1H-Pyrazolo[4,3-d]pyrimidine compounds as toll-like receptor 7 (TLR7) agonists

Abstract

The following formula (I): [Formula 1] The compound represented by TIFF2023512227000242.tif4969 is useful as an agonist of Toll-like receptor 7 (TLR7). Such compounds can be used in cancer therapy, particularly in combination with anti-cancer immunotherapeutic agents, or as a vaccine adjuvant.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 966,111, filed on January 27, 2020, under 35 U.S.C. § 119(e), the disclosure of which is incorporated herein by reference.

Background Art

[0002] The present disclosure relates to Toll-like receptor 7 (「TLR7」) agonists and complexes thereof, methods of preparation, and uses of such agonists and complexes thereof.

[0003] Toll-like receptors (「TLRs」) are receptors that recognize pathogen-associated molecular patterns (「PAMPs」), which are small molecule motifs conserved among specific types of pathogens. TLRs can be present either on the surface of cells or intracellularly. Activation of TLRs by binding of cognate PAMPs signals the presence of relevant pathogens within the host - i.e., infection - and stimulates the host immune system to fight the infection. There are 10 TLRs in humans, named TLR1, TLR2, TLR3, etc.

[0004] Activation of TLRs by agonists - that of TLR7 has been most studied - can have a beneficial effect on the action of vaccines and immunotherapeutic agents by stimulating the immune response overall in the treatment of various pathologies other than actual pathogen infection. Therefore, there is great interest in the use of TLR7 agonists as vaccine adjuvants or as enhancers in cancer immunotherapy. See, for example, Vasilakos and Tomai 2013, Sato-Kaneko et al. 2017, Smits et al. 2008, and Ota et al. 2019.

[0005] TLR7 is an intracellular receptor located on the membrane of endosomes and recognizes PAMPs associated with single-stranded RNA viruses. Its activation induces the secretion of type I interferons such as IFNα and IFNβ (Lund et al. 2004). TLR7 has two binding sites, one for single-stranded RNA ligands (Berghoefer et al. 2007) and one for small molecules such as guanosine (Zhang et al. 2016).

[0006] TLR7 may bind to and be activated by guanosine-like synthetic agonists such as imiquimod, resiquimod, and gardiquimod, which are based on the 1H-imidazo[4,5-c]quinoline skeleton. For a review of small molecule TLR7 agonists, see Cortez and Va 2018.

Chemical formula

[0007] Synthetic TLR7 agonists based on the pteridinone molecular skeleton are also known, such as besatrimod (Desai et al. 2015).

Chemical formula

[0008] Other synthetic TLR7 agonists based on purine-like skeletons have been disclosed and often have the following general formula (A):

Chemical formula

[0009] Disclosures of bioactive molecules having a purin-like skeleton and their use in the treatment of conditions such as fibrosis, inflammatory diseases, cancer, or pathogenic infections include: Akinbobuyi et al. 2015 and 2016; Barberis et al. 2012; Carson et al. 2014; Ding et al. 2016, 2017a, and 2017b; Graupe et al. 2015; Hashimoto et al. 2009; He et al. 2019a and 2019b; Holldack et al. 2012; Isobe et al. 2009a and 2012; Poudel et al. 2019a and 2019b; Pryde 2010; and Young et al. 2019.

[0010] The group R” can be pyridyl: Bonfanti et al. 2015a and 2015b; Halcomb et al. 2015; Hirota et al. 2000; Isobe et al. 2002, 2004, 2006, 2009a, 2009b, 2011, and 2012; Kasibhatla et al. 2007; Koga-Yamakawa et al. 2013; Musmuca et al. 2009; Nakamura 2012; Ogita et al. 2007; and Yu et al. 2013.

[0011] There is a disclosure of related molecules in which the 6,5-fused ring system - pyrimidine 6-membered ring and imidazole 5-membered ring of formula (A) are fused and modified. (a) Dellaria et al. 2007, Jones et al. 2010 and 2012, and Pilatte et al. 2017 disclose compounds in which the pyrimidine ring is substituted with a pyridine ring. (b) Chen et al. 2011, Coe et al. 2017, Poudel et al. 2020a and 2020b, and Zhang et al. 2018 disclose compounds in which the imidazole ring is substituted with a pyrazole ring. (c) Cortez et al. 2017 and 2018; Li et al. 2018; and McGowan et al. 2016a, 2016b, and 2017 disclose compounds in which the imidazole ring is substituted with a pyrrole ring.

[0012] Bonfanti et al. 2015b and 2016 and Purandare et al. 2019 disclose TLR7 modulators in which two rings of the purine moiety are bridged by a macrocyclic molecule.

[0013] TLR7 agonists may be bound to a partner molecule, which can be, for example, a phospholipid, poly(ethylene glycol) ("PEG"), an antibody, or another TLR (generally TLR2). Representative disclosures include: Carson et al. 2013, 2015, and 2016, Chan et al. 2009 and 2011, Cortez et al. 2017, Gadd et al. 2015, Lioux et al. 2016, Maj et al. 2015, Vernejoul et al. 2014, and Zurawski et al. 2012. The main binding site is the R" group of formula (A).

[0014] Jensen et al. 2015 disclose the use of cationic lipid vehicles for the delivery of TLR7 agonists.

[0015] Some TLR7 agonists, such as resiquimod, are TLR7 / TLR8 dual agonists. See, for example, Beesu et al. 2017, Embrechts et al. 2018, Lioux et al. 2016, and Vernejoul et al. 2014.

[0016] Full citations for the documents cited herein by first author or inventor and year of publication are set forth at the end of this specification. SUMMARY OF THE INVENTION

[0017] This specification relates to compounds having activity as TLR7 agonists that have a 1H-pyrazolo[4,3-d]pyrimidine aromatic system.

Chemical Formula

[0018] In one aspect, the following formula (I):

Chemical Formula

Chemical Formula

Chemical formula

Chemical formula

[0019] The compounds disclosed herein have activity as TLR7 agonists and some may be conjugated to antibodies for targeted delivery to the target tissue or organ of the desired action. They may be PEGylated and their pharmaceutical properties may also be modulated.

[0020] The compounds, or complexes or PEGylated derivatives thereof, disclosed herein may be used to treat patients suffering from conditions suitable for treatment by activation of the immune system by administering a therapeutically effective amount of such a compound or complex or PEGylated derivative thereof, particularly in combination with a vaccine or cancer immunotherapeutic agent. [Mode for Carrying Out the Invention]

[0021] Compound In one aspect, the compounds of the present disclosure are represented by the following formula (Ia), wherein R 1 , R 2 , R 5 , and W are as defined for formula (I): [Chemical formula] R 2 is preferably OMe.

[0022] In another aspect, the compounds of the present disclosure are represented by the following formula (Ib), wherein R 1 、R 2 、R 3 、and R 5 are as defined for formula (I):

Chemical formula

[0023] In another aspect, the compounds of the present disclosure are represented by the following formula (Ic), wherein R 1 、R 2 、R 4 、and R 5 are as defined for formula (I):

Chemical formula

[0024] In another aspect, the present disclosure provides a compound having the structure represented by the following formula (Id):

Chemical formula

Chemical formula

Chemical formula

[0025] In another aspect, the present disclosure provides a compound having the structure represented by the following formula (Ie):

Chemical formula

[0026] Specific examples of W’ include [Chemical formula] are included.

[0027] Suitable group R 1 Examples of are: [Chemical formula] are included.

[0028] Preferably, R 1 is [Chemical formula] selected from the group consisting of.

[0029] R 2 is preferably OMe or OCHF2, more preferably OMe.

[0030] R 5 is preferably H, CH2OH, or Me, more preferably H.

[0031] When W is [Chemical formula] and n is 1, examples include [Chemical formula] are included.

[0032] Preferably, [Chemical formula] is [Chemical formula] selected from the group consisting of.

[0033] When W is [Chemical formula] Examples where it is [Chemical formula] [Chemical formula] include.

[0034] Preferably, [Chemical formula] is [Chemical formula] selected from the group consisting of.

[0035] In one embodiment, W is [Chemical formula] is.

[0036] In one embodiment, W is [Chemical formula] is.

[0037] In another embodiment, R 3 is H, halo, OH, CN, NH2, NH[C(=O)] 0-1 (C1-C5 alkyl), N(C1-C5 alkyl)2, NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C4-C 10 (bicycloalkyl), NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C5-C 10 (spiroalkyl), N(C3-C6 cycloalkyl)2, N[C1-C3 alkyl]C(=O)(C1-C6 alkyl), a 6-membered aromatic or heteroaromatic moiety, a 5-membered heteroaromatic moiety, or the following structure:

Chemical formula

[0038] In one embodiment, each of R 1 and W contains a spiroalkyl or spiroalkanediyl moiety.

[0039] In one embodiment, R 1 contains a spiroalkyl moiety and W contains a bicycloalkyl or bicycloalkanediyl moiety.

[0040] In one embodiment, R 1 contains a spiroalkyl moiety and W does not contain a spiroalkyl or spiroalkanediyl moiety.

[0041] In one embodiment, W contains a spiroalkyl or spiroalkanediyl moiety and R 1 does not contain a spiroalkyl moiety.

[0042] By way of illustration and not limitation, the following formula:

Chemical formula

Chemical formula

[0043] By way of illustration and not limitation, the spiroalkyl group includes:

Chemical formula

[0044] By way of illustration and not limitation, the following formula:

Chemical formula

Chemical formula

[0045] By way of illustration and not limitation, the bicycloalkyl group includes:

Chemical formula

[0046] By way of illustration and not limitation, the following formula:

Chemical formula

Chemical formula

[0047] The above representative spiroalkyl and bicycloalkyl groups and the following formula:

Chemical formula

[0048] Specific examples of the compounds disclosed herein are shown in Table A below. The table also provides data regarding biological activity: data on the human TLR7 reporter assay and / or induction of the CD69 gene in human whole blood, determined through the procedures provided below. Analytical data (mass spectrum, LC / MS retention time, and NMR) are described in the rightmost column. In one embodiment, the compounds of the present disclosure have (a) an EC 50 value of less than 1,000 nM in the human TLR7 (hTLR7) reporter assay and (b) an EC 50 value of less than 1,000 nM for CD69 induction in human whole blood (hWB). (When the assay is performed multiple times, the reported value is the average value.) [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

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Table 22

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Table 24

Table 25

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Table 27

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Table 30

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Table 33

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Table 35

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Table 40

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Table 49

Table 50

Table 51

Table 52

Table 53

Table 54

Table 55

Table 56

Table 57

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Table 59

Table 60

Table 61

Table 62

Table 63

Table 64

Table 65

Table 66

Table 67

Table 68

Table 69

Table 70

Table 71

Table 72

Table 73

Table 74

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Table 76

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Table 79

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Table 99

Table 100

Table 101

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Table 104

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Table 106

Table 107

Table 108

[0049] Pharmaceutical Composition and Administration In another aspect, there is provided a pharmaceutical composition comprising a compound as disclosed herein, or a complex thereof, formulated with a pharmaceutically acceptable carrier or additive. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as biological agents or small molecule agents. The pharmaceutical composition may be administered in combination therapy with another therapeutic agent, particularly an anti-cancer agent.

[0050] The pharmaceutical composition may contain one or more additives. Additives that may be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizing agents, coloring agents, flavoring agents, coatings, disintegrants, lubricants, sweetening agents, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable additives are described in Gennaro, Remington: The Science and Practice of Pharmacy, 20th Edition (Lippincott Williams & Wilkins 2003).

[0051] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated with a substance and protected from the action of acids and other natural conditions that may inactivate the compound. The term "parenteral administration" usually means administration by injection, other than enteral and topical administration, and examples include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions. Alternatively, the pharmaceutical composition may be administered by a non-parenteral route, such as a topical, epithelial or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.

[0052] The pharmaceutical composition can be in the form of a sterile aqueous solution or a sterile aqueous dispersion. They may also be formulated in microemulsions, liposomes, or other ordered structures suitable for achieving high drug concentrations. The composition may also be provided in the form of a lyophilized product to be reconstituted with water before administration.

[0053] The amount of active ingredient that can combine with the carrier substance to produce a single dosage form will vary depending on the patient being treated and the particular method of administration, and will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 0.01 percent to about 99 percent, preferably from about 0.1 percent to about 70 percent, and most preferably from about 1 percent to about 30 percent of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0054] The dosing schedule is adjusted to provide a therapeutic response. For example, a single bolus dose may be administered, the dose may be divided into several portions and administered over time, or the dose may be proportionally increased or decreased depending on the urgency of the situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. "Dosage unit form" refers to physically discrete units suitable as a single dose for the patient being treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic response, together with the necessary pharmaceutical carrier.

[0055] The dose ranges from about 0.0001 to 100 mg / kg, more generally from 0.01 to 5 mg / kg, based on the body weight of the host. For example, the dose may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight, or may be in the range of 1-10 mg / kg, or 0.1 to 5 mg / kg. Representative treatment regimens are administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferred dosing schedules include the following dosing schedules: (i) administer six doses every four weeks, then administer every three months; (ii) administer every three weeks; (iii) administer once at 3 mg / kg body weight, followed by intravenous administration at 1 mg / kg body weight or 3 mg / kg body weight using one of the following: administer every three weeks at 1 mg / kg body weight. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / mL, and in some methods about 25-300 μg / mL.

[0056] The "therapeutically effective amount" of the compounds of the present invention preferably results in a reduction in the severity of the symptoms of the disease, an increase in the number and duration of asymptomatic periods of the disease, or the prevention of functional or physical impairment due to the pain of the disease. For example, for the treatment of a patient having cancer, the "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, even more preferably at least about 80%, compared to a patient not receiving treatment. The therapeutically effective amount of the therapeutic compound may reduce the size of the tumor or otherwise alleviate the symptoms in the patient, who is generally a human, but may also be another mammal. When two or more therapeutic agents are administered in combination therapy, the "therapeutically effective amount" refers to the effectiveness of the combination as a whole, not as individual agents.

[0057] The pharmaceutical composition can be a controlled release or sustained release formulation such as an implant, a transdermal patch, and a microencapsulation delivery system. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by J.R. Robinson, Marcel Dekker, New York, 1978.

[0058] The therapeutic composition can be administered using medical devices such as (1) a needleless subcutaneous injector; (2) a microinfusion pump; (3) a transdermal device; (4) an infusion device; and (5) an osmotic device.

[0059] In certain embodiments, the pharmaceutical composition may be formulated to ensure appropriate distribution in vivo. For example, to ensure that the therapeutic compounds of the present invention cross the blood-brain barrier, they may be formulated in liposomes, which may further contain targeting moieties and may enhance selective transport to specific cells or organs.

[0060] Industrial Applicability and Use The TLR7 agonist compounds disclosed in this specification can be used for the treatment of diseases or conditions that can be alleviated by the activation of TLR7.

