHIRALCEL OD(10 μm, 30×250 mm) column, mobile phase: [0.1% NH3H2O EtOH], B%: 40% - 40%, 3.2 min, 25 min) to obtain 6 - amino - 2 - [(R) - ethyl(methyl)phosphoryl] - 9 - [[6 - (4 - methylpiperazin - 1 - yl) - 3 - pyridyl]methyl] - 7H - purin - 8 - one(108.3 mg, Example 13 - A) as a white solid. Measured MS value(ESI + )[(M + H) + : 431.2. 1 1H NMR(400 MHz, DMSO - d6)δ ppm 10.41(s, 1H), 8.15(d, J = 2.4 Hz, 1H), 7.54(dd, J = 8.8, 2.4 Hz, 1H), 6.78(d, J = 8.8 Hz, 1H), 6.67(brs, 2H), 4.81(s, 2H), 3.43(t, J = 4.8 Hz, 4H), 2.37(t, J = 4.8 Hz, 4H), 2.21(s, 3H), 2.05 - 1.98(m, 2H), 1.63(d, J = 13.2 Hz, 3H), 1.04(dt, J = 17.6, 7.6 Hz, 3H).

[0115] Example 13-B was prepared in the same manner as Example 13-A 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 found (ESI + ) [(M+H) + : 431.2. 1 HNMR (400 MHz, DMSO-d6) δ ppm 10.41 (s, 1H), 8.14 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 8.8, 2.4 Hz, 1H), 6.77 (d, J = 8.8 Hz, 1H), 6.67 (brs, 2H), 4.80 (s, 2H), 3.42 (t, J = 4.8 Hz, 4H), 2.34 (t, J = 4.8 Hz, 4H), 2.18 (s, 3H), 2.05 - 1.90 (m, 2H), 1.62 (d, J = 12.8 Hz, 3H), 1.03 (dt, J = 17.6, 7.6 Hz, 3H).

[0116] Examples 14-A and 14-B 6-Amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 14-A) and 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (Example 14-B) A solution of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (250.0 mg, Compound 13g-A, 0.680 mmol, 1 equiv) and 1-methylsulfonylpiperazine (1.1 g, 6.82 mmol, 10 equiv) in THF (2 mL) was heated to 170 °C. The solvent was evaporated and the residue was stirred at 170 °C for 8 h. The mixture was purified by reverse-phase flash chromatography and preparative HPLC to give 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazin-1-yl)-3-pyridyl]methyl]-7H-purin-8-one (19.8 mg, Example 14-A) as a white solid. MS found (ESI + ) [(M + H) + : 495.4. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 10.63 (brs, 1H), 8.19 (t, J = 2.4 Hz, 1H), 7.60 (dd, J = 8.8, 2.4 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 6.71 (brs, 2H), 4.83 (s, 2H), 3.59 (t, J = 4.8 Hz, 4H), 3.15 (t, J = 4.8 Hz, 4H), 2.88 (s, 3H), 2.06 - 1.88 (m, 2H), 1.65 (d, J = 13.2 Hz, 3H), 1.04 (dt, J = 17.2, 7.6 Hz, 3H).

[0117] Example 14-B was prepared in the same manner as Example 14-A 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 found (ESI+ )[(M+H) + :495.4. 1 HNMR(400MHz, DMSO-d6) δ ppm 10.63(brs, 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(brs, 2H), 4.81(s, 2H), 3.59(t, J = 4.8Hz, 4H), 3.15(t, J = 4.8Hz, 4H), 2.87(s, 3H), 2.03 - 1.85(m, 2H), 1.64(d, J = 13.2Hz, 3H), 1.04(dt, J = 17.2, 7.6Hz, 3H).

[0118] Example 15 - A and Example 15 - B 6 - Amino - 9 - [[6 - [2 - (dimethylamino)ethylamino]-3 - pyridyl]methyl]-2 - [(R)-ethyl(methyl)phosphoryl]-7H - purin - 8 - one (Example 15 - A) and 6 - Amino - 9 - [[6 - [2 - (dimethylamino)ethylamino]-3 - pyridyl]methyl]-2 - [(S)-ethyl(methyl)phosphoryl]-7H - purin - 8 - one (Example 15 - B) TIFF2025522978000053.tif601706 - Amino - 9 - [(6 - chloro - 3 - pyridyl)methyl]-2 - dimethylphosphoryl - 7H - purin - 8 - one (Compound 1g) was replaced by 6 - Amino - 9 - [(6 - chloro - 3 - pyridyl)methyl]-2 - [(R)-ethyl(methyl)phosphoryl]-7H - purin - 8 - one (Compound 13g - A), and Example 15 - A was prepared in the same manner as Step 8 of Example 1. 6 - Amino - 9 - [[6 - [2 - (dimethylamino)ethylamino]-3 - pyridyl]methyl]-2 - [(R)-ethyl(methyl)phosphoryl]-7H - purin - 8 - one (1.1g, Example 15 - A) was obtained as a white solid. MS measured value (ESI + )[(M+H) + :419.2. 11H NMR (400 MHz, DMSO-d6) δ ppm 10.46 (brs, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 8.8, 2.4 Hz, 1H), 6.69 (brs, 2H), 6.41 (d, J = 8.8 Hz, 1H), 6.36 (t, J = 5.6 Hz, 1H), 4.74 (s, 2H), 3.27 (q, J = 6.4 Hz, 2H), 2.35 (t, J = 6.4 Hz, 2H), 2.14 (s, 6H), 2.08 - 1.92 (m, 2H), 1.65 (d, J = 13.2 Hz, 3H), 1.03 (dt, J = 17.6, 7.6 Hz, 3H).

[0119] Example 15-B was prepared in the same manner as Example 15-A 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 found (ESI + )[(M + H) + : 419.2. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 10.46 (brs, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 8.8, 2.4 Hz, 1H), 7.02 (brs, 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 configuration of the stereocenter of Example 15-B was confirmed by X-ray crystallographic analysis.

[0120] Examples 16-A and 16-B 6-Amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Example 16-A) and 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Example 16-B) TIFF2025522978000054.tif591706-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g) was replaced with 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A), and Example 16-A was prepared in the same manner as Example 2. 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 found (ESI + )[(M+H) + :418.1. 1 H 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).

[0121] Example 16-B was prepared in the same manner as Example 16-A, except that 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-B) was used 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 found (ESI + )[(M+H) + : 418.1. 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.45 (brs, 1H), 8.18 (d, J = 2.4 Hz, 1H), 7.68 (dd, J = 8.4, 2.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.70 (s, 2H), 4.89 (s, 2H), 4.30 (t, J = 5.6 Hz, 2H), 2.57 (t, J = 5.6 Hz, 2H), 2.17 (s, 6H), 2.05 - 1.86 (m, 2H), 1.62 (d, J = 13.2 Hz, 3H), 1.02 (dt, J = 18.0, 7.6 Hz, 3H).

[0122] Example 17 6-Amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one TIFF2025522978000055.tif30170

[0123] Step 1: Preparation of tert-butyl N-[2-[[5-[[6-amino-2-[(R)-ethyl(methyl)phosphoryl]-8-oxo-7H-purin-9-yl]methyl]-2-pyridyl]amino]ethyl]-N-methylcarbamate (Compound 17a) TIFF2025522978000056.tifInstead of 301706-amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g), 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one (Compound 13g-A) was used, and instead of N’,N’-dimethylethane-1,2-diamine, tert-butyl N-(2-aminoethyl)-N-methyl-carbamate was used. The title compound was prepared in the same manner as in Step 8 of Example 1. tert-Butyl N-[2-[[5-[[6-amino-2-[(R)-ethyl(methyl)phosphoryl]-8-oxo-7H-purin-9-yl]methyl]-2-pyridyl]amino]ethyl]-N-methyl-carbamic acid (50 mg, Compound 17a) was obtained as a white solid. MS found (ESI + )[(M+H) + :505.2.

[0124] Step 2: Preparation of 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (Example 17) TIFF2025522978000057.tifTo a solution of tert-butyl N-[2-[[5-[[6-amino-2-[(R)-ethyl(methyl)phosphoryl]-8-oxo-7H-purin-9-yl]methyl]-2-pyridyl]amino]ethyl]-N-methyl-carbamate (30.0 mg, Compound 17a, 0.060 mmol, 1 equiv) in DCM (2 mL) was added TFA (0.5 mL). The reaction mixture was stirred at 20 °C for 2 h and then concentrated in vacuo. The residue was purified by preparative HPLC to give 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (7.2 mg, Example 17) as a white solid. MS found (ESI + )[(M+H) + :405.2. 1HNMR(400MHz,CD3OD)δppm8.26(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).

[0125] Example 18-A and Example 18-B 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-A) and 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-B) TIFF2025522978000058.tif60170

[0126] Step 1: Preparation of 1-methylphosphonoylpropane (Compound 18a) TIFF2025522978000059.tif13170The title compound was prepared in the same manner as in Step 2 of Example 13 by using n-propylmagnesium bromide instead of ethylmagnesium bromide. 1-Methylphosphonoylpropane (crude product, 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).

[0127] Step 2: Preparation of N,9-bis[(4-methoxyphenyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (Compound 18b) TIFF2025522978000060.tif52170 By using 1-methylphosphonylpropane (Compound 18a) instead of methylphosphonylmethane, the title compound was prepared in the same manner as in Step 3 of Example 1. N,9-bis[(4-methoxyphenyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (crude product, 35 g, Compound 18b) was obtained as a black oil, which was used in the next step without further purification. MS found (ESI + )[(M+H) + :480.1.