[0061] In one embodiment, the TLR7 agonist is used in combination with an anti-cancer immunotherapeutic agent - also known as an immune anti-cancer agent. Anti-cancer immunotherapeutic agents exert their effects, particularly through the activation of T cells, by stimulating the body's immune system to attack and destroy cancer cells. The immune system has a number of checkpoint (regulatory) molecules that help maintain the balance between its legitimate attack on target cells and the inhibition of its attack on healthy and normal cells. Some are stimulatory (upregulatory) molecules, and their involvement means promoting T cell activation and enhancing the immune response. Others are inhibitory (downregulatory or brake) molecules, and their involvement means inhibiting T cell activation and weakening the immune response. The binding of agonist immunotherapeutic agents to stimulatory checkpoint molecules can result in the activation of the latter and the enhancement of the immune response against cancer cells. Alternatively, the binding of antagonist immunotherapeutic agents to inhibitory checkpoint molecules can prevent the downregulation of the immune system by the latter and help maintain an active response against cancer cells. Examples of stimulatory checkpoint molecules are B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H. Examples of inhibitory checkpoint molecules are CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, CD96, and TIM-4.

[0062] In the mechanism of action of either anti-cancer immunotherapeutic agent, its efficacy can be enhanced by upregulation of the systemic immune system such as activation of TLR7. Therefore, in one embodiment, the present specification provides a method for treating cancer, characterized by administering to a patient suffering from cancer a therapeutically effective combination of an anti-cancer immunotherapeutic agent and a TLR7 agonist as disclosed herein. The timing of administration may be simultaneous, sequential, or alternating. The administration method may be systemic or local. The TLR7 agonist may be delivered using a complex in a targeted manner.

[0063] Cancers that can be treated by the combination therapy as described above include acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, anal cancer, appendiceal cancer, teratoid / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative tumor, colon cancer, colorectal cancer, craniopharyngioma, cholangiocarcinoma, endometrial cancer, epithelioma, esophageal cancer, neuroblastoma, Ewing's sarcoma, eye cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, lip and oral cavity cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0064] Anticancer immunotherapeutic agents that can be used in combination therapies as disclosed herein include AMG 557, AMP-224, atezolizumab, avelumab, BMS 936559, semipramab, CP-870893, dacetuzumab, durvalumab, enoblituzumab, galiximab, IMP321, ipilimumab, lucatumumab, MEDI-570, MEDI-6383, MEDI-6469, muromonab-CD3, nivolumab, pembrolizumab, pidilizumab, spartalizumab, tremelimumab, urelumab, utomilumab, balstilimab, bonlerolizumab. Their alternative names (trade names, former names, research codes, or synonyms) and their respective target checkpoint molecules are shown in Table B below.

Table 109

[0065] In one embodiment of the combination therapy with a TLR7 agonist, the anticancer immunotherapeutic agent is an antagonist anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody. The cancer can be lung cancer (including non-small cell lung cancer), pancreatic cancer, kidney cancer, head and neck cancer, lymphoma (including Hodgkin lymphoma), skin cancer (including melanoma and Merkel cell carcinoma), urothelial cancer (including bladder cancer), gastric cancer, hepatocellular carcinoma, or colorectal cancer.

[0066] In another embodiment of the combination therapy with a TLR7 agonist, the anticancer immunotherapeutic agent is an antagonist anti-CTLA-4 antibody, preferably ipilimumab.

[0067] In another embodiment of the combination therapy with a TLR7 agonist, the anticancer immunotherapeutic agent is an antagonist anti-PD-1 antibody, preferably nivolumab or pembrolizumab.

[0068] The TLR7 agonists disclosed herein are also useful as vaccine adjuvants.

[0069] The implementation of the present invention can be further understood by referring to the following examples provided as illustrations and not limitations.

Example

[0070] Analysis Procedure NMR The following conditions were used to obtain proton nuclear magnetic resonance (NMR) spectra: NMR spectra were obtained using either DMSO-d6 or CDCl3 as the solvent and internal standard, on either a 400 MHz or 500 MHz Bruker instrument. The raw NMR data was analyzed by using either ACD Spectrus version 2015-01 from ADC Labs or MestReNova software.

[0071] Chemical shifts are reported in parts per million (ppm) on the low-field side, referenced from internal tetramethylsilane (TMS) or the position of TMS inferred by the deuterated NMR solvent. Apparent multiplicities are reported as: singlet - s, doublet - d, triplet - t, quartet - q, or multiplet - m. Peaks showing broadening are further represented as br. Integration values are approximate. It should be noted that integration intensity, peak shape, chemical shift, and coupling constants can depend on solvent, concentration, temperature, pH, and other factors. Additionally, peaks that overlap with water or solvent peaks in the NMR spectrum, or those where exchange occurs, may not provide reliable integration intensity. In some cases, NMR spectra may be obtained using water peak suppression, but overlapping peaks may become invisible, or their shape and / or integration values may change.

[0072] Liquid Chromatography The following preparative and analytical (LC / MS) liquid chromatography methods were used:

[0073] LCMS Procedure A: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile Phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Temperature: 50 °C; Gradient: 0% B to 100% B over 3 minutes, then hold at 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm)

[0074] LCMS Procedure B: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: 5:95 acetonitrile: water containing 0.1% TFA; Mobile Phase B: 95:5 acetonitrile: water containing 0.1% TFA; Temperature: 50 °C; Gradient: 0% B to 100% B over 3 minutes, then hold at 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (220 nm)

[0075] LCMS Procedure C: Column: Waters XBridge BEH C18 XP (50 x 2.1 mm) 2.5 μm; Mobile Phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile Phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Temperature: 50 °C; Gradient: 0 - 100% B over 3 minutes; Flow Rate: 1.1 mL / min

[0076] LCMS Procedure D: Column: Ascentis Express C18 (50 x 2.1 mm) 2.7 μm; Mobile Phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile Phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Temperature: 50 °C; Gradient: 0 - 100% B over 3 minutes; Flow Rate: 1.1 mL / min

[0077] LCMS Procedure E: Column: BEH C18 2.1 x 50 mm; Mobile Phase A: water containing 0.05% TFA; Mobile Phase B: acetonitrile containing 0.05% TFA; Temperature: 50 °C; Gradient: 2 - 98% B over 1.7 minutes; Flow Rate: 0.8 mL / min

[0078] LCMS procedure F: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Temperature: 50 °C; Gradient: 0% B to 100% B over 3 minutes, then hold at 100% B for 0.50 minutes; Flow rate: 1 mL / min; Detection: MS and UV (220 nm). This method is an ultra-fast liquid chromatography (UPLC (trademark) method).

[0079] Synthesis - General Procedure Generally, the procedures disclosed herein result in a mixture of positional isomers alkylated at the 1H or 2H position of the pyrazolopyrimidine ring system (also referred to as the N1 and N2 positional isomers, respectively, referring to the alkylated nitrogen). For the sake of simplicity, the N2 positional isomer is not shown for convenience but is present in the initially formed mixture and is separated later, for example, by preparative HPLC.

Chemical formula

[0080] The mixture of positional isomers may be separated at the initial stage of the synthesis and the remaining synthesis steps may be carried out using the 1H positional isomer, or alternatively, if necessary, the synthesis may be advanced using the mixture of positional isomers and the separation may be carried out at a later stage.

[0081] The compounds of the present disclosure can be prepared by a number of methods well known to those skilled in the art of organic synthetic chemistry. These methods include the methods described below or variations thereof. Preferred methods include, but are not limited to, the methods described in the following schemes. Scheme 1

Chemical formula

[0082] R aIn Scheme 1 and in other cases where it appears, for example,

Chem.

[0083] Compound 11 can be prepared by the synthetic sequence illustrated in Scheme 1 above. Reduction of nitropyrazole 1 gives compound 2, which is then cyclized with 1,3-bis(methoxycarbonyl)-2-methyl-2-thiourea to give hydroxypyrazolopyrimidine 3. Amine R a NH2 is introduced using BOP / DBU coupling conditions, and then bromo or iodo-pyrazolopyrimidine 5 is obtained by bromination using NBS or iodination using NIS (step 4). Alkylation with benzyl halide 6 gives a mixture of N1 and N2 products, and separation gives N1 intermediate 7. After catalytic hydrogenation (step 6), intermediate alcohol 9 is obtained by performing LiAlH4 reduction and carbamate hydrolysis in one pot. Alcohol 9 is converted to benzyl chloride and then substituted with an appropriate amine to give compound 11 (alkylation of brominated intermediate 5 in step 5 gives a more favorable ratio of N1 / N2 products compared to alkylation of non-brominated intermediate 4). Scheme 2

Chem.

[0084] Alternatively, intermediate 9 may be obtained using the route described in Scheme 2 above. Intermediate 3 is brominated or iodinated using NBS or NIS, and then alkylated to obtain intermediate ester 12. Next, intermediate 7 is obtained by amination using BOP coupling conditions. After catalytic hydrogenation, intermediate 9 is obtained by reduction with LiAlH4 to alcohol and deprotection of methyl carbamate. Scheme 3

Chemical Structure

[0085] An alternative route to intermediate 8 begins with the alkylation of nitropyrazole 1 using benzyl halide 6, giving benzylpyrazole 13. After reduction of the nitro group, cyclization with 1,3-bis(methoxycarbonyl)-2-methyl-2-thiourea gives hydroxypyrazolopyrimidine 15, which is converted to the appropriate amine derivative 8 using BOP / DBU conditions. This is illustrated in Scheme 3 above. Scheme 4

Chemical Structure

[0086] Another alternative route to the target compound is shown in Scheme 4 above. Reduction of the ester group of intermediate 15 and removal of the methyl carbamate using NaOH gives alcohol 16. Conversion of alcohol 16 to the chloride and subsequent substitution with the appropriate amine gives 17, and then amination using BOP / DBU conditions gives the target molecule 11. Scheme 5

Chemical Structure

[0087] In the above scheme 5, hydrolysis of the methyl ester in 7 / 8 or 15 followed by amide formation can give the corresponding amides 7a / 8a or 15a. Catalytic hydrogenation of 7a followed by carbamate deprotection gives compound 7b. Carbamate deprotection of 8a gives compound 8b. Finally, introduction of an amine into 15a followed by carbamate deprotection gives compound 15b.

[0088] Synthesis - Specific Examples To further illustrate the above, the following non-limiting representative synthetic schemes are included. Variations of these examples within the scope of the claims are within the scope of those skilled in the art and are considered to be within the scope of the present disclosure. The reader, being a person skilled in the relevant art to which the present disclosure is provided, will recognize that the compounds disclosed herein can be prepared and used without an exhaustive list of examples.

[0089] Analytical data for compounds numbered 100 and above can be found in Table A. Example 1 - Compound 101

Chemical formula

[0090] (S)-3-((1-(4-((2,6-Diazaspiro[3.3]heptan-2-yl)methyl)-2-methoxybenzyl)-5-amino-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)hexan-1-ol 1 (US 2020 / 0038403 A1; 31 mg, 0.065 mmol) in DMF (1 mL) was treated with Ac2O (6.09 μL, 0.065 mmol) and stirred at RT for 1 h. The solvent was evaporated and the residue was dissolved in DMF (1 mL). The crude residue was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 4% B for 0 min, 4 - 44% B over 20 min then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give 5 mg of Compound 101.

[0091] The following compounds were prepared analogously: Compound 106, Compound 107, Compound 215 (prepared by reductive amination of Compound 1 with formaldehyde), and Compound 216 (prepared by reductive amination of Compound 1 with acetone). Example 2 - Compound 110

Chemical formula

[0092] A solution of 1-(4-((2,6-diazaspiro[3.3]heptan-2-yl)methyl)-2-methoxybenzyl)-N7-butyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine 2 (US 2020 / 0038403 A1; 32 mg, 0.073 mmol) and cyclobutanecarboxylic acid (7.01 μL, 0.073 mmol) in DMF (0.5 mL) was treated with Hunig's base (0.064 mL, 0.366 mmol) and HATU (33.4 mg, 0.088 mmol) and stirred for 30 minutes. The base was evaporated and syringe filtered. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:10 mM NH4OAc; mobile phase B: water containing 95:5 acetonitrile:10 mM NH4OAc; gradient: hold at 12% B for 0 minutes, 12 - 52% over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 110.

[0093] The following compounds were prepared similarly: Compound 104, Compound 105, and Compound 111. Example 3 - Compound 102

Chemical Structure

[0094] A 2 mL DMF solution of N7-butyl-1-(4-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine 3 (US 2020 / 0038403 A1; 15 mg, 0.04 mmol) was treated with 6,6-difluoro-2-azaspiro[3.3]heptane (10.6 mg, 0.08 mml) and heated at 80 °C for 1 hour. LCMS indicated the completion of the reaction. The reaction mixture was syringe filtered. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; gradient: hold at 21% B for 0 min, 21 - 61% B over 20 min, then hold at 100% B for 4 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the target compound were combined and dried by centrifugal evaporation to give Compound 102.

[0095] Compound 103 was prepared similarly. Example 4 - Compound 112

Chemical formula

[0096] Methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate 4 (US 2020 / 0038403; 20 mg, 0.028 mmol) in 1 mL of DMF solution was treated with 1-oxa-6-azaspiro[3.3]heptane (13 mg, 0.14 mmol) and heated at 80 °C for 1 hour. The reaction mixture was treated with triethylamine-trihydrofluoride (23 μL, 0.14 mmol) and stirred at RT for 3 hours. The crude product was treated with NaOH (112 μL, 0.559 mmol) and heated at 80 °C for 2 hours. The reaction mixture was neutralized to pH 7 with 6M aqueous HCl. The solvent was evaporated in a rotary evaporator. The residue was dissolved in 1 mL of DMF and the crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 9% B for 0 min, 9 - 49% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 112.

[0097] Compound 113 and Compound 114 were prepared similarly. Example 5 - Compound 108

Chemical Structure

[0098] Step 1. A solution of methyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate 5 (US 2020 / 0038403 A1; 300 mg, 0.835 mmol), spiro[2.3]hexan-5-ylmethanamine hydrochloride (139 mg, 1.252 mmol) in DMSO (2 mL) was treated with DBU (0.378 mL, 2.505 mmol). BOP (554 mg, 1.252 mmol) was added. The reaction mixture was heated at 40 °C for 1 h. The reaction mixture was treated with NaOH (0.835 mL, 4.17 mmol) and heated at 80 °C for 2 h. The product was directly purified by reverse-phase ISCO using a 50 g C-18 column, eluting with 0-50% water / MeCN (0.05% TFA), and the fractions were lyophilized to give Compound 166 as a white solid.

[0099] Step 2. A solution of [[4-((5-amino-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxyphenyl]methanol 166 (300 mg, 0.760 mmol) in THF (2 mL) was treated with SOCl2 (0.111 mL, 1.521 mmol) and stirred at RT for 30 minutes. The solvent was evaporated in a V-10 evaporator, and 30 mg of the crude chloride was dissolved in DMSO (0.5 mL) and treated with 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethan-1-one (51 mg, 0.363 mmol) and Hunig's base (0.127 mL, 0.727 mmol). The reaction mixture was heated at 80 °C for 3 hours. The excess base was evaporated, and the crude product was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 12% B for 0 minutes, increase to 12 - 52% B over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 109 were combined and dried by centrifugal evaporation to give compound 108.

[0100] The following compounds were prepared similarly: compound 109 (in step 1, spiro[2.2]pentan-1-ylmethanamine was used instead of spiro[2.3]hexan-5-ylmethanamine), compound 129, compound 130, compound 131, compound 132, compound 133, compound 134, compound 135, compound 145, compound 146, compound 147, compound 148, compound 152 (in step 1, (3-cyclopropylcyclobutyl)methanamine was used instead of spiro[2.3]hexan-5-ylmethanamine), compound 167, compound 168, compound 169, compound 170, compound 183, and compound 241. Example 6 - Compound 115

Chemical formula

[0101] Step 1. A solution of methyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate 7 (US 2020 / 0038403 A1; 100 mg, 0.278 mmol), (1-fluorospiro[2.3]hexan-5-yl)methanamine (71.9 mg, 0.557 mmol) in DMSO (2 mL) was treated with DBU (0.126 mL, 0.835 mmol). BOP (185 mg, 0.417 mmol) was added. The reaction mixture was heated at 40 °C for 1 h. The reaction mixture was treated with NaOH (0.278 mL, 1.391 mmol) and heated at 80 °C for 2 h. The product was directly purified by reverse-phase ISC using a 50 g C-18 column, eluting with 0-50% water / MeCN (0.05% TFA) to give 84 mg of compound 8 as a white solid, as a mixture of diastereomers. LC / MS [M+H] + =469.1 1 H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.88 (s, 1H), 7.75 (d, J = 1.8 Hz, 1H), 6.98 (s, 1H), 6.86 - 6.74 (m, 2H), 5.71 (s, 2H), 4.67 - 4.58 (m, 1H), 4.45 (d, J = 3.6 Hz, 3H), 3.75 (d, J = 3.2 Hz, 5H), 2.80 (s, 1H), 2.18 (q, J = 9.1 Hz, 1H), 2.05 - 1.90 (m, 2H), 1.85 - 1.76 (m, 1H), 0.74 (ddd, J = 21.0, 11.2, 5.9 Hz, 2H)

[0102] Step 2. Thionyl chloride (0.030 mL, 0.407 mmol) was added to a solution of compound 8 (84 mg, 0.204 mmol) in THF (1 mL). The reaction mixture was stirred at RT for 1 hour. The solvent was evaporated in a V-10 evaporator to give the crude chloride, which was carried on to the next step without further purification. A 0.5 mL DMF solution of 12 mg of the chloride and cyclobutylamine (3.96 mg, 0.056 mmol) in a 20 mL sealed vial was heated at 70 °C for 1 hour. The excess base was evaporated and the crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; gradient: hold at 2% B for 0 min, 2 - 42% B over 23 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give 6 mg of compound 115 as a mixture of diastereomers.

[0103] The following compounds were prepared analogously: compound 116, compound 117, compound 118, compound 119, compound 120, compound 121, compound 124, compound 125, compound 126, compound 127, and compound 128. Example 7 - Compound 136

Chemical formula

[0104] Step 1. A solution of compound 7 (200 mg, 0.557 mmol), (1,1-difluorospiro[2.3]hexan-5-yl)methanamine (164 mg, 1.113 mmol) in DMSO (2 mL) was treated with DBU (0.252 mL, 1.670 mmol). BOP (369 mg, 0.835 mmol) was added. The reaction mixture was heated at 40 °C for 1 h. The reaction mixture was treated with NaOH (0.557 mL, 2.78 mmol) and heated at 80 °C for 2 h. The reaction was directly purified on a reversed-phase ISC using a 50 g C-18 column eluting with 0-50% water / acetonitrile and the fractions were lyophilized to give the desired product as a white solid. LC / MS Prediction C 21 H 24 F2N6O2=431.4 Actual measurement [M+H] + =431.2

[0105] Step 2. A solution of compound 10 (142 mg, 0.330 mmol) in tetrahydrofuran (2 mL) was treated with SOCl2 (0.048 mL, 0.660 mmol) and stirred for 1 h. The solvent was evaporated in a V-10 evaporator and the crude product was carried on to the next step. A mixture of the crude chloride and cyclobutylamine (11.8 mg, 0.167 mmol) in 0.5 mL DMF was heated at 80 °C for 1 h. The excess amine was evaporated and the crude material was purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water with NHOAc; Mobile phase B: 95:5 acetonitrile: water with NHOAc; Gradient: 15% B at 0 min hold, 15-55% B over 20 min, then 100% B at 0 min hold; Flow rate: 20 mL / min; Column temperature: 25° C. Fraction collection was triggered by the MS signal. Fractions containing the product were combined and dried by centrifugal evaporation to give 4.2 mg of compound 136, isolated as a mixture of diastereomers.

[0106] The following compounds were prepared similarly: Compound 122, Compound 123, Compound 137, Compound 138, Compound 139, Compound 140, Compound 141, Compound 142, Compound 143, and Compound 144. Example 8 - Compound 173 [Chemical formula]

[0107] Step 1. A solution of Compound 11 (US 2020 / 0038403 A1; 350 mg, 0.904 mmol) and spiro[2.3]hexan-5-ylmethanamine hydrochloride (151 mg, 1.355 mmol) in DMSO (2 mL) was treated with DBU (0.409 mL, 2.71 mmol). BOP (599 mg, 1.355 mmol) was added. The reaction mixture was heated at 40 °C for 1 hour. The reaction mixture was treated with NaOH (0.904 mL, 4.52 mmol) and heated at 80 °C for 2 hours. The reaction product was directly purified by reverse-phase ISCO using a 50 g C-18 column, eluting with 0 - 50% water / acetonitrile (0.05% TFA), and the fractions were lyophilized to give Compound 12 as a white solid. LC / MS [M+H] + = 395.2 1 H NMR (400 MHz, DMSO-d6) 12.34 (s, 1H), 8.32 (t, J = 5.7 Hz, 1H), 7.86 (s, 1H), 7.80 (s, 1H), 7.53 - 7.43 (m, 2H), 6.79 (d, J = 7.9 Hz, 1H), 5.81 (s, 2H), 3.84 (s, 3H), 3.72 (t, J = 6.5 Hz, 3H), 2.77 - 2.65 (m, 1H), 1.82 (dd, J = 12.0, 6.3 Hz, 3H), 1.66 (s, 1H), 0.36 (s, 4H)

[0108] Step 2. A 0.5 mL DMF solution of compound 12 (40 mg, 0.098 mmol) and 2-methyl-2,6-diazaspiro[3.3]heptane (11 mg, 0.098 mmol) was treated with Hunig's base (1 μL, 0.294 mmol) and HATU (44 mg, 0.118 mmol). The reaction mixture was stirred at RT for 30 minutes. The excess base was evaporated and the crude product was purified by reverse phase ISCO using a 50 g C-18 column, eluting with 0 - 50% water / acetonitrile (0.05% TFA), and the fractions were lyophilized to give compound 173 as a white solid.

[0109] The following compounds were prepared analogously: compound 171, compound 172, compound 174, compound 175, compound 176, compound 177, compound 178, compound 179, compound 184, compound 190, compound 192, compound 193, compound 194, compound 195, compound 196, compound 197, compound 201, compound 222, compound 225, compound 226, compound 227, compound 228, compound 230, compound 231, compound 234, compound 235, compound 236, compound 237, compound 238, compound 239, and compound 240. Example 9 - Compound 149.

Chemical formula

[0110] Step 1. A DMSO (2 mL) solution of compound 11 (100 mg, 0.258 mmol) and (1,1-difluorospiro[2.3]hexan-5-yl)methanamine (76 mg, 0.516 mmol) was treated with DBU (0.117 mL, 0.774 mmol). BOP (171 mg, 0.387 mmol) was added. The reaction mixture was heated at 40 °C for 1 hour, treated with NaOH (0.258 mL, 1.291 mmol), heated at 80 °C for 2 hours, and directly purified by reverse phase ISC using a 50 g C-18 column, eluting with 0 - 50% water / acetonitrile (0.05% TFA), and the fractions were lyophilized to give 91 mg of compound 14 as a white solid. LC / MS [M-H] + =443.2

[0111] Step 2. A 0.5 mL DMF solution of compound 14 (15 mg, 0.034 mmol) and 2-methyl-2,6-diazaspiro[3.3]heptane (3.8 mg, 0.034 mmol) was treated with Hunig's base (18 μL, 0.1 mmol) and HATU (15.4 mg, 0.041 mmol). The reaction was stirred at RT for 20 minutes. The excess amine was evaporated and the crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 8% B for 0 minutes, 8 - 48% B over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 149 as a white solid.

[0112] The following compounds were prepared similarly: compound 150, compound 151, compound 159, compound 160, compound 161, compound 162, compound 163, compound 164, and compound 165. Example 10 - Compound 153

Chemical Structure

[0113] Step 1. A solution of compound 7 (100 mg, 0.278 mmol) and spiro[3.3]heptan-2-ylmethanamine (69.7 mg, 0.557 mmol) in DMSO (2 mL) was treated with DBU (0.126 mL, 0.835 mmol). BOP (185 mg, 0.417 mmol) was added. The reaction mixture was heated at 40 °C for 1 h, treated with NaOH (0.278 mL, 1.391 mmol), heated at 80 °C for 2 h, and directly purified by reverse-phase ISC eluting with 0 - 50% water / MeCN (0.05% TFA) using a 50 g C-18 column. The fractions were lyophilized to give compound 16 as a white solid. LC / MS [M+H] + =409.3

[0114] Step 2. A solution of compound 16 (190 mg, 0.465 mmol) in THF (1 mL) was treated with SOCl2 (0.068 mL, 0.930 mmol) and stirred for 30 min. The solvent was evaporated and the crude chloride was carried on to the next step. A solution of the chloride (15 mg, 0.035 mmol) and cyclobutylamine (12 mg, 0.176 mmol) in 0.5 mL of DMF was dissolved and heated at 70 °C for 1 h. Cyclobutylamine was evaporated and the crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.05% TFA; mobile phase B: acetonitrile containing 95:5 acetonitrile:0.05% TFA; gradient: hold at 9% B for 0 min, 9 - 49% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give 7.9 mg of compound 153.

[0115] Through this example, the following compounds were prepared analogously: Compound 154, Compound 155, Compound 156, Compound 157, Compound 158, Compound 185, and Compound 186. In the examples of Compound 185 and Compound 186, (6,6-difluorospiro[3.3]heptan-2-yl)methanamine was used instead of spiro[3.3]heptan-2-ylmethanamine in Step 1. Example 11 - Compound 200 [Chemical formula]

[0116] Step 1. A solution of Compound 7 (100 mg, 0.258 mmol) and spiro[3.3]heptan-2-ylmethanamine (48.5 mg, 0.387 mmol) in DMSO (2 mL) was treated with DBU (0.117 mL, 0.774 mmol). BOP (171 mg, 0.387 mmol) was added. The reaction mixture was heated at 40 °C for 1 hour, treated with NaOH (0.258 mL, 1.291 mmol), and heated at 80 °C for 2 hours. The reaction product was directly purified by reverse-phase ISC using a 50 g C-18 column, eluting with 0 - 50% water / acetonitrile (0.05% TFA), and the fractions were lyophilized to obtain Compound 18 as a white solid. LC / MS [M+H] + =422.3 11H NMR (500 MHz, DMSO-d6) δ 7.59 (s, 1H), 7.22 (s, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.46 (s, 0H), 6.37 (d, J = 7.8 Hz, 1H), 5.66 (d, J = 12.0 Hz, 4H), 4.31 (s, 2H), 4.08 (s, 2H), 3.89 (s, 3H), 3.53 (s, 1H), 3.38 (t, J = 6.4 Hz, 1H), 3.31 (d, J = 7.6 Hz, 1H), 3.24 (d, J = 7.5 Hz, 1H), 3.01 (d, J = 4.4 Hz, 0H), 2.31 (q, J = 7.7 Hz, 1H), 2.17 (s, 3H), 1.93 - 1.86 (m, 2H), 1.85 (td, J = 12.7, 11.5, 3.7 Hz, 4H), 1.80 (d, J = 7.3 Hz, 3H), 1.72 (q, J = 7.7 Hz, 2H), 1.57 - 1.50 (m, 2H)

[0117] Step 2. A solution of compound 18 (20 mg, 0.047 mmol) in DMF (0.5 mL) was treated with 2-methyl-2,6-diazaspiro[3.3]heptane (5.31 mg, 0.047 mmol), followed by HATU (21.60 mg, 0.057 mmol) and Hunig's base (0.025 mL, 0.142 mmol). LCMS indicated completion of the reaction after 30 minutes. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 11% B for 0 minutes, increase from 11 - 51% B over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 200.

[0118] The following compounds were prepared similarly: Compound 180, Compound 181, Compound 182, Compound 187, Compound 188, Compound 189, Compound 202, Compound 203, Compound 204, and Compound 205. In the example of Compound 187, (6,6-difluorospiro[3.3]heptan-2-yl)methanamine was used instead of spiro[3.3]heptan-2-ylmethanamine in Step 1. Example 12 - Compound 210 [Chemical formula]

[0119] Step 1. A solution of Compound 7 (100 mg, 0.278 mmol) and (5-methylisoxazol-3-yl)methanamine (62 mg, 0.557 mmol) in DMSO (2 mL) was treated with DBU (0.210 mL, 1.391 mmol). BOP (185 mg, 0.417 mmol) was added. The reaction mixture was heated at 40 °C for 1 hour, treated with NaOH (0.278 mL, 1.391 mmol), and heated at 80 °C for 2 hours. The reaction mixture was directly purified by reverse-phase ISC using a 50 g C-18 column, eluting with 0 - 50% water / acetonitrile (0.05% TFA). The fractions were lyophilized to obtain Compound 20 (white solid). LC / MS [M+H]+ = 396.1 1 H NMR (400 MHz, DMSO-d6) δ 8.80 (t, J = 5.9 Hz, 1H), 7.84 (s, 1H), 7.70 (s, 1H), 6.88 (s, 1H), 6.81 - 6.71 (m, 2H), 6.11 (s, 1H), 5.62 (s, 2H), 4.73 (d, J = 5.8 Hz, 2H), 4.39 (s, 2H), 4.13 (d, J = 5.9 Hz, 0H), 3.61 (s, 3H), 2.29 (d, J = 4.0 Hz, 3H), 1.56 - 1.43 (m, 1H), 0.59 - 0.50 (m, 1H)

[0120] Step 2. A solution of compound 20 (70 mg, 0.177 mmol) in THF (0.5 mL) was treated with SOCl2 (0.026 mL, 0.354 mmol) and stirred at RT for 30 minutes. The solvent was evaporated in a V-10 evaporator and the crude chloride was advanced to the next step. The crude chloride (18 mg, 0.043 mmol) and 2,6-diazaspiro[3.3]heptane (21 mg, 0.217 mmol) were mixed in 0.5 mL of DMSO and the reaction mixture was heated at 80 °C for 1 hour. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 5% B for 0 minutes, 5 - 45% B over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give 7.4 mg of compound 210 as a white solid.