[0128] Step 3: Preparation of 2-[methyl(propyl)phosphoryl]-9H-purin-6-amine (Compound 18c) TIFF2025522978000061.tif24170 By using N,9-bis[(4-methoxyphenyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (Compound 18b) instead of 2-dimethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 1c), the title compound was prepared in the same manner as in Step 4 of Example 1. 2-[methyl(propyl)phosphoryl]-9H-purin-6-amine (crude product, 16 g, Compound 18c) was obtained as a red oil, which was used in the next step without further purification. MS found (ESI + )[(M+H) + :240.0.

[0129] Step 4: Preparation of 9-[(6-chloro-3-pyridyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (Compound 18d) TIFF2025522978000062.tif29170 By using 2-[methyl(propyl)phosphoryl]-9H-purin-6-amine (Compound 18c) instead of 2-dimethylphosphoryl-9H-purin-6-amine (Compound 1d), the title compound was prepared in the same manner as in Step 5 of Example 1. 9-[(6-chloro-3-pyridyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (15 g, Compound 18d) was obtained as a pale yellow solid. MS found (ESI+ )[(M+H) + :365.1. 1 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.53 - 8.52 (m, 2H), 7.87 (dd, J = 8.4, 2.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 5.49 (s, 2H), 2.06 - 1.93 (m, 2H), 1.71 (d, J = 13.2 Hz, 3H), 1.51 - 1.40 (m, 2H), 0.92 (t, J = 7.6 Hz, 3H).

[0130] Step 5: Preparation of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-A) and 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-B) TIFF2025522978000063.tif591709 - [(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1e) was replaced with 9-[(6-chloro-3-pyridyl)methyl]-2-[methyl(propyl)phosphoryl]purin-6-amine (Compound 18d). The title compounds were prepared in the same manner as in Step 6 of Example 1. After preparative HPLC purification, the racemic product was separated by chiral SFC to obtain 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purin-6-amine (faster elution, 6.0 g, Compound 18e-A) as a brown solid, and 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]purin-6-amine (slower elution, 6.0 g, Compound 18e-B) as a brown solid. Chiral SFC conditions: DAICEL CHIRALPAK AD (10 μm, 50 × 250 mm) column, mobile phase: Phase A for CO2, Phase B for iPrOH (0.1% NH3H2O), gradient elution: 40% - 40% iPrOH (0.1% NH3H2O) in CO2, flow rate: 3 mL / min, detector: PDA.

[0131] Compound 18e-A: Measured MS value (ESI + )[{ 35 Cl& 79 Br}(M+H) + : 444.9. 1 H 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 configuration of the stereocenter of Compound 18e-A was confirmed by X-ray crystallographic analysis.

[0132] Compound 18e-B: Measured MS value (ESI + )[{ 35 Cl& 79 Br}(M+H) + : 444.9. 1 H 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), 0.92 (t, J = 7.2 Hz, 3H).

[0133] Step 6: Preparation of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Compound 18f-A) and 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Compound 18f-B) By using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-A) instead of 591708-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1f), Compound 18f-A was prepared in the same manner as in Step 7 of Example 1. 6-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (3.6 g, Compound 18f-A) was obtained as a brown solid. MS found (ESI + )[{ 35 Cl}(M+H) + : 381.1. 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.70 (brs, 1H), 8.43 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 8.4, 2.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 6.80 (brs, 2H), 4.99 (s, 2H), 2.00 - 1.83 (m, 2H), 1.60 (d, J = 13.2 Hz, 3H), 1.52 - 1.38 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H).

[0134] By using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-B) instead of 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-[(S)-methyl(propyl)phosphoryl]purin-6-amine (Compound 18e-A), Compound 18f-B was prepared in the same manner as Compound 18f-A. 6-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (5.0 g, Compound 18f-B) was obtained as a brown solid. MS found (ESI + )[{ 35 Cl}(M+H) + : 381.1. 11H NMR (400 MHz, DMSO-d6) δ ppm 10.51 (brs, 1H), 8.43 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 8.4, 2.4 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 6.73 (brs, 2H), 4.99 (s, 2H), 2.03 - 1.80 (m, 2H), 1.60 (d, J = 13.2 Hz, 3H), 1.53 - 1.35 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H).

[0135] Project 7: Preparation of 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-A) and 6-Amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one (Example 18-B) TIFF2025522978000065.tif60170N,N'-dimethylethane-1,2-diamine (30.0 mL, 32.83 mmol, 5.0 equivalents) was mixed with 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 equivalent), and the mixture 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% NH3H2O iPrOH], B%: 55% - 55%, 6.6 minutes) to obtain 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one (1.0 g, Example 18-A) as a white solid. Measured MS value (ESI + )[(M + H) + : 433.2. 11H NMR (400 MHz, DMSO-d6) δ ppm 8.01 (d, J = 2.0 Hz, 1H), 7.38 (dd, J = 8.8, 2.4 Hz, 1H), 6.90 (brs, 2H), 6.40 (d, J = 8.6 Hz, 1H), 6.32 (t, J = 5.4 Hz, 1H), 4.74 (s, 2H), 3.26 (q, J = 6.4 Hz, 2H), 2.34 (t, J = 6.8 Hz, 2H), 2.13 (s, 6H), 2.04 - 1.87 (m, 2H), 1.63 (d, J = 13.2 Hz, 3H), 1.56 - 1.42 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H).

[0136] Example 18 - B was prepared in the same manner as Example 18 - A, 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 found (ESI + ) [(M + H) + : 433.2. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 8.01 (d, J = 2.0 Hz, 1H), 7.37 (dd, J = 8.8, 2.4 Hz, 1H), 6.68 (brs, 2H), 6.41 (d, J = 8.6 Hz, 1H), 6.36 (t, J = 5.4 Hz, 1H), 4.74 (s, 2H), 3.28 (q, J = 6.4 Hz, 2H), 2.33 (t, J = 6.8 Hz, 2H), 2.13 (s, 6H), 2.04 - 1.87 (m, 2H), 1.63 (d, J = 13.2 Hz, 3H), 1.56 - 1.42 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H).

[0137] Example 19 6-Amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one TIFF2025522978000066.tif29170

[0138] Step 1: Preparation of 1-ethylphosphonoylethane (Compound 19a) TIFF2025522978000067.tif13170Ethylmagnesium bromide (144.8 mL, 3 M in THF, 434.47 mmol, 3 eq) was added dropwise to a solution of diethyl phosphite (20.0 g, 144.8 mmol, 1 eq) dissolved in THF (120 mL) over 30 minutes at 0 °C under N2. The mixture was then warmed to 25 °C and stirred for 12 hours. After cooling the reaction mixture to 0 °C, a solution of potassium carbonate (60.05 g, 434.47 mmol, 3 eq) dissolved in water (40 mL) was added dropwise to the stirred solution. The mixture was diluted with DCM (3 × 50 mL) and filtered. The filtrate was collected, dried over Na2SO4, and concentrated under reduced pressure to give 1-ethylphosphonoylethane (crude product, 15.0 g, Compound 19a) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3) δ ppm 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).

[0139] Step 2: Preparation of 2-diethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (Compound 19b) TIFF2025522978000068.tif52170The title compound was prepared in the same manner as in Step 3 of Example 1 by using 1-ethylphosphonoylethane (Compound 19a) instead of methylphosphonoylmethane. 2-Diethylphosphoryl-N,9-bis[(4-methoxyphenyl)methyl]purin-6-amine (23 g, Compound 19b) was obtained as a red oil. MS found (ESI + )[(M + H) + : 480.2.

[0140] Step 3: Preparation of 2 - Diethylphosphoryl - 9H - purin - 6 - amine (Compound 19c) TIFF2025522978000069.tifThe title compound was prepared in the same manner as in Step 4 of Example 1 by using 2 - Diethylphosphoryl - N,9 - bis[(4 - methoxyphenyl)methyl]purin - 6 - amine (Compound 19b) instead of 202 - Dimethylphosphoryl - N,9 - bis[(4 - methoxyphenyl)methyl]purin - 6 - amine (Compound 1c). 2 - Diethylphosphoryl - 9H - purin - 6 - amine (crude product, 20 g, Compound 19c) was obtained as a red oil. MS found (ESI + )[(M + H) + : 240.0.

[0141] Step 4: Preparation of 9 - [(6 - Chloro - 3 - pyridyl)methyl] - 2 - diethylphosphoryl - purin - 6 - amine (Compound 19d) TIFF2025522978000070.tifThe title compound was prepared in the same manner as in Step 5 of Example 1 by using 2 - Diethylphosphoryl - 9H - purin - 6 - amine (Compound 19c) instead of 202 - Dimethylphosphoryl - 9H - purin - 6 - amine (Compound 1d). 9 - [(6 - Chloro - 3 - pyridyl)methyl] - 2 - diethylphosphorylpurin - 6 - amine (20 g, Compound 19d) was obtained as a pale yellow oil. MS found (ESI + )[{ 35 Cl}(M + H) + : 365.0.

[0142] Step 5: Preparation of 8 - Bromo - 9 - [(6 - chloro - 3 - pyridyl)methyl] - 2 - diethylphosphoryl - purin - 6 - amine (Compound 19e) TIFF2025522978000071.tif The title compound was prepared in the same manner as in Step 6 of Example 1 by using 9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-purin-6-amine (Compound 19d) instead of 291709-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1e). 8-Bromo-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphorylpurin-6-amine (14 g, Compound 19e) was obtained as a pale yellow solid. MS found (ESI + )[{ 35 Cl& 79 Br}(M+H) + : 444.9. 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.46 (d, J = 2.4 Hz, 1H), 7.76 (brs, 2H), 7.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 5.43 (s, 2H), 2.02 - 1.92 (m, 4H), 1.05 - 0.97 (m, 6H).