[0121] The following compounds were prepared analogously: compound 211, compound 212, and compound 213. Example 13 - Compound 214

Chemical formula

[0122] Step 1. A solution of methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (300 mg, 0.774 mmol) in DMSO (3.9 mL) was treated with (5-methylisoxazol-3-yl)methanamine (174 mg, 1.55 mmol), BOP (411 mg, 0.929 mmol) and DBU (233 μL, 1.549 mmol). The reaction mixture was stirred at RT for 2 h, diluted with EtOAc and washed with H2O (3 times). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (353 mg, yield 95%). 1 1H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 7.99 - 7.93 (m, 1H), 7.77 (t, J = 5.9 Hz, 1H), 7.49 (d, J = 1.5 Hz, 1H), 7.45 (dd, J = 7.8, 1.5 Hz, 1H), 6.62 (d, J = 7.9 Hz, 1H), 6.10 (d, J = 0.9 Hz, 1H), 5.80 (s, 2H), 4.73 (d, J = 5.9 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 3.64 (s, 3H), 2.31 (s, 3H) LC / MS conditions: Column: Aquity UPLC BEH C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 100% water containing 0.05% TFA; Mobile phase B: 100% acetonitrile containing 0.05% TFA; Gradient: from 2% B to 98% B over 1 min, then hold at 98% B for 0.50 min; Flow rate: 0.8 mL / min LC RT: 0.67 min LC / MS (M + H) 482.3

[0123] Step 2. A solution of methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (125 mg, 0.260 mmol) in dioxane (1.3 mL) was treated with NaOH (10 M aqueous solution, 0.2 mL, 2.0 mmol) and heated to 75 °C. After 2 hours, the reaction mixture was cooled to RT and treated with HCl (4 M in dioxane, 0.52 mL, 2.1 mmol). The resulting solution was concentrated under reduced pressure. The residue was redissolved in MeOH / DCM and concentrated again under reduced pressure to give crude 4-((5-amino-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid. A solution of this crude product (40 mg) in DMF (469 μL) was treated with 2-methyl-2,6-diazaspiro[3.3]heptane·2HCl (17 mg, 0.094 mmol), DIEA (57 μl, 0.33 mmol) and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (50% EtOAc solution, 55.8 μL, 0.094 mmol). The reaction mixture was stirred at RT for 1 hour. The reaction mixture was diluted with DMF (1 mL) and H2O (0.2 mL) and filtered through a PTFE frit. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; gradient: hold at 5% B for 0 min, 5 - 45% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 214 (13.7 mg, 58% yield). Example 14 - Compound 198 [Chemical Structure]

[0124] Step 1. Cs2CO3 (11.42 g, 35.1 mmol) was added to a stirred solution of methyl 4-nitro-1H-pyrazole-5-carboxylate (5 g, 29.2 mmol) in DMF (30 mL). After cooling in an ice bath, a solution of methyl 4-(bromomethyl)-3-methoxybenzoate (7.57 g, 29.2 mmol) in DMF (20 mL) was added portionwise over 5 minutes in several portions. The reaction mixture was left to warm slowly to room temperature and stirred overnight, then poured into water (150 mL) and extracted with EtOAc (3 x 70 mL). The combined organic phases were washed with brine (4 x 50 mL), dried (MgSO4), filtered, and concentrated. Flash chromatography (220 g SiO2 column, 0 to 50% EtOAc in hexane) gave methyl 1-(2-methoxy-4-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate (1.012 g, 2.90 mmol, 9.92% yield) as a solid. LC-MS(ES,m / z):[M+H] + 350.1 1 H NMR(400MHz,DMSO-d6)δ 8.40(s,1H),7.57(d,J=7.6 Hz,1H),7.50(s,1H),7.27(d,J=7.9 Hz,1H),5.53(s,2H),3.96(s,3H),3.86(s,3H),3.82(s,3H)

[0125] Step 2. Methyl 1-(2-methoxy-4-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate (2 g, 5.73 mmol) was suspended in ethanol (100 mL). 10% palladium on carbon (100 mg) was added, the reaction vessel was evacuated, and purged with hydrogen six times. The reaction mixture was stirred overnight under a hydrogen atmosphere and filtered through CELITE (trademark) while washing with EtOH (100 mL). The filtrate was evaporated to dryness to obtain methyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.764 g, 5.52 mmol, 96% yield) as a solid. LC-MS(ES,m / z):[M+H] + 320.1 1 H NMR(400MHz,DMSO-d6)δ 7.50(s,1H),7.46(d,J=7.7 Hz,1H),7.18(s,1H),6.42(d,J=7.9 Hz,1H),5.55(s,2H),5.14(s,2H),3.91(s,3H),3.84(s,3H),3.70(s,3H)

[0126] Step 3. Methyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.75 g, 5.48 mmol) was suspended in MeOH (60 mL). 1,3-Bis(methoxycarbonyl)-2-methyl-2-thioureido (1.243 g, 6.03 mmol) was then added, followed by HOAc (1.882 mL, 32.9 mmol). The reaction mixture was stirred at RT for 1 h. 2 mL of TFA was added and the reaction mixture was stirred overnight. NaOMe (23.69 g, 110 mmol, 25% by weight) was added and then the mixture was stirred at RT for 4 h. The precipitate was filtered and suspended in MeOH (50 mL). NaOMe (3 g, 13.88 mmol, 25% by weight) was added and the reaction was stirred at RT for 1 h. The reaction mixture was acidified with AcOH, stirred for 10 min, then the reaction was filtered, washed with MeOH, and the solid methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (670 mg, 1.730 mmol, 32% yield) was obtained. LC-MS (ES, m / z): [M+H] + 388.1 1 H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1H), 7.52 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 6.70 (d, J = 7.7 Hz, 1H), 5.76 (s, 2H), 3.90 (s, 3H), 3.85 (s, 3H), 3.76 (s, 3H)

[0127] Step 4. 4-(Hydroxymethyl)-3-methoxybenzoic acid methyl ester (180 mg, 0.465 mmol), spiro[2.3]hexan-5-ylmethanamine hydrochloride (103 mg, 0.697 mmol), BOP (308 mg, 0.697 mmol) and DMSO (1 mL) were placed in a 20 mL scintillation vial. DBU (0.245 mL, 1.626 mmol) was added. The reaction mixture was stirred at 60 °C for 2 h, cooled, filtered, and purified by reverse phase flash chromatography (50 g C 18 column, 0.05% formic acid in water, 0 to 65% MeCN) to give methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (165 mg, 0.343 mmol, 73.9% yield, white solid). LC-MS (ES, m / z): [M+H] + 481.2

[0128] Step 5. Methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (165 mg, 0.343 mmol) was dissolved in dioxane (4 mL). NaOH (1.030 mL, 5.15 mmol) was added and the reaction was heated at 80 °C for 2 h. After cooling, the reaction mixture was acidified with HCl and evaporated to dryness and the product was used next without purification. LC-MS (ES, m / z): [M+H] + 409.3

[0129] Step 6. 4-((5-Amino-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid (100 mg, 0.086 mmol), HBTU (39.0 mg, 0.103 mmol), 1-methylpiperidin-4-amine (19.57 mg, 0.171 mmol) and DMF (2 mL) were placed in a 20 mL scintillation vial. DIPEA (0.045 mL, 0.257 mmol) was added. The reaction mixture was stirred at RT for 1 hour, filtered and purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: held at 0% B for 0 minutes, 0 - 40% B over 25 minutes, then held at 100% B for 0 minutes; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. Fraction Compound 198 (16.4 mg, 0.032 mmol, 38% yield)

[0130] Compound 199 was prepared similarly. Example 15 - Compound 207, ditrifluoroacetate

Chemical formula

[0131] Step 1. 10% Palladium on carbon (0.622 g, 0.584 mmol) was added to a stirred solution of methyl 4-nitro-1H-pyrazole-5-carboxylate (10 g, 58.4 mmol) in EtOH (100 mL). The reaction was evacuated, purged with hydrogen 6 times, and then stirred under a hydrogen atmosphere for 2 days. The reaction mixture was filtered through CELITE (trademark) and washed with EtOH (100 mL). The filtrate was evaporated to dryness, triturated with ether / hexane, and methyl 4-amino-1H-pyrazole-5-carboxylate (8.012 g, 56.8 mmol, 97% yield) was obtained as a solid. LC-MS(ES,m / z): [M+H] + 142.1

[0132] Step 2. Methyl 4-amino-1H-pyrazole-5-carboxylate (4 g, 28.3 mmol) was dissolved in MeOH (75 mL), and 1,3-bis(methoxycarbonyl)-2-methyl-2-thiourea (6.43 g, 31.2 mmol) was added, followed by acetic acid (6.49 mL, 113 mmol). The reaction mixture was stirred at RT for 5 h. NaOMe (36.7 g, 170 mmol, 25% by weight) was added. The reaction mixture was stirred at RT overnight, acidified with AcOH, filtered, and washed with water (100 mL), THF (100 mL), and ether (100 mL) to obtain methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (5.098 g, 24.37 mmol, 86% yield) as a solid. LC-MS(ES,m / z): [M+H] + 210.0

[0133] Step 3. Methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (5.1 g, 24.38 mmol) was suspended in DMF (100 mL). NBS (4.34 g, 24.38 mmol) was added and the reaction was stirred at RT for 1 h. The reaction mixture was quenched with water (100 mL), stirred for 10 min, then filtered and washed with water (100 mL), THF (2 x 50 mL), and ether (2 x 50 mL) to obtain methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (8.32 g, 23.11 mmol, 95% yield) as a solid. LC-MS(ES,m / z): [M+H] + 288.0, 290.0

[0134] Step 4. To a stirred solution of methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (2.50 g, 8.68 mmol) in DMF (35 mL) was added Cs2CO3 (3.11 g, 9.55 mmol), followed by a stirred solution of methyl 4-(bromomethyl)-3-methoxybenzoate (2.249 g, 8.68 mmol) in DMF (15 mL). The reaction mixture was stirred overnight at RT, quenched with water (400 mL), and extracted with EtOAc (3 x 150 mL). The combined organic phases were washed with brine (4 x 100 mL), dried (MgSO4), filtered, and concentrated. Trituration with DCM / ether / hexane gave methyl 4-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (1.791 g, 3.07 mmol, 35.4% yield). According to LCMS, the product was 80% pure (the remaining 20% was the N2 positional isomer). LC-MS(ES,m / z):[M+H] + 466.1,468.1

[0135] Step 5. To a 20 mL microwave vial were added methyl 4-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (500 mg, 1.072 mmol) (about 80% pure and contaminated with the N2 positional isomer), 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriphosphorinane (TMB, 269 mg, 2.145 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (235 mg, 0.322 mmol), K2CO3 (296 mg, 2.145 mmol), dioxane (12 mL), and water (3 mL). The reaction mixture was heated in a microwave oven at 120 °C for 1 hour and evaporated to dryness. DMSO (3 mL) was added to the residue. The mixture was filtered, and subjected to reverse-phase flash chromatography (100 g C 18Purified using a column (in water containing 0.05% TFA, 0 to 50% acetonitrile), methyl 4-((5-amino-7-hydroxy-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (117 mg, 0.341 mmol, yield 31.8%) was obtained as an off-white solid. LC-MS (ES, m / z): [M+H] + 344.1

[0136] Step 6. Methyl 4-((5-amino-7-hydroxy-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (130 mg, 0.379 mmol), spiro[2.3]hexan-5-ylmethanamine hydrochloride (84 mg, 0.568 mmol), BOP (251 mg, 0.568 mmol) and DMSO (2 mL) were placed in a 20 mL scintillation vial. DBU (0.200 mL, 1.325 mmol) was added. The reaction mixture was stirred at 50 °C for 1 hour, cooled, diluted with water (1 mL), filtered, and purified by reverse-phase flash chromatography (50 g C 18 column, in water containing 0.05% TFA, 0 to 60% acetonitrile), and methyl 4-((5-amino-3-methyl-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (80 mg, 0.183 mmol, yield 48.4%) was obtained as a solid. LC-MS (ES, m / z): [M+H] + 437.3 11H NMR (400 MHz, DMSO-d6) δ 8.29 (br t, J = 5.6 Hz, 1H), 7.80 (br s, 2H), 7.53 - 7.46 (m, 2H), 6.79 (d, J = 7.7 Hz, 1H), 5.74 (s, 2H), 3.85 (s, 6H), 3.71 (br t, J = 6.5 Hz, 2H), 2.78 - 2.64 (m, 1H), 2.31 (s, 3H), 2.03 - 1.93 (m, 2H), 1.82 (dd, J = 12.1, 6.4 Hz, 2H), 0.35 (s, 4H)

[0137] Step 7. 4-((5-Amino-3-methyl-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid (75 mg, 0.172 mmol), dioxane (2 mL) and NaOH (0.412 mL, 2.062 mmol) were placed in a 20 mL scintillation vial. The reaction mixture was heated at 80 °C for 2 h, cooled, neutralized with 5N HCl, evaporated to dryness, and 4-((5-amino-3-methyl-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid (190 mg, 0.157 mmol, 92% yield) was obtained as a solid and used without purification. LC-MS (ES, m / z): [M+H] + 423.3

[0138] Step 8. 4-((5-Amino-3-methyl-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid (100 mg, 0.083 mmol, purity 35%), HATU (37.8 mg, 0.099 mmol), 1-methylpiperidin-4-amine (18.92 mg, 0.166 mmol) and DMF (2 mL) were placed in a 20 mL scintillation vial. DIPEA (0.043 mL, 0.249 mmol) was added. The reaction mixture was stirred at RT for 1 hour, filtered and purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; Mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; Gradient: held at 9% B for 0 min, 9 - 49% B over 20 min, then held at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the target compound were combined and dried by centrifugal evaporation to give compound 207 (43.3 mg, 0.058 mmol, yield 70%).