[0143] Step 6: Preparation of 6-amino-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-7H-purin-8-one (Compound 19f) TIFF2025522978000072.tif The title compound was prepared in the same manner as in Step 7 of Example 1 by using 8-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-purin-6-amine (Compound 19e) instead of 291708-bromo-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-purin-6-amine (Compound 1f). 6-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-7H-purin-8-one (11 g, Compound 19f) was obtained as a purple solid. MS found (ESI + )[{ 35 Cl}(M+H) + : 381.1. 11H NMR (400 MHz, DMSO-d6) δ ppm 8.42 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 8.0, 2.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 6.83 (brs, 2H), 4.98 (s, 2H), 1.97 - 1.87 (m, 4H), 1.02 - 0.94 (m, 6H).

[0144] Step 7: Preparation of 6-Amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (Example 19) TIFF2025522978000073.tif291706-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-dimethylphosphoryl-7H-purin-8-one (Compound 1g) was replaced with 6-Amino-9-[(6-chloro-3-pyridyl)methyl]-2-diethylphosphoryl-7H-purin-8-one (Compound 19f). The title compound was prepared in the same manner as in Step 8 of Example 1. 6-Amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one (3.2 g, Example 19) was obtained as a pink solid. MS found (ESI + ) [(M + H) + : 433.2. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 8.00 (d, J = 2.4 Hz, 1H), 7.38 (dd, J = 8.4, 2.4 Hz, 1H), 6.72 (brs, 2H), 6.41 (d, J = 8.4 Hz, 1H), 6.36 (t, J = 5.6 Hz, 1H), 4.74 (s, 2H), 3.27 (q, J = 6.4 Hz, 2H), 2.35 (t, J = 6.4 Hz, 2H), 2.14 (s, 6H), 2.14 - 1.95 (m, 4H), 1.05 (t, J = 7.6 Hz, 3H), 1.01 (t, J = 7.6 Hz, 3H).

[0145] Reference compound Compound RO14, which is Example 14 disclosed in International Publication No. WO 2016 / 180695 of the prior art, was selected as a reference compound for comparison. TIFF2025522978000074.tif44170

[0146] Example 20 Activity of the compound of the present invention in the HEK293-hTLR7 assay HEK293-Blue-hTLR7 cell assay: A stable HEK293-Blue-hTLR7 cell line was purchased from InvivoGen (Catalog number: hkb-htlr7, San Diego, CA, USA). These cells were designed to study the stimulation of human TLR7 by monitoring the activation of NF-κB. 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. SEAP was induced by activating NF-κB and AP-1 by stimulating HEK-Blue hTLR7 cells with a TLR7 ligand. Thus, reporter expression was regulated by the NF-κB promoter upon stimulation of human TLR7 for 20 hours. The activity of the SEAP reporter in the cell culture supernatant was measured at a wavelength of 640 nm using the QUANTI-Blue™ kit (Catalog number: rep-qb1, Invivogen, San Diego, CA, USA), and the detection medium changed to purple or blue in the presence of alkaline phosphatase.

[0147] HEK293-Blue-hTLR7 cells were incubated in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g / L glucose, 50 U / mL penicillin, 50 mg / mL streptomycin, 100 mg / mL normocin, 2 mM L-glutamine, and 10% (v / v) heat-inactivated fetal bovine serum at a density of 250,000 - 450,000 cells / mL in a volume of 180 μL in a 96-well plate for 24 hours. Then, HEK293-Blue-hTLR7 cells were incubated with the addition of 20 μL of the test compound by serial dilution in the presence of 1% final DMSO and incubated at 37 °C in a CO2 incubator for 20 hours. Then, 20 μL of the supernatant from each well was incubated with 180 μL of Quanti-blue substrate solution at 37 °C for 2 hours, and the absorbance was read at 620 - 655 nm using a spectrophotometer. The signaling pathway by which TLR7 activation leads to downstream NF-κB activation is widely accepted, and thus, a similar reporter assay has also been widely used to evaluate TLR7 agonists (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329.sci, Hiroaki Hemmi et al, Nature Immunology 3, 196 - 200 (2002)).

[0148] The compounds of the present invention were tested for their TLR7 agonism activity as described herein in the HEK293-hTLR7 assay, and the results are listed in Table 1. The examples had an EC 50 It was found to have. Certain compounds of the present invention had an EC 50 which was much better than the reference compound RO14.

Table 1

[0149] Example 21 Solubility: Lyophilized Solubility Assay (LYSA) and Thermodynamic Solubility Assay (THESA) Water solubility is an important physicochemical property for any potential drug candidate and is routinely measured in both drug discovery and development. Poor solubility is detrimental to absorption following oral administration and can mask compound activity in bioassays in various ways, including underestimated activity, decreased hit rates in high-throughput screening (HTS), variable data output, inaccurate structure-activity relationships (SAR), and inaccurate in vitro absorption, distribution, metabolism, excretion, and toxicity (ADMET) test results. The importance of solubility data in early-stage drug profiling during development has also been widely debated in order to predict oral absorption. Therefore, solubility limits ideally need to be identified as early as possible before performing functional assays. Thus, early assessment of solubility in the drug discovery process is extremely important.

[0150] The solubility of a substance can be broadly defined as the maximum amount of the substance that can dissolve in a given volume of solvent. However, it is important to understand that the solubility of a compound can vary dramatically depending on the conditions of the solvent (e.g., temperature and pH) and the physicochemical properties of the compound (e.g., ionization and crystallinity). These important factors need to be considered when measuring solubility in order to generate high-quality solubility data that are useful for improving compounds throughout the discovery and development stages. The kinetic solubility of a compound is the maximum solubility of the fastest precipitating species of that compound, which is often measured using a stock solution of the compound dissolved in an organic solvent, typically dimethyl sulfoxide (DMSO), as the starting material. The value of the kinetic solubility strongly depends on time and method, and thus, reproducibility between different laboratories using different protocols cannot be expected. The precipitate formed is rarely identified during evaluation and can be any combination of the various possible solid states of the compound. Considering the level of supersaturation that can occur when an organic solvent is diluted with water, the value of the kinetic solubility is typically higher than the corresponding thermodynamic (equilibrium) solubility value. In solubility assays during the early discovery process, the kinetic solubility is often measured. The lyophilization solubility assay (LYSA) is a high-throughput kinetic solubility assay for the early drug discovery process.

[0151] Lyophilization Solubility Assay (LYSA) method Samples were prepared in duplicate from a 10 mM DMSO stock solution. After evaporating DMSO using a centrifugal vacuum evaporator, the compound was dissolved in 0.05 M phosphate buffer (pH 6.5), stirred for 1 hour, and shaken for 2 hours. After overnight, the solution was filtered using a microtiter filter plate. Next, the filtrate and its 1 / 10 dilution were analyzed by HPLC-UV. Additionally, a four-point calibration curve was created from the 10 mM stock solution and used for measuring the solubility of the compound. The results were in units of μg / mL. When the percentage of the sample measured in the post-evaporation solution, divided by the maximum value of the calculated sample amount, was greater than 80%, the solubility was reported as greater than this value. Note that since a metastable solid phase can form during the evaporation process, this method can yield higher solubility values than assays that use a stable polymorph as the starting material.

[0152] 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 the excess undissolved material. Thermodynamic solubility is measured by dispensing a solid compound into a solvent. This is often regarded as the true solubility of the compound and is important information in pharmaceutical development. The Thermodynamic Solubility Assay (THESA) is a tool widely used for measuring the equilibrium solubility in later drug discovery processes and is considered the gold standard of solubility assays.

[0153] Thermodynamic Solubility Assay (THESA) method Approximately 1 - 2 mg of each compound was added to 300 μL of 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 shaken for 2 hours. All suspensions were left standing overnight (about 16 hours). The next day, the samples were filtered through a micro - nic filter plate into a V - bottom plate to separate the solid material from the solution. Then, all solutions were diluted in a 96 - well V - bottom microplate and analyzed by UPLC - UV. The calibration curve was established by UPLC using five different dilutions (concentration approximately 1 mg / mL) of the compound in DMSO stock solution prepared in a microtube. Dilution was carried out using DMSO. From this regression equation, the solubility of the compound was determined. When the drug was completely dissolved in the buffer, the value of the equilibrium solubility was assumed to be higher than the value determined by UPLC and was reported as such.

[0154] This result clearly showed that both the kinetic solubility (LYSA) and the thermodynamic (equilibrium) solubility (THESA) of the compounds of the present invention were significantly improved compared to the reference compound RO14.

Table 2

[0155] Example 22 Dissolution test: Fasted - state simulated intestinal fluid (FaSSIF), Fed - state simulated intestinal fluid (FeSSIF) Drug dissolution in the physiological environment of the gastrointestinal tract is a major step in the oral absorption process from pharmaceutical dosage forms. Only the dissolved drug can permeate the mucosa at the absorption sites of the digestive tract, so both the solubility of the drug and its dissolution rate are important for its in - vivo behavior. It is important to conduct dissolution tests under conditions that closely resemble the important parameters of human gastrointestinal physiology. In addition to the selection of appropriate equipment and appropriate equipment parameters, the use of physiologically relevant dissolution media is very important.

[0156] Biorelevant gastrointestinal media that mimic fasting 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 characteristics of several classes of drugs to assist in predicting in vivo absorption behavior. Biorelevant in vitro dissolution tests are useful for qualitatively predicting the effects of formulations and foods on the dissolution and availability of orally administered drugs. It has been observed that biorelevant media can provide a more accurate simulation of pharmacokinetic profiles than simulated gastric or intestinal fluids. The formulations and preparation instructions for biorelevant media are detailed below.

[0157] Fasted-state simulated intestinal fluid (FaSSIF) Step 1: Preparation of blank FaSSIF 0.420 g of NaOH (pellets), 3.954 g of NaH2PO4 (monohydrate) and 6.186 g of NaCl were dissolved in approximately 0.9 L of purified water. The pH was adjusted to 6.5 using either 1 N NaOH or 1 N HCl. The total volume of the mixture was made up to 1.0 L with purified water at room temperature.