[0139] Compound 208 and compound 217 were prepared similarly. Example 16 - Compound 218

Chemical Structure

[0140] Step 1. An aqueous solution of potassium hydroxide (5N, 24.07 mL, 120 mmol) was added to a cooled (ice bath) solution of methyl 3-hydroxy-4-methylbenzoate (4 g, 24.07 mmol) in acetonitrile (150 mL). After stirring at 0 °C for 5 minutes, diethyl (bromodifluoromethyl)phosphonate (12.85 g, 48.1 mmol) was added. The reaction mixture was left as such and stirred for 16 hours until it slowly warmed to RT. Further KOH solution (5N, 16 mL, 80 mmol) was added. The reaction mixture was stirred at RT for an additional 30 minutes, diluted with water (200 mL), and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine (2 x 50 mL), dried (MgSO4), filtered, and concentrated. Methyl 3-(difluoromethoxy)-4-methylbenzoate (2.552 g, 11.80 mmol, 49.0% yield) was obtained as an oil by flash chromatography (SiO2 column, in hexane, 0 to 10% EtOAc). LC-MS(ES,m / z):[M+H] + 217.1 1 H NMR(400MHz,DMSO-d6)δ 7.76(dd,J=7.8,1.7 Hz,1H),7.68(br.s,1H),7.51-7.10(m,2H),3.87(s,3H),2.31(s,3H)

[0141] Step 2. NBS (1.811 g, 10.18 mmol) and benzoyl peroxide (0.448 g, 1.850 mmol) were added to a stirred solution of methyl 3-(difluoromethoxy)-4-methylbenzoate (2 g, 9.25 mmol) in carbon tetrachloride (20 mL). The reaction was stirred at 75 °C for 4 hours and then at RT overnight. The reaction mixture was evaporated to dryness and purified using flash chromatography (SiO2 column, in hexane, 0 to 15% EtOAc) to give methyl 4-(bromomethyl)-3-(difluoromethoxy)benzoate (1.561 g, 5.29 mmol, 57.2% yield) as an oil. LC-MS(ES,m / z):[M+H] +295.0,297.0 1 1H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 8.1, 1.5 Hz, 1H), 7.80 (s, 1H), 7.52 (d, J = 8.1 Hz, 1H), 6.64 (t, J = 73.0 Hz, 1H), 4.57 - 4.51 (m, 2H), 3.98 - 3.90 (m, 3H)

[0142] Step 3. A stirred suspension of methyl (3 - bromo - 7 - hydroxy - 1H - pyrazolo[4,3 - d]pyrimidin - 5 - yl)carbamate (1.269 g, 4.41 mmol) and Cs2CO3 (1.579 g, 4.85 mmol) in DMF (30 mL) was cooled in an ice bath. A solution of methyl 4 - (bromomethyl) - 3 - (difluoromethoxy)benzoate (1.3 g, 4.41 mmol) in DMF (5 mL) was added. The reaction mixture was left as it was and stirred for 3 hours until it warmed slowly to RT. The reaction mixture was poured into water (400 mL) and extracted with EtOAc (3 x 150 mL). The combined organic phases were washed with brine (4 x 80 mL), dried (MgSO4), filtered, concentrated, and methyl 4 - ((3 - bromo - 7 - hydroxy - 5 - ((methoxycarbonyl)amino) - 1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl) - 3 - (difluoromethoxy)benzoate (1.69 g, 3.37 mmol, 76% yield) was obtained as a solid. LC - MS (ES, m / z): [M + H] + 502.1,504.0 1 1H NMR (400 MHz, DMSO - d6) δ 11.72 (br s, 1H), 11.45 (br s, 1H), 7.80 (dd, J = 7.9, 1.3 Hz, 1H), 7.74 (s, 1H), 7.35 (t, J = 73.2 Hz, 1H), 7.26 - 7.18 (m, 1H), 5.82 (s, 2H), 3.87 (s, 3H), 3.76 (s, 3H)

[0143] Step 4. 10% palladium on carbon (0.16 g) was added to a stirred suspension of methyl 4-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoate (1.6 g, 3.19 mmol) in ethanol (150 mL). The reaction mixture was evacuated, purged with hydrogen six times, stirred under a hydrogen atmosphere for 24 hours, and filtered through CELITE™. Since most of the product clogged the CELITE™ together with the palladium, all the solid material was scraped off the CELITE™ and put into water (100 mL), and extracted with EtOAc (3 x 70 mL). The combined organic phases were washed with brine (2 x 50 mL), filtered through CELITE™ again. This filtrate was combined with the first filtrate, dried (MgSO4), filtered, concentrated, and methyl 3-(difluoromethoxy)-4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (1.2 g, 2.83 mmol, 89% yield) was obtained as an off-white solid. LC-MS(ES,m / z):[M+H] + 424.1 1 H NMR(400MHz,DMSO-d6)δ 11.16(br s,1H),7.93(s,1H),7.77(d,J=8.5 Hz,1H),7.73(s,1H),7.36(t,J=73.2 Hz,1H),7.04(d,J=7.9 Hz,1H),5.84(s,2H),3.87(s,3H),3.76(s,3H)

[0144] Step 7. In a 20 mL scintillation vial, methyl 3-(difluoromethoxy)-4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (1.250 g, 2.95 mmol), spiro[2.3]hexan-5-ylmethanamine hydrochloride (0.654 g, 4.43 mmol), BOP (1.959 g, 4.43 mmol) and DMSO (15 mL) were added. DBU (1.558 mL, 10.33 mmol) was added and the reaction mixture was stirred at 50 °C for 3 h. The reaction mixture was poured into saturated NaHCO3 solution (100 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine (4 x 50 mL), dried (MgSO4), filtered and concentrated. Methyl 3-(difluoromethoxy)-4-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (338 mg, 0.654 mmol, 22.16% yield) was obtained as a solid by flash chromatography (80 g SiO2 column, packed with CELITE™, in hexane, 0 to 100% EtOAc). LC-MS(ES,m / z):[M+H] + 517.3

[0145] Step 6. To a stirred solution of methyl 3-(difluoromethoxy)-4-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (330 mg, 0.639 mmol) in dioxane (3600 μL) was added NaOH (1278 μL, 6.39 mmol). The reaction mixture was stirred at 80 °C for 2 h. After cooling, the reaction mixture was neutralized with 5N HCl (1.28 mL) and evaporated to dryness. The residue was suspended in DMSO (2 mL), water (35 mL) was added, the product was filtered off and washed with water (30 mL) to give 4-((5-amino-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoic acid (126 mg, 0.284 mmol, 44.4% yield) as a solid. LC-MS (ES, m / z): [M+H] + 445.2 1 H NMR (400 MHz, DMSO-d6) δ 7.71 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.63 (s, 1H), 7.37 (t, J = 73.5 Hz, 1H), 6.80 (br t, J = 5.4 Hz, 1H), 6.55 (d, J = 7.9 Hz, 1H), 5.92 (br s, 2H), 5.81 (s, 2H), 3.57 - 3.51 (m, 2H), 2.72 - 2.57 (m, 1H), 1.98 - 1.88 (m, 2H), 1.77 - 1.69 (m, 2H), 0.37 - 0.25 (m, 4H)

[0146] Step 7. In a 20 mL scintillation vial, 4-((5-amino-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoic acid (30 mg, 0.068 mmol), HATU (30.8 mg, 0.081 mmol), (3aR,6aS)-2-methyloctahydropyrrolo[3,4-c]pyrrole (12.78 mg, 0.101 mmol) and DMF (2 mL) were added. DIPEA (0.035 mL, 0.203 mmol) was added. The reaction mixture was stirred overnight at RT, filtered, and purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: Hold at 10% B for 0 min, increase from 10 - 50% B over 25 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 218 (30.3 mg, 81% yield).

[0147] The following compounds were prepared analogously: Compound 219, Compound 220, Compound 221, and Compound 224. Example 17 - Compound 223

Chemical formula

[0148] Step 1. Cs2CO3 (1329 mg, 4.08 mmol) was added to a stirred solution of methyl (3-bromo-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (700 mg, 2.040 mmol) in DMF (5 mL). After cooling in an ice bath, a solution of methyl 4-(bromomethyl)-3-(difluoromethoxy)benzoate (572 mg, 1.938 mmol) in DMF (2 mL) was added. The reaction mixture was left as such until it warmed to RT and stirred for 3 h. Water (20 mL) was added and the reaction mixture was extracted with EtOAc (3 x 5 mL). The combined organic phases were washed with brine (4 x 10 mL), dried (MgSO4), filtered, and concentrated. Methyl 4-((3-bromo-7-(butylamino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoate (275 mg, 0.493 mmol, 24.19% yield) was obtained as a solid by flash chromatography (SiO2 column, packed with DCM, 0 to 60% EtOAc in hexane). LC-MS (ES, m / z): [M+H] + 557.1, 559.1 1 H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.82 - 7.69 (m, 2H), 7.61 - 7.14 (m, 2H), 6.87 (d, J = 7.9 Hz, 1H), 5.88 (s, 2H), 3.87 (s, 3H), 3.64 (s, 3H), 3.54 - 3.45 (m, 2H), 1.58 - 1.46 (m, 2H), 1.19 (dq, J = 15.0, 7.4 Hz, 2H), 0.83 (t, J = 7.3 Hz, 3H)

[0149] Step 2. Methyl 4-((3-bromo-7-(butylamino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoate (275 mg, 0.493 mmol) was dissolved in ethanol (15 mL). 10% Pd / C (27 mg) was added. The reaction vessel was evacuated and purged with hydrogen six times. The reaction mixture was stirred under a H2 atmosphere for 2 hours, filtered, and evaporated to dryness. The residue was dissolved in dioxane (2 mL). NaOH (0.564 mL, 2.82 mmol) was added. The reaction mixture was stirred at 80 °C for 2 hours, cooled, neutralized with 5N HCl, and evaporated to dryness. The residue was dissolved in MeOH / water (1:1, 8 mL). Methanol was removed by evaporation. The remaining aqueous suspension was filtered, washed with water, and 4-((5-amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoic acid (54 mg, 0.133 mmol, 27% yield) was obtained as a solid. LC-MS(ES,m / z):[M+H] + =407.22 1 H NMR(400MHz,DMSO-d6)δ 8.50(br s,1H),7.84(s,2H),7.79-7.68(m,2H),7.63-7.05(t,J=73.2 Hz 1H),6.97(d,J=7.9 Hz,1H),5.94(s,2H),3.54(q,J=6.4 Hz,2H),1.54(quin,J=7.2 Hz,2H),1.19(dq,J=14.9,7.3 Hz,2H),0.84(t,J=7.3 Hz,3H)

[0150] Step 3. 4-((5-Amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoic acid (50 mg, 0.123 mmol), HATU (56.1 mg, 0.148 mmol), tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (24.39 mg, 0.123 mmol), and DMF (2 mL) were placed in a 20 mL scintillation vial. DIPEA (0.064 mL, 0.369 mmol) was added. The reaction mixture was stirred at RT for 1 h, quenched with saturated NaHCO3 solution (10 mL), and extracted with EtOAc (3 x 5 mL). The combined organic phases were washed with brine (4 x 5 mL), dried (MgSO4), filtered, and concentrated. The residue was dissolved in DCM (1.5 mL), and TFA (0.5 mL) was added. The reaction was stirred at RT for 30 min and then evaporated to dryness. The crude material was dissolved in DMF (2 mL), filtered, and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water containing NH4OAc; mobile phase B: 95:5 acetonitrile:water containing NH4OAc; gradient: hold at 0% B for 0 min, 0 - 37% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 223 (20.9 mg, 0.043 mmol, 35% yield). Example 18 - Compound 242, tri-TFA salt

Chemical formula

[0151] Step 1. 20 mL of scintillation vial was charged with methyl 4-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoate (750 mg, 1.493 mmol), spiro[2.3]hexan-5-ylmethanamine hydrochloride (500 mg, 2.370 mmol), BOP (991 mg, 2.240 mmol) and DMSO (7.5 mL). DBU (0.788 mL, 5.23 mmol) was added. The reaction mixture was stirred at 50 °C overnight, poured into saturated NaHCO3 solution (100 mL), and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine (4 x 50 mL), dried (MgSO4), filtered, and concentrated. Methyl 4-((3-bromo-5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoate (286 mg, 0.480 mmol, yield 32.2%) was obtained as a solid by flash chromatography (80 g SiO2 column, in hexane, 0 to 60% EtOAc). LC-MS(ES,m / z):[M+H] + 595.1,597.1 1 H NMR(400MHz,DMSO-d6)δ 9.91(s,1H),7.78-7.71(m,2H),7.44(t,J=5.4 Hz,1H),7.38(t,J=73.2 Hz,1H),6.86(d,J=7.9 Hz,1H),5.89(s,2H),3.86(s,3H),3.70-3.59(m,5H),2.76(br t,J=7.2 Hz,1H),2.15-2.03(m,2H),1.80(dd,J=12.1,6.4 Hz,2H),0.32(s,4H)

[0152] Step 2. To a stirred solution of methyl 4-((3-bromo-5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-(difluoromethoxy)benzoate (286 mg, 0.480 mmol) in ethanol (15 mL) was added 10% palladium on carbon (28 mg). The reaction mixture was evacuated and purged with hydrogen six times, then stirred under a hydrogen atmosphere for 1 hour. The reaction mixture was filtered and evaporated to dryness to give methyl 3-(difluoromethoxy)-4-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (290 mg, 0.477 mmol, 99% yield) as a white solid. LC-MS(ES,m / z):[M+H] + 517.3 1 H NMR(400MHz,DMSO-d6)δ 11.93(br s,1H),8.92(br s,1H),8.17(s,1H),7.80(d,J=7.9 Hz,1H),7.77(s,1H),7.42(t,J=73.0 Hz,1H),7.11(d,J=7.9 Hz,1H),6.04(s,2H),3.92(s,3H),3.90(s,3H),3.82(br t,J=6.5 Hz,2H),2.89-2.75(m,1H),2.03(dd,J=12.1,8.4 Hz,2H),1.92(dd,J=12.2,6.7 Hz,2H),0.40(s,4H)

[0153] Step 3. To a stirred solution of methyl 3-(difluoromethoxy)-4-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (250 mg, 0.484 mmol) in THF (10 mL) at 0 °C was added LiAlH4 (1.065 mL, 1.065 mmol) portionwise over 10 minutes. The reaction mixture was stirred at 0 °C for 30 minutes and then quenched with Rochelle salt (10 mL, 20 w / v). After stirring for 10 minutes, the reaction mixture was transferred to a separatory funnel containing 50 mL of water and extracted with EtOAc (3 x 30 mL). The combined organics were washed with brine (3 x 30 mL), dried (MgSO4), filtered, and concentrated. Flash chromatography (24 g SiO2 column, packed with DCM, 0 to 10% MeOH in DCM) gave methyl (1-(2-(difluoromethoxy)-4-(hydroxymethyl)benzyl)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (117 mg, 0.240 mmol, 49.5% yield) as a solid. LC-MS(ES,m / z):[M+H] + 489.2 1 H NMR(400MHz,DMSO-d6)δ 9.65(s,1H),7.88(s,1H),7.43-6.98(m,4H),6.62(d,J=7.9 Hz,1H),5.79(s,2H),5.29(t,J=5.6 Hz,1H),4.46(d,J=5.5 Hz,2H),3.68-3.59(m,5H),2.78(dt,J=15.0,7.3 Hz,1H),2.00(dd,J=12.0,8.5 Hz,2H),1.83(dd,J=12.2,6.3 Hz,2H),0.35(s,4H)

[0154] Step 4. Methyl (1-(2-(difluoromethoxy)-4-(hydroxymethyl)benzyl)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (55 mg, 0.113 mmol) was dissolved in DCM (2 mL), and SOCl2 (0.025 mL, 0.338 mmol) was added. The reaction mixture was stirred at RT for 30 min and then evaporated to dryness. The residue was dissolved in DMF (2 mL), and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (33.5 mg, 0.169 mmol) and then DIPEA (0.059 mL, 0.338 mmol) were added. The reaction mixture was stirred at 50 °C for 4 h and then at RT overnight, quenched with saturated NaHCO3 solution (10 mL), and extracted with EtOAc (3 x 4 mL). The combined organic phases were washed with brine (3 x 5 mL), dried (MgSO4), filtered, and concentrated. The residue was dissolved in DCM (2 mL), and TFA (0.4 mL) was added. The reaction was stirred at RT for 1 h and then evaporated to dryness and redissolved in dioxane (2 mL). NaOH (0.338 mL, 1.689 mmol, 5N) was added, and the reaction was stirred at 80 °C for 1 h, cooled, neutralized with 5N HCl, and evaporated to dryness. The residue was dissolved in DMF (2 mL), filtered, and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; gradient: hold at 2% B for 0 min, 2 - 42% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 242, 3TFA salt (21.1 mg, 0.025 mmol, yield 21.7%).