[0158] Step 2: Preparation of FaSSIF To approximately 50 mL of blank FaSSIF buffer, 0.2240 g of SIF Powder Original (manufactured by Biorelevant.com Ltd, product code: FFF03, UK) was added 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.

[0159] Fed-state simulated intestinal fluid (FeSSIF) Step 1: Preparation of blank FeSSIF 4.040 g of NaOH (pellets), 8.650 g of glacial acetic acid and 11.874 g of NaCl were dissolved in approximately 0.9 L of purified water. The pH was adjusted to 5 using either 1 N NaOH or 1 N HCl. The total volume of the mixture was made up to 1.0 L with purified water at room temperature.

[0160] Step 2: Preparation of FeSSIF To approximately 50 mL of blank FeSSIF buffer, 1.120 g of SIF Powder Original (manufactured by Biorelevant.com Ltd, product code: FFF03, UK) was added, and the mixture was stirred until the powder was completely dissolved. The total volume of the mixture was adjusted to 100 mL using blank FeSSIF buffer at room temperature.

[0161] Dissolution test method Approximately 1 - 2 mg of each compound was added in excess to 300 μL of buffer at room temperature. Each sample was placed in a microanalysis glass tube, sonicated for 1 hour, and stirred for 2 hours. All suspensions were left overnight (about 16 hours). The next day, all pH values were measured with a pH meter, and the samples were filtered through a micro - nic filter plate into a V - bottom plate to separate the solid matter from the solution. Then, all solutions were diluted in a 96 - well V - bottom microplate and analyzed by UPLC - UV.

[0162] The calibration curve was established by UPLC using five different dilutions (concentration approximately 1 mg / mL) of the compound in DMSO stock solution prepared in microtubes. Dilution was carried out with DMSO (in special circumstances, acetonitrile or 80% HCl 0.1 N / 20% acetonitrile was used). The solubility of the compound was determined from this regression equation. When the compound was completely dissolved in the buffer, the value of the equilibrium solubility was assumed to be higher than the value determined by UPLC and was reported as such.

[0163] This result clearly showed that the dissolution of the compounds of the present invention in fasted - state simulated intestinal fluid (FaSSIF) and fed - state simulated intestinal fluid (FeSSIF) was significantly improved compared to the reference compound RO14. Table 3. FaSSIF and FeSSIF of the compounds of the present invention

Table 3

Claims

1. Compound of formula (I), (In the formula, R １ C １～６ It is alkyl, R ２ C １～６ It is alkyl, R ３ is ((C １～６ alkyl)) ２ amino)C １～６ alkoxy, ((C １～６ alkyl)) ２ amino)C １～６ alkylamino, ((C １～６ alkyl)) ２ amino)C １～６ alkylpyrrolidinyl, (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]undecanyl, C １～６ alkylpiperazinyl, C １～６ alkylsulfonylpiperazinyl, pyrrolidinyl C １～６ alkylamino or pyridinyl substituted with tetrahydropyranyl amino) or a pharmaceutically acceptable salt thereof.

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

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

4. R ２ The compound according to claim 1 or 2, wherein the compound is methyl, ethyl, or propyl.

5. R ３ but And R ４ However, ((C １～６ Alkyl) ２ Amino) C １～６ Alkoxy, ((C １～６ Alkyl) ２ Amino) C １～６ Alkylamino, ((C １～６ Alkyl) ２ Amino) C １～６ Alkylpyrrolidinyl, (C １～６ Alkyl) ２ Piperazinil, (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]undecanyl, C １～６ Alkylpiperazinyl, C １～６ Alkylsulfonylpiperazinyl, pyrrolidinyl C １～６ The compound according to claim 1 or 2, wherein it is an alkylamino or tetrahydropyranylamino.

6. R ３ but And R ４ However, ((C １～６ Alkyl) ２ Amino) C １～６ Alkylamino or (C １～６ (Alkylamino) C １～６ The compound according to claim 1 or 2, wherein it is an alkylamino compound.

7. R ３ The compound according to claim 1 or 2, wherein the compound is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino.

8. R １ However, C １～６ It is alkyl, R ２ However, C １～６ It is alkyl, R ３ but And R ４ However, ((C １～６ Alkyl) ２ Amino) C １～６ Alkylamino or (C １～６ (Alkylamino) C １～６ The compound according to claim 1 or 2, which is an alkylamino compound. or a pharmaceutically acceptable salt thereof.

9. R １ However, it is methyl, R ２ However, it is methyl, ethyl, or propyl, R ３ but And R ４ The compound according to claim 8, wherein the compound is 2-(dimethylamino)ethylamino or 2-(methylamino)ethylamino. or a pharmaceutically acceptable salt thereof.