[0155] Compound 243 was prepared similarly. Example 19 - Compound 206 [Chemical formula]

[0156] Step 1. DBU (0.856 mL, 5.68 mmol) was added to a suspension of methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (550 mg, 1.420 mmol; refer to Step 6 of Example 2 before NaOH treatment) and (S)-3-aminohexan-1-ol hydrochloride 2 (327 mg, 2.130 mmol) in DMSO (5 mL). When the reaction mixture was stirred at RT for 10 minutes, the mixture became a clear solution. BOP (1256 mg, 2.84 mmol) was added, and the reaction mixture was stirred at 70 °C for 2 hours and then subjected to LCMS. No starting material was detected. This solution was treated with 5M NaOH (5 mL, 25.00 mmol) solution and stirred at 70 °C for 0.5 hour. After cooling, the reaction mixture was filtered through a syringe filter disk. The filtrate was purified on a preparative reverse-phase C18 column (150 g) eluting with acetonitrile:water (containing 0.05% TFA modifier) = 0 - 50%, and the desired fraction was frozen and lyophilized to give (S)-4-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid (860.8 mg, 1.246 mmol, 88% yield). LCMS ESI: Calculated for C 20 H 27 N6O4 = 415.2 (M+H + )、Found 415.2 (M+H + )

[0157] Step 2. A mixture of (S)-4-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid (60 mg, 0.145 mmol), 2-methyl-2,6-diazaspiro[3.3]heptane, 2HCl (53.6 mg, 0.290 mmol) in DMF (1 mL) was treated with Hunig's base (0.126 mL, 0.724 mmol), followed by BOP (96 mg, 0.217 mmol). The reaction mixture was stirred at RT for 3 h. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 5% B for 0 min, 5 - 45% B over 25 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 206 (15.5 mg, 0.030 mmol, yield 20.88%).

[0158] The following compounds were prepared analogously: compound 209, compound 229, compound 232, and compound 233. Example 20 - Compound 244

Chemical Structure

[0159] Step 1. A solution of tert-butyl hydrazinecarboxylate (12.75 g, 96 mmol) and DIPEA in DMF (24 mL) was treated at RT by dropping methyl 4-(bromomethyl)-3-methoxybenzoate (5 g, 19.30 mmol) in 24 mL of DMF over 1 hour via a dropping funnel. The reaction mixture was stirred overnight at RT. EtOAc (135 mL) and H2O (75 mL) were added, and the biphasic mixture was stirred for 30 minutes. The reaction mixture was poured into a separatory funnel, and the aqueous layer was removed. The organic layer was washed twice with H2O (75 mL) and twice with 10% LiCl solution (75 mL), dried over Na2SO4, and concentrated. Column chromatography (Isco, 220 g SiO2, 0% CH2Cl2 (5 minutes), then 15% EtOAc-CH2Cl2) gave tert-butyl 2-(2-methoxy-4-(methoxycarbonyl)benzyl)hydrazine-1-carboxylate as a clear oil (3.85 g). LC / MS (M+H) 311.2; LC RT = 0.80 min (Procedure E) 1 H NMR (400 MHz, chloroform-d) δ 7.64 (dd, J = 7.7, 1.5 Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 6.08 - 5.87 (m, 1H), 4.07 (s, 2H), 3.94 (d, J = 4.6 Hz, 6H), 1.50 - 1.40 (m, 9H)

[0160] Step 2. tert-Butyl 2-(2-methoxy-4-(methoxycarbonyl)benzyl)hydrazine-1-carboxylate (25.4 g, 82 mmol) was dissolved in MeOH (164 mL) at RT. 4N HCl-dioxane (123 ml, 59.5 mmol) was added, and the reaction was stirred overnight at RT. The white precipitate was collected by filtration and dried to give methyl 4-(hydrazinylmethyl)-3-methoxybenzoate, dihydrochloride (20 g). LC / MS (M+H) 211.1; LC RT = 0.51 min (Procedure F) 11H NMR (400 MHz, DMSO-d6) δ 9.12 (br s), 7.62 - 7.55 (m, 1H), 7.53 - 7.47 (m, 2H), 4.10 (s, 2H), 3.88 (s, 3H), 3.87 (s, 3H)

[0161] Step 3. A solution of (E)-N,N-dimethyl-2-nitroethene-1-amine (46.4 g, 400 mmol) and pyridine (420 ml, 5195 mmol) in CH2Cl2 (799 ml) was cooled to -10 °C and treated slowly with ethyl 2-chloro-2-oxoacetate (51.4 ml, 460 mmol). The reaction mixture was left for 2 hours until it warmed to 25 °C and stirred overnight. CH2Cl2 was removed by rotary evaporation and methyl 4-(hydrazinylmethyl)-3-methoxybenzoate dihydrochloride (31.7 g, 112 mmol) was added all at once. The solution was stirred at RT for 2 hours and the solvent was removed under vacuum. The residue was washed with water, 1N aqueous HCl and extracted with EtOAc. The organic layer was dried over Na2SO4 and concentrated. The residue was dissolved in CH2Cl2, passed through a short silica gel column and recrystallized from ethanol to give ethyl 1-(2-methoxy-4-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate (29.4 g). LC / MS (M+Na) 386.0; LC RT = 0.98 min (Procedure F) 1 1H NMR (400 MHz, chloroform-d) δ 8.06 (s, 1H), 7.64 (dd, J = 7.9, 1.5 Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 5.53 (s, 2H), 4.45 (q, J = 7.2 Hz, 2H), 3.94 (s, 3H), 3.88 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H)

[0162] Step 4. Ammonium formate (1.41 g, 22.4 mmol) and zinc (0.915 g, 14.0 mmol) were added to a solution of ethyl 1-(2-methoxy-4-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate (2.03 g, 5.60 mmol) in THF (4.67 ml) / MeOH (4.7 ml) at RT. The reaction mixture was stirred at RT for 2 h, and additional amounts of ammonium formate (0.353 g, 5.60 mmol) and zinc (0.229 g, 4.67 mmol) were added. After 1 h, the reaction mixture was filtered through a pad of CELITE™, and the filtrate was concentrated under reduced pressure to give a white solid. The solid was suspended in EtOAc, stirred for 30 min, and filtered. Next, the organic filtrate was concentrated under reduced pressure to give ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.83 g, 98%). 1 1H NMR (400 MHz, DMSO-d6) δ 7.50 - 7.49 (m, 1H), 7.48 - 7.44 (m, 1H), 7.18 (s, 1H), 6.39 (d, J = 7.8 Hz, 1H), 5.53 (s, 2H), 5.10 (s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.90 (s, 3H), 3.83 (s, 3H), 1.13 (t, J = 7.1 Hz, 3H) LC / MS conditions: Column: Aquity UPLC BEH C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 100% water containing 0.05% TFA; Mobile phase B: 100% acetonitrile containing 0.05% TFA; Gradient: 2% B to 98% B over 1 min, then hold at 98% B for 0.50 min; Flow rate: 0.8 mL / min LC RT: 0.86 min LCMS (M + H) = 334.2

[0163] Step 5. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.65 g, 4.95 mmol) was dissolved in CHCl3 (49.5 ml) and cooled to 0 °C. NBS (0.925 g, 5.20 mmol) was added to the mixture all at once. After 15 minutes, the reaction was diluted with CHCl3 and vigorously stirred with 10% aqueous Na2S2O3 for 10 minutes. The organic phase was separated, washed with H2O, dried over MgSO4, and concentrated. The crude product was purified by column chromatography (80 g SiO2, eluting with a 0 to 50% EtOAc-hexane gradient) to give ethyl 4-amino-3-bromo-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.32 g) as a white solid. LC / MS (M+H) 412.2 / 414.2; LC RT = 1.02 min (Procedure E) 1 H NMR (400 MHz, DMSO-d6) δ 7.61 - 7.41 (m, 2H), 6.55 (d, J = 8.3 Hz, 1H), 5.56 (s, 2H), 5.02 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.90 (s, 3H), 3.85 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H)

[0164] Step 6. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (741.2 mg, yield 67.1%), K2CO3 (1.098 g, 7.94 mmol) and 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (3.5 M in THF) (1.816 ml, 6.36 mmol) were suspended in dioxane (26.5 ml):water (5.30 ml) (5:1). The reaction mixture was bubbled with a stream of N2 for 5 minutes, then PdCl2(dppf)-CH2Cl2 adduct (0.052 g, 0.064 mmol) was added and bubbling was continued for a further 4 minutes, after which the reaction was sealed and heated to 90 °C. After 3 hours, an additional amount of 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (3.5 M in THF) (0.908 ml, 3.18 mmol) and PdCl2(dppf)-CH2Cl2 adduct (0.052 g, 0.064 mmol) were added and the reaction was stirred at 100 °C for 16 hours. The cooled reaction mixture was diluted with 100 mL of EtOAc, filtered through CELITE™ and washed further with EtOAc. The crude product was concentrated on 4 g of CELITE™. Column chromatography (80 g SiO2, eluting with a 0 to 30% EtOAc-CH2Cl2 gradient) gave the expected product, ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (741 mg) as a cream solid. LC / MS (M+H) 348.2; LC RT = 0.89 min (procedure E) 1 H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 1.5 Hz, 1H), 7.46 (dd, J = 7.9, 1.5 Hz, 1H), 6.40 (d, J = 7.8 Hz, 1H), 5.48 (s, 2H), 4.94 - 4.86 (m, 2H), 4.14 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 2.10 (s, 3H), 1.15 - 1.08 (m, 3H)

[0165] Step 7. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (742 mg, 2.136 mmol) was suspended in MeOH (10.800 mL), gently heated with vigorous stirring to solubilize the substance. 1,3-Bis-(methoxycarbonyl)-2-methyl-2-thioureido (661 mg, 3.20 mmol) was then added, followed by AcOH (0.611 mL, 10.68 mmol). The reaction mixture was stirred at RT for 16 h. An additional amount of AcOH was added (0.049 mL, 0.854 mmol), and then the mixture was stirred at RT for a further 72 h, after which NaOMe (in 25% wt MeOH) was added (5.69 mL, 25.6 mmol). After stirring for 3 h, the reaction mixture was acidified again with AcOH. The product was collected by filtration, air-dried for 10 min and dried completely in a laboratory dryer to give methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (722.0 mg) as a cream-colored solid. LC / MS (M+H) 402.3; LC RT = 0.86 min (procedure E) 1 H NMR (400 MHz, DMSO-d6) δ 11.58 - 11.17 (m, 2H), 7.51 (d, J = 1.4 Hz, 1H), 7.49 - 7.42 (m, 1H), 6.67 (d, J = 7.9 Hz, 1H), 5.67 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.71 (s, 3H), 2.31 (s, 3H)

[0166] Step 8. Methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (200 mg, 0.498 mmol) and BOP (331 mg, 0.747 mmol) were suspended in DMF (2491 μl) at RT. After adding butan-1-amine (64.0 μl, 0.648 mmol), followed by DBU (3 eq) (225 μl, 1.495 mmol), the reaction mixture became homogeneous. The reaction mixture was stirred at 40 °C for 16 h. An additional amount of butan-1-amine (64.0 μl, 0.648 mmol), BOP (331 mg, 0.747 mmol) and DBU (3 eq) (225 μl, 1.495 mmol) were added to the reaction and stirred at 40 °C for 2 h, then cooled to RT. The reaction mixture was partitioned between EtOAc and saturated NaHCO3. The organic layer was removed and the aqueous phase was extracted further with EtOAc in three portions. The combined organics were washed with 10% LiCl solution, dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (24 g SiO2, 0 to 80% EtOAc-hexane gradient elution) to give methyl 4-((7-(butylamino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (117.4 mg). LC / MS (M+H) 457.4; LC RT = 0.84 min (Procedure E) 1 1H NMR (400 MHz, chloroform-d) δ 7.66 (d, J = 1.3 Hz, 1H), 7.64 (dd, J = 8.0, 1.4 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 5.64 (s, 2H), 4.04 (s, 3H), 3.94 (s, 3H), 3.86 (s, 3H), 3.54 - 3.44 (m, 2H), 2.43 (s, 3H), 1.50 (quin, J = 7.3 Hz, 2H), 1.32 - 1.19 (m, 2H), 0.94 - 0.87 (m, 3H)

[0167] Step 9. Methyl 4-((7-(butylamino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (117 mg, 0.256 mmol) was dissolved in THF (854 μl) at RT. LiAlH4 (1 M in THF) (256 μl, 0.256 mmol) was added dropwise and the reaction was stirred at RT for 20 minutes. LiAlH4 (1 M in THF) (256 μl, 0.256 mmol) was further added and the reaction was stirred for an additional 20 minutes. The reaction mixture was quenched with MeOH, diluted with Rochelle salt and EtOAc, and stirred for 16 hours. The organic layer was separated and the aqueous phase was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, concentrated, and methyl (7-(butylamino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (86.6 mg) was obtained. LC / MS (M+H) 429.4; LC RT = 0.74 min (Procedure E) 1 H NMR (400 MHz, chloroform-d) δ 7.04 (s, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.91 - 6.86 (m, 1H), 5.58 (s, 3H), 4.70 (s, 2H), 3.97 (s, 3H), 3.81 (s, 3H), 3.50 (td, J = 6.9, 5.6 Hz, 2H), 2.54 (s, 3H), 1.54 - 1.43 (m, 2H), 1.31 - 1.22 (m, 2H), 0.94 - 0.88 (m, 3H)

[0168] Step 10. Methyl (7-(butylamino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (86 mg, 0.201 mmol) was dissolved in THF (1004 μl) at RT. SOCl2 (73.2 μl, 1.004 mmol) was added. The reaction mixture was stirred at RT for 1 hour and concentrated to give methyl (7-(butylamino)-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (57.1 mg). LC / MS(M+H) 447.4; LC RT = 0.89 min (Procedure E)

[0169] Step 11. Methyl (7-(butylamino)-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (28 mg, 0.063 mmol) and 1-(2,6-diazaspiro[3.3]heptan-2-yl)ethan-1-one hydrochloride (33.2 mg, 0.188 mmol) were dissolved in acetonitrile (626 μl) at RT. DIPEA (32.8 μl, 0.188 mmol) was added and the reaction mixture was heated at 50 °C for 16 h. The reaction mixture was concentrated and the residue was redissolved in dioxane (0.7 mL), and NaOH solution (10 M, 125 μl, 1.253 mmol) was added. The reaction mixture was heated at 80 °C for 3 h, cooled to RT, and concentrated. The residue was dissolved in DMF:H2O:AcOH (6:2:2, 1 mL), filtered through a PTFE frit, and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 10% B for 0 min, 10 - 50% B over 25 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 244 (4.7 mg) containing 0.8 eq of AcOH. Example 21 - Compound 269 [Chemical Structure]

[0170] Step 1. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.65 g, 4.95 mmol) was dissolved in CHCl3 (49.5 ml) and cooled to 0 °C. NBS (0.925 g, 5.20 mmol) was added in one portion. After 15 minutes, the reaction was diluted with CHCl3 and vigorously stirred with 10% aqueous Na2S2O3 for 10 minutes. The organic phase was separated, washed with H2O, dried over MgSO4 and concentrated. The crude product was purified by column chromatography (80 g SiO2, eluting with a 0 to 50% EtOAc - hexane gradient) to give ethyl 4-amino-3-bromo-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.32 g) as a white solid. LC / MS (M + H) 412.2 / 414.2; LC RT = 1.02 min (Method A) 1 H NMR (400 MHz, DMSO-d6) δ 7.61 - 7.41 (m, 2H), 6.55 (d, J = 8.3 Hz, 1H), 5.56 (s, 2H), 5.02 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.90 (s, 3H), 3.85 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H)

[0171] Step 2. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (741.2 mg, yield 67.1%), K2CO3 (1.098 g, 7.94 mmol) and 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (3.5 M in THF) (1.816 ml, 6.36 mmol) were suspended in dioxane (26.5 ml):water (5.30 ml) (5:1). The reaction mixture was bubbled with a stream of N2 for 5 minutes, then PdCl2(dppf)-CH2Cl2 adduct (0.052 g, 0.064 mmol) was added and bubbling was continued for an additional 4 minutes, after which the reaction vessel was sealed and heated to 90 °C. After 3 hours, an additional amount of 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (TMB, 3.5 M in THF; 0.908 ml, 3.18 mmol) and PdCl2(dppf)-CH2Cl2 adduct (0.052 g, 0.064 mmol) were added and the reaction mixture was stirred at 100 °C for 16 hours. The cooled reaction mixture was diluted with 100 mL of EtOAc, filtered through CELITE™ and washed further with EtOAc. The crude product was concentrated on 4 g of CELITE™. Column chromatography (80 g SiO2, eluting with a 0 to 30% EtOAc-CH2Cl2 gradient) gave the expected product, ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (741 mg) as a cream solid. LC / MS (M+H) 348.2; LC RT = 0.89 min (method A) 1 H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 1.5 Hz, 1H), 7.46 (dd, J = 7.9, 1.5 Hz, 1H), 6.40 (d, J = 7.8 Hz, 1H), 5.48 (s, 2H), 4.94 - 4.86 (m, 2H), 4.14 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 2.10 (s, 3H), 1.15 - 1.08 (m, 3H)

[0172] Step 3. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (742 mg, 2.136 mmol) was suspended in MeOH (10.800 mL) and gently heated with vigorous stirring to solubilize the substance. 1,3-Bis-(methoxycarbonyl)-2-methyl-2-thioureido (661 mg, 3.20 mmol) was then added, followed by AcOH (0.611 mL, 10.68 mmol). The reaction mixture was stirred at RT for 16 h. An additional amount of AcOH was added (0.049 mL, 0.854 mmol) and the reaction was stirred at RT for a further 72 h, after which NaOMe (in 25% wt MeOH) was added (5.69 mL, 25.6 mmol). After stirring for 3 h, the reaction mixture was acidified again with AcOH until acidic. The product was collected by filtration, air dried for 10 min and dried completely in a laboratory dryer to give methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (722.0 mg) as a cream solid. LC / MS (M+H) 402.3; LC RT = 0.86 min (Method A) 1 H NMR (400 MHz, DMSO-d6) δ 11.58 - 11.17 (m, 2H), 7.51 (d, J = 1.4 Hz, 1H), 7.49 - 7.42 (m, 1H), 6.67 (d, J = 7.9 Hz, 1H), 5.67 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.71 (s, 3H), 2.31 (s, 3H)

[0173] Step 4. Methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (300 mg, 0.747 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine, HCl (381 mg, 0.972 mmol) and BOP (496 mg, 1.121 mmol) were suspended in DMF (3737 μl) at RT. After addition of DBU (4 eq) (451 μl, 2.99 mmol), the reaction mixture became homogeneous and this was heated to 40 °C. After 15 min, an additional amount of DBU (2 eq) (225 μl, 1.495 mmol) was added and the reaction was stirred at 40 °C for 16 h. (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine, HCl (381 mg, 0.972 mmol), BOP (496 mg, 1.121 mmol) and DBU (4 eq) (451 μl, 2.99 mmol) were added and the reaction was stirred for a further 48 h. The reaction mixture was diluted with EtOAc and washed with H2O (2x) and 10% LiCl solution (1x). The organic phase was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (24 g SiO2, 0 to 80% EtOAc-CH2Cl2 gradient elution) and then further purified (12 g SiO2, 0 to 70% EtOAc-hexane gradient elution) to give methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (270.6 mg). LC / MS (M+H) 739.7; LC RT = 1.04 min (Method A)

[0174] Step 5. To a solution of methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (500 mg, 0.677 mmol) in dry THF (10 mL) and MeOH (3 mL) was added LiBH4 (1.692 mL, 3.38 mmol) under a nitrogen atmosphere. The reaction mixture was heated at 45 °C for 24 h. The reaction mixture was partitioned between aqueous NH4Cl and EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by CombiFlash chromatography (60 - 120 silica gel; 10 - 100% ethyl acetate in petroleum ether) to give (S)-(4-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxyphenyl)methanol (150 mg, 0.230 mmol, 34.0% yield) as a pale yellow solid. LC / MS (M+H) 653.4

[0175] Step 6: To a stirred solution of (S)-(4-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxyphenyl)methanol (150 mg, 0.230 mmol) in THF (0.5 mL) was added SOCl2 (0.1 ml, 1.370 mmol). The reaction mixture was stirred at 0 °C for 1 h under a nitrogen atmosphere and then concentrated under reduced pressure to give (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine as a pale yellow solid, which was carried on to the next step without further purification. LC / MS (M+H) 671.4

[0176] Step 7: To a stirred solution of (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (150 mg, 0.223 mmol) in DMF (2 mL) was added 2-methyl-2-azaspiro[3.3]heptan-6-amine, HCl (72.7 mg, 0.447 mmol) and K2CO3 (61.8 mg, 0.447 mmol). The reaction mixture was stirred at 50 °C for 3 hours and then filtered. The filtrate was concentrated under reduced pressure to give (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(2-methoxy-4-(((2-methyl-2-azaspiro[3.3]heptan-6-yl)amino)methyl)benzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine as a pale brownish oil, which was carried on to the next step without further purification. LC / MS (M+H) 761.5

[0177] Step 8: HCl (0.3 mL, 9.87 mmol) was added to a stirred solution of (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(2-methoxy-4-(((2-methyl-2-azaspiro[3.3]heptan-6-yl)amino)methyl)benzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (150 mg, 0.197 mmol) in MeOH (3 mL). The reaction mixture was stirred at 0 °C to RT for 2 h under a nitrogen atmosphere and then concentrated under reduced pressure. The residue was purified by preparative HPLC using the following conditions (column: Ascentis Express C18 (50 x 2.1 mm), 2.7 μm; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; temperature: 50 °C; gradient: 0 - 100% B over 3 min; flow rate: 1.1 mL / min injection 2 conditions: column: Ascentis Express C18 (50 x 2.1 mm), 2.7 μm; mobile phase A: 5:95 acetonitrile: water containing 0.1% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.1% TFA; temperature: 50 °C; gradient: 0 - 100% B over 3 min; flow rate: 1.1 mL / min), to give Compound 269 (14.6 mg, 0.027 mmol, 13.75% yield). Example 22 - Compound 245

Chemical Structure

[0178] Step 1. Methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (2 g, 9.56 mmol) and Selectfluor™ (10.16 g, 28.7 mmol) were suspended in MeCN (20 mL). Acetic acid (2 mL) was added. The reaction mixture was stirred at 70 °C for 24 h, cooled, and poured into water (100 mL). The resulting mixture was left standing in a freezer (-20 °C) for 30 min. The precipitated product was collected by filtration, washed with water (40 mL), and methyl (3-fluoro-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (1311 mg, 5.77 mmol, 60.4% yield) was obtained as a solid. LC-MS(ES,m / z):[M+H] + =228.2 1 H NMR(400MHz,DMSO-d6) δ 13.69(s,1H),11.63(s,1H),11.26(s,1H),3.76(s,3H)

[0179] Step 2. A stirred suspension of methyl (3-fluoro-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (1.311 g, 5.77 mmol) and Cs2CO3 (2.257 g, 6.93 mmol) in DMF (5 mL) was cooled in an ice bath. A solution of methyl 4-(bromomethyl)-3-methoxybenzoate (1.495 g, 5.77 mmol) in DMF (5 mL) was added. The reaction mixture was left as such and slowly warmed to RT and stirred overnight, then filtered. The filtrate was evaporated in a Genevac apparatus. The precipitate was washed with THF (100 mL) and water (100 mL), and the filtrates were collected separately. The final precipitate was combined with the dried materials from the DMF filtrate and the THF filtrate, evaporated on silica, and then purified using flash chromatography (80 g SiO2 column, in DCM, 0 to 10% MeOH) to give methyl 4-((3-fluoro-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (1.03 g, 2.54 mmol, 44.0% yield) as a solid. LC-MS(ES,m / z):[M+H] + =406.1

[0180] Step 3. 40 mL of a scintillation vial was charged with methyl 4-((3-fluoro-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (1013 mg, 2.499 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (1333 mg, 3.75 mmol), BOP (1658 mg, 3.75 mmol), DBU (1.13 mL, 7.5 mmol) and DMSO (10 mL). The reaction mixture was stirred at 60 °C for 2 h, cooled, poured into saturated NaHCO3 solution (150 mL) and extracted into EtOAc (3 x 60 mL). The combined organic phases were washed with brine (4 x 50 mL), dried (MgSO4), filtered and concentrated. The crude material was purified using flash chromatography (80 g SiO2 column, in hexane, 0 to 70% EtOAc) to give methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-fluoro-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (493 mg, 0.664 mmol, 26.6% yield) as an oil. LC-MS(ES,m / z):[M+H] + =743.3

[0181] Step 4. A solution of methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-fluoro-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (493 mg, 0.664 mmol) in THF (50 mL) was cooled in an ice bath. LiAlH4 (0.697 mL, 1.394 mmol) was added. The reaction mixture was stirred at 0 °C for 15 minutes. Rochelle salt (20 mL, 20 w / v) was added and the mixture was stirred at RT for 15 minutes, then the reaction mixture was poured into water (100 mL) and extracted into EtOAc (3 x 50 mL). The combined organic phases were washed with brine (3 x 40 mL), dried (MgSO4), filtered, and concentrated. The crude material was purified using flash chromatography (24 g SiO2 column, in hexane, 0 to 100% EtOAc) to give methyl (S)-(3-fluoro-7-((1-hydroxyhexan-3-yl)amino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (125 mg, 0.262 mmol, 39.5% yield) as a solid. LC-MS(ES,m / z):[M+H] + =477.2

[0182] Step 5. To a stirred solution of methyl (S)-(3-fluoro-7-((1-hydroxyhexan-3-yl)amino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (40 mg, 0.084 mmol) in DCM (2 mL) were added DIPEA (0.044 mL, 0.252 mmol) and methanesulfonyl chloride (0.013 mL, 0.168 mmol). The reaction mixture was stirred at RT for 30 min and then evaporated to dryness. The residue was dissolved in DMF (2 mL), and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (33.3 mg, 0.168 mmol) and DIPEA (0.044 mL, 0.252 mmol) were added. The reaction mixture was stirred at 80 °C for 2 h, cooled, quenched with saturated NaHCO3 solution (10 mL), and extracted into EtOAc (3 x 5 mL). The combined organic phases were washed with brine (4 x 5 mL), dried (MgSO4), filtered, and concentrated. The residue was dissolved in DCM (2 mL), and TFA (0.4 mL) was added. The reaction mixture was stirred overnight at RT and then evaporated to dryness. Next, the residue was dissolved in dioxane (2 mL). NaOH (0.420 mL, 2.099 mmol) was added. The reaction mixture was stirred at 80 °C for 2 h, cooled, acidified with 5N HCl, and evaporated to dryness. The crude material was dissolved in DMF (2 mL), filtered, and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 4% B for 0 min, 4 - 44% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 245 (6.1 mg, 0.012 mmol, 14.14% yield). Example 23 - Compound 246 [Chemical Structure Diagram]

[0183] Step 1. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.65 g, 4.95 mmol) was dissolved in CHCl3 (49.5 ml) and cooled to 0 °C. NBS (0.925 g, 5.20 mmol) was added to the reaction mixture in one portion. After 15 minutes, the reaction was diluted with CHCl3 and vigorously stirred with 10% aqueous Na2S2O3 for 10 minutes. The organic phase was separated, washed with H2O, dried over MgSO4 and concentrated. The crude product was purified by column chromatography (80 g SiO2, eluting with a 0 to 50% EtOAc - hexane gradient) to give ethyl 4-amino-3-bromo-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.32 g) as a white solid. LC / MS (M+H) 412.2 / 414.2; LC RT = 1.02 min (Method A) 1 H NMR (400 MHz, DMSO-d6) δ 7.61 - 7.41 (m, 2H), 6.55 (d, J = 8.3 Hz, 1H), 5.56 (s, 2H), 5.02 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.90 (s, 3H), 3.85 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H)

[0184] Step 2. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (741.2 mg, yield 67.1%), K2CO3 (1.098 g, 7.94 mmol) and 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (in 3.5 M THF) (1.816 ml, 6.36 mmol) were suspended in dioxane (26.5 ml):water (5.30 ml) (5:1). After the reaction mixture was aerated with a stream of N2 for 5 minutes, PdCl2(dppf)-CH2Cl2 adduct (0.052 g, 0.064 mmol) was added, and aeration was continued for a further 4 minutes. The reaction vessel was then sealed and heated to 90 °C. After 3 hours, an additional amount of 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (in 3.5 M THF) (0.908 ml, 3.18 mmol) and PdCl2(dppf)-CH2Cl2 adduct (0.052 g, 0.064 mmol) were added, and the reaction mixture was stirred at 100 °C for 16 hours. The cooled reaction mixture was diluted with 100 mL of EtOAc, filtered through CELITE™, and further washed with EtOAc. The crude product was concentrated on 4 g of CELITE™. Column chromatography (80 g SiO2, eluting with a 0 to 30% EtOAc-CH2Cl2 gradient) gave the expected product, ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (741 mg) as a cream-colored solid. LC / MS (M+H) 348.2; LC RT = 0.89 min (Method A) 1 1H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 1.5 Hz, 1H), 7.46 (dd, J = 7.9, 1.5 Hz, 1H), 6.40 (d, J = 7.8 Hz, 1H), 5.48 (s, 2H), 4.94 - 4.86 (m, 2H), 4.14 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 2.10 (s, 3H), 1.15 - 1.08 (m, 3H)

[0185] Step 3. Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-3-methyl-1H-pyrazole-5-carboxylate (742 mg, 2.136 mmol) was suspended in MeOH (10.800 mL) and gently heated with vigorous stirring to solubilize the substance. 1,3-Bis-(methoxycarbonyl)-2-methyl-2-thiourea (661 mg, 3.20 mmol) was then added, followed by AcOH (0.611 mL, 10.68 mmol). The reaction mixture was stirred at RT for 16 h. An additional amount of AcOH was added (0.049 mL, 0.854 mmol) and the reaction was stirred at RT for a further 72 h, after which NaOMe (in 25% wt MeOH) was added (5.69 mL, 25.6 mmol). After stirring for 3 h, the reaction mixture was acidified again with AcOH until acidic. The product was collected by filtration, air dried for 10 min and dried completely in a laboratory dryer to give methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (722.0 mg) as a cream solid. LC / MS (M+H) 402.3; LC RT = 0.86 min (Method A) 1 H NMR (400 MHz, DMSO-d6) δ 11.58 - 11.17 (m, 2H), 7.51 (d, J = 1.4 Hz, 1H), 7.49 - 7.42 (m, 1H), 6.67 (d, J = 7.9 Hz, 1H), 5.67 (s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.71 (s, 3H), 2.31 (s, 3H)