10. 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one, 6-amino-9-[[6-(3-aminopyrrolidine-1-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one, 6-amino-2-dimethylphosphoryl-9-[[6-(3,3-dimethylpiperazine-1-yl)-3-pyridyl]methyl]-7H-purine-8-one, 6-amino-9-[[6-(2,5-diazabicyclo[2.2.2]octan-2-yl)-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one, 9-[[6-(2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrole-5-yl)-3-pyridyl]methyl]-6-amino-2-dimethylphosphoryl-7H-purine-8-one, 6-amino-2-dimethylphosphoryl-9-[[6-(2-pyrrolidine-1-ylethylamino)-3-pyridyl]methyl]-7H-purine-8-one, 6-amino-2-dimethylphosphoryl-9-[[6-(tetrahydropyran-4-ylamino)-3-pyridyl]methyl]-7H-purin-8-one, 6-amino-9-[[6-[3-[(dimethylamino)methyl]pyrroridine-1-yl]-3-pyridyl]methyl]-2-dimethylphosphoryl-7H-purine-8-one, 6-amino-2-dimethylphosphoryl-9-[[6-(4-methyl-1-oxa-4,9-diazaspiro[5.5]undecane-9-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-amino-2-dimethylphosphoryl-9-[[6-[(1-methylpyrrolidine-3-yl)amino]-3-pyridyl]methyl]-7H-purine-8-one, 6-amino-2-dimethylphosphoryl-9-[[6-[2-[ethyl(methyl)amino]ethylamino]-3-pyridyl]methyl]-7H-purin-8-one, 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazine-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylpiperazine-1-yl)-3-pyridyl]methyl]-7H-purin-8-one, 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazine-1-yl)-3-pyridyl]methyl]-7H-purine-8-one, 6-amino-2-[(S)-ethyl(methyl)phosphoryl]-9-[[6-(4-methylsulfonylpiperazine-1-yl)-3-pyridyl]methyl]-7H-purine-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(R)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethoxy]-3-pyridyl]methyl]-2-[(S)-ethyl(methyl)phosphoryl]-7H-purin-8-one, 6-amino-2-[(R)-ethyl(methyl)phosphoryl]-9-[[6-[2-(methylamino)ethylamino]-3-pyridyl]methyl]-7H-purin-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(S)-methyl(propyl)phosphoryl]-7H-purin-8-one, 6-amino-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-2-[(R)-methyl(propyl)phosphoryl]-7H-purin-8-one, and 6-amino-2-diethylphosphoryl-9-[[6-[2-(dimethylamino)ethylamino]-3-pyridyl]methyl]-7H-purine-8-one, A compound selected from, or a pharmaceutically acceptable salt thereof.

11. A method for preparing the compound according to claim 1 or 2, comprising the following steps: a) Compound of formula (III), and amine or alcohol HR ４ (II) Reaction in solvent-free, or in the presence of an inorganic base (the base is sodium hydride), or in the presence of an organometallic catalyst system (the catalyst system is Pd ２ (dba) ３ / RuPhos / t-BuONa,Pd ２ (dba) ３ (Selected from / BretPhos / t-BuONa and Pd-PEPPSI-IPentCl / t-BuOK), in the formula, R1 is a C1-6 alkyl group. R2 is a C1-6 alkyl group. R4 is ((C1-6 alkyl)2 amino)C1-6 alkoxy, ((C1-6 alkyl)2 amino)C1-6 alkylamino, ((C1-6 alkyl)2 amino)C1-6 alkylpyrrolidinyl, (C1-6 alkyl)2 piperazinyl, (C1-6 alkylamino)C1-6 alkylamino, (C1-6 alkylpyrrolidinyl)amino, 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl, 2,5-diazabicyclo[2.2.2]octanyl, aminopyrrolidinyl, C1-6 alkyl-1-oxa-4,9-diazaspiro[5.5]undecanyl, C1-6 alkylpiperazinyl, C1-6 A method comprising a reaction involving alkylsulfonylpiperazinyl, pyrrolidinyl C1-6 alkylamino, or tetrahydropyranylamino.

12. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, as produced according to the method of claim 11.

13. A pharmaceutical composition comprising the compound according to claim 1 or 2 and a pharmaceutically acceptable additive.

14. A compound or pharmaceutically acceptable salt according to claim 1 or 2 for use as a therapeutically active substance.

15. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, 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.

16. Use of the compound according to claim 1 or 2 as a TLR7 agonist.

17. Use of the compound according to claim 1 or 2 for the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, 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.

18. Use of the compound according to claim 1 or 2 for preparing a medicament for the treatment of cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, 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.

19. The use according to claim 17, wherein the cancer is selected from pancreatic ductal adenocarcinoma and colorectal cancer.

20. A method for treating cancer, wherein the cancer is selected from pancreatic ductal adenocarcinoma, colorectal cancer, melanoma, hepatocellular carcinoma, cholangiocarcinoma, 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, and the method comprises administering a therapeutically effective amount of the compound according to claim 1 or 2.

21. The invention as previously described herein.

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