[0186] Step 4. Methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (300 mg, 0.747 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine, HCl (381 mg, 0.972 mmol) and BOP (496 mg, 1.121 mmol) were suspended in DMF (3737 μl) at RT. After addition of DBU (4 eq) (451 μl, 2.99 mmol), the reaction mixture became homogeneous and was heated to 40 °C. After 15 minutes, an additional amount of DBU (2 eq) (225 μl, 1.495 mmol) was added and the reaction was stirred at 40 °C for 16 h. (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine, HCl (381 mg, 0.972 mmol), BOP (496 mg, 1.121 mmol) and DBU (4 eq) (451 μl, 2.99 mmol) were added and the reaction was stirred for an additional 48 h. The reaction mixture was diluted with EtOAc and washed with H2O (2x) and 10% LiCl solution (1x). The organic phase was dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (24 g SiO2, 0 to 80% EtOAc-CH2Cl2 gradient elution) and then further purified (12 g SiO2, 0 to 70% EtOAc-hexane gradient elution) to give methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (270.6 mg). LC / MS (M+H) 739.7; LC RT = 1.04 min (Method A)

[0187] Step 5. Methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (270 mg, 0.365 mmol) was dissolved in THF (3654 μl) at RT. LiAlH4 (731 μl, 0.731 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at RT for 15 minutes and quenched with MeOH and Rochelle salt. EtOAc was added and the mixture was stirred for 3 hours until the layers became clear. The organic phase was removed and the aqueous layer was extracted three times with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4 and concentrated. Column chromatography (12 g SiO2, 0 to 100% EtOAc-hexane gradient elution, then 0 to 20% MeOH-CH2Cl2) gave the expected substance, methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (61.7 mg). LC / MS (M+H) 711.4; LC RT = 1.08 minutes (Method A)

[0188] Step 6. Methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (60 mg, 0.084 mmol) was dissolved in CH2Cl2 (844 μl) at RT. SOCl2 (30.8 μL, 0.422 mmol) was added and the reaction was stirred for [time not specified] hours. It was concentrated to give the expected product, methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (56.6 mg). LC / MS (M+H) 729.3; LC RT = 1.18 minutes (Method A)

[0189] Step 7. Methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (45 mg, 0.02 mmol) was dissolved in acetonitrile (620 μL) at RT. tert-Butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate, HCl (29.0 mg, 0.123 mmol), followed by DIPEA (21.55 μl, 0.123 mmol) was added. The reaction mixture was heated at 80 °C for 16 hours and concentrated. The residue was purified by column chromatography (4 g SiO2, 0 - 5% MeOH-CH2Cl2 gradient). Some by-products were passed through the column to give partially purified tert-butyl (S)-6-(4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (42 mg). LC / MS (M+H) 892.7; LC RT = 0.995 minutes (Method A)

[0190] Step 8. tert-Butyl (S)-6-(4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (42 mg, 0.047 mmol) was dissolved in CH2Cl2 (471 μl) at RT. TFA (100 μL) was added. After 2 h, the reaction was concentrated under a stream of N2 and redissolved in dioxane (470 μL). Triethylamine trihydrofluoride (16.79 μL, 0.103 mmol) was added and the reaction was heated to 70 °C. After 45 min, 10 M aqueous NaOH (61.3 μl, 0.613 mmol) was added. The reaction mixture was stirred at 70 °C for 16 h, quenched with AcOH (54 μL), concentrated under a stream of N2, diluted with DMF-H2O, filtered through a PTFE frit, and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 1% B for 0 min, 1-41% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. The fractions - collection of which was triggered by the MS signal - containing the product were combined and dried by centrifugal evaporation to give Compound 246 (2.9 mg). Example 24 - Compound 247 [Chemical formula]

[0191] Step 1. A solution of methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (510 mg, 1.32 mmol; US 2020 / 0038403 A1, Figure 2A, Compound 16) in DMSO (6.6 mL) was treated with (5-methyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (236 mg, 1.58 mmol), BOP (698 mg, 1.58 mmol) and DBU (595 μL, 3.95 mmol). The reaction was stirred at RT. After 16 h, additional amounts of (5-methyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (50 mg, 0.33 mmol), BOP (50 mg, 0.11 mmol) and DBU (200 μL, 1.33 mmol) were added and the reaction was stirred at RT for 2 h. The reaction mixture was diluted with EtOAc and washed with H2O (4x). The organic layer was absorbed onto CELITE™ and purified by column chromatography (100 g C18 gold column; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; flow rate: 60 mL / min, 20-60% gradient). The fractions containing the desired product were combined, treated with HCl (2 mL, 2 mmol in 1M H2O) and concentrated in vacuo to give methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (382 mg, 60% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 9.72-9.70 (m, 1H), 7.96-7.94 (m, 1H), 7.83-7.76 (m, 1H), 7.49 (d, J = 1.4 Hz, 1H), 7.46 (dd, J = 7.8, 1.5 Hz, 1H), 6.74 (d, J = 7.8 Hz, 1H), 5.79 (s, 2H), 4.86 (d, J = 5.8 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.60 (s, 3H), 2.54 (s, 3H) LC RT: 0.64 min LC / MS [M+H] + 483.3 (Method E)

[0192] Step 2. A solution of methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (382 mg, 0.791 mmol) in dioxane (9.0 mL) was treated with NaOH (10 M aqueous solution, 0.32 mL, 3.2 mmol) and heated to 40 °C. After 30 minutes, the temperature was raised to 60 °C. An additional amount of NaOH (10 M aqueous solution, 450 μL, 3 mmol) and MeOH (1 mL) were added to the reaction mixture over 6 hours. The reaction mixture was cooled to RT, neutralized with HOAc, and concentrated under reduced pressure. The crude product was dissolved in MeOH, filtered through a PTFE frit, and purified by preparative HPLC using the following conditions: Column: Axia C18 100 mm x 30 mm, 5 μm particles; Mobile phase A: methanol: water containing 0.1% TFA in a ratio of 10:90; Mobile phase B: methanol: water containing 0.1% TFA in a ratio of 90:10; Gradient: held at 15% B for 0 minutes, increased from 15 - 30% B over 10 minutes, then held at 30% B for 4 minutes; Flow rate: 40 mL / min; UV detection at 220 nm; Column temperature: 25 °C. The fractions containing the desired product were combined, treated with HCl (1 M in H2O, 2 mL, 2 mmol), and concentrated under reduced pressure to obtain 4-((5-amino-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid·HCl (98.9 mg, yield 28%). 11H NMR (400 MHz, DMSO-d6) δ 13.23 - 12.93 (m, 1H), 12.67 - 12.43 (m, 1H), 9.06 - 8.92 (m, 1H), 8.03 - 7.87 (m, 2H), 7.83 (s, 1H), 7.51 - 7.46 (m, 2H), 6.98 (d, J = 8.2 Hz, 1H), 5.80 (s, 2H), 4.91 (d, J = 5.7 Hz, 2H), 3.82 (s, 3H), 2.57 (s, 3H) LC RT: 0.52 min LC / MS [M+H] + 411.3 (Method E)

[0193] Step 3. A solution of 4-((5-amino-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoic acid·HCl (25 mg, 0.056 mmol) in DMF (0.6 mL) was treated with 2-methyl-2,6-diazaspiro[3.3]heptane·2HCl (20.7 mg, 0.112 mmol), DIEA (68 μL, 0.39 mmol) and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (50% solution in EtOAc, 67 μL, 0.11 mmol). The reaction was stirred at RT. After 16 h, the reaction mixture was diluted with DMF (1 mL) and H2O (0.2 mL) and filtered through a PTFE frit. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 26 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 247 as the bis TFA salt (20.5 mg, 72% yield).

[0194] Compound 248 was prepared similarly. Example 25 - Compound 254 [Chemical formula]

[0195] Step 1: A solution of methyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (700 mg, 1.95 mmol; US 2020 / 0038403 A1; Figure 7, Compound 64) in DMSO (9.7 mL) was treated with (5-methyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (379 mg, 2.53 mmol), BOP (129 mg, 2.92 mmol) and DBU (1.0 mL, 6.8 mmol). The reaction mixture was stirred at RT for 2 h, diluted with DCM and washed with H2O. The organic layer was washed with H2O (6x), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was dissolved in DCM / MeOH, adsorbed onto CELITE™ and purified by column chromatography (100 g C18 gold column; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; flow rate: 60 mL / min, 10 - 50% gradient). The purified product was dissolved in DCM and washed with saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (372 mg, 42% yield). 11H NMR (400 MHz, DMSO-d6) δ 9.69 - 9.66 (m, 1H), 7.89 (s, 1H), 7.76 (t, J = 5.8 Hz, 1H), 6.95 (s, 1H), 6.81 - 6.77 (m, 1H), 6.76 - 6.70 (m, 1H), 5.69 (s, 2H), 5.17 (t, J = 5.7 Hz, 1H), 4.89 (d, J = 5.7 Hz, 2H), 4.45 (d, J = 5.8 Hz, 2H), 3.77 (s, 3H), 3.60 (s, 3H), 2.56 (s, 3H) LC RT: 0.56 min LC / MS [M+H] + 455.3 (Method E)

[0196] Step 2: A solution of methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (372 mg, 0.818 mmol) in DCM (8.2 mL) was treated with SOCl2 (179 μL, 2.46 mmol). The reaction mixture was stirred at RT for 10 min and concentrated under reduced pressure. The residue was redissolved in DCM and concentrated under reduced pressure to give methyl (1-(4-(chloromethyl)-2-methoxybenzyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (387 mg, 100%). 1 1H NMR (400 MHz, DMSO-d6) δ 11.82 - 11.60 (m, 1H), 9.40 - 9.21 (m, 1H), 8.12 - 8.08 (m, 1H), 7.10 (s, 1H), 7.04 - 6.95 (m, 2H), 5.81 (s, 2H), 5.02 (br d, J = 5.3 Hz, 2H), 4.74 (s, 2H), 3.80 (s, 3H), 3.75 (s, 3H), 2.60 (s, 3H) LC RT: 0.70 min LC / MS [M+H] + = 473.3 (Method E)

[0197] Step 3: A solution of methyl (1-(4-(chloromethyl)-2-methoxybenzyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (45 mg, 0.095 mmol) in DMF (1.9 mL) was treated with DIEA (83 μL, 0.48 mmol) and 2-thia-6-azaspiro[3.3]heptane 2,2-dioxide·HCl (26.2 mg, 0.143 mmol). The reaction mixture was stirred at 60 °C for 6 h and concentrated under reduced pressure. The residue was dissolved in dioxane (0.7 mL), treated with NaOH (10 M aqueous solution, 76 μL, 0.76 mmol), and heated at 60 °C for 5 h. The reaction mixture was neutralized with HOAc at RT and concentrated under reduced pressure. The crude product was dissolved in DMF and H2O, filtered through a PTFE frit, and purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Gradient: Hold at 1% B for 0 min, increase to 1 - 41% B over 20 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation. The separated product was further purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; Mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; Gradient: Hold at 0% B for 0 min, increase to 0 - 40% B over 20 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 254 (11.7 mg, 16%). Example 26 - Compound 263 [Chemical formula]

[0198] Step 1. A solution of methyl 4-((5-((tert-butoxycarbonyl)amino)-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (685 mg, 1.59 mmol; US 2020 / 0038403 A1, Figure 8, compound 71) in THF (16 mL) was cooled to 0 °C and treated with LiAlH4 (2.8 mL, 2.8 mmol in 1 M THF). The reaction mixture was stirred at 0 °C for 15 minutes, quenched with H2O and Rochelle salt (saturated aqueous solution), and stirred at RT for 3 hours. The organic layer was absorbed onto CELITE™ and purified by column chromatography (24 g SiO2; elution with a 0 to 20% MeOH-DCM gradient) to give tert-butyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (460 mg, 72% yield). 1 H (400 MHz, DMSO-d6) δ 11.69 - 11.43 (m, 1H), 10.95 - 10.62 (m, 1H), 7.87 - 7.79 (m, 1H), 6.97 (s, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.59 (d, J = 7.8 Hz, 1H), 5.66 (s, 2H), 5.16 (t, J = 5.8 Hz, 1H), 4.45 (d, J = 5.8 Hz, 2H), 3.79 (s, 3H), 1.49 (s, 9H) LC RT: 0.77 min LC / MS [M+H] + = 402.2 (Method E)

[0199] Step 2. A solution of tert-butyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (460 mg, 1.15 mmol) in DMSO (5.7 mL) was treated with (5-methyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (223 mg, 1.49 mmol), BOP (760 mg, 1.72 mmol) and DBU (0.69 mL, 4.6 mmol). The reaction mixture was stirred at RT for 2 h, diluted with EtOAc and washed with H2O (2x). The organic layer was absorbed onto CELITE™ and purified by column chromatography (100 g C18 gold column; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; flow rate: 60 mL / min, 30 - 50% gradient). The purified product was dissolved in DCM and washed with saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give tert-butyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (190 mg, 33% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.24 - 9.15 (m, 1H), 7.87 (s, 1H), 7.72 (t, J = 5.8 Hz, 1H), 6.95 (s, 1H), 6.82 - 6.75 (m, 1H), 6.73 - 6.68 (m, 1H), 5.68 (s, 2H), 5.17 (t, J = 5.7 Hz, 1H), 4.87 (d, J = 5.7 Hz, 2H), 4.44 (d, J = 5.7 Hz, 2H), 3.76 (s, 3H), 2.55 (s, 3H), 1.43 (s, 9H) LC RT: 0.75 min LC / MS [M+H] + = 497.2 (Method E)

[0200] Step 3. A solution of tert-butyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (91.5 mg, 0.184 mmol) in dioxane (0.6 mL) was treated with HCl (4M in dioxane, 0.69 mL, 2.8 mmol), stirred at 40 °C for 90 minutes, and concentrated. The residue was dissolved in DCM and concentrated under reduced pressure to give (4-((5-amino-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxyphenyl)methanol (73.1 mg, yield 100%). LC RT: 0.65 min LC / MS [M+H] + = 397.1 (Method E)

[0201] Step 4. A solution of 1-(4-(chloromethyl)-2-methoxybenzyl)-N7-((5-methyl-1,2,4-oxadiazol-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (27 mg, 0.065 mmol) in DMSO (1.3 mL) was treated with DIEA (57 μL, 0.33 mmol) and 2-isopropyl-2,6-diazaspiro[3.3]heptane (14 mg, 0.098 mmol). The reaction mixture was stirred at 65 °C for 30 minutes, diluted with DMSO, filtered through a PTFE frit, and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; gradient: hold at 0% B for 0 minutes, 0 - 40% B over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 263 (13.7 mg, 35%) as the acetate.

[0202] Compound 264 was prepared similarly. Example 27 - Compound 249

Chem.

[0203] In DMF (1 mL), a mixture of compound 835 (20 mg, 0.042 mmol) and acetaldehyde (183 mg, 0.083 mmol) was treated with acetic acid (0.024 mL, 0.416 mmol) and 20 mg of 4 Å molecular sieves, followed by sodium triacetoxyborohydride (35.3 mg, 0.166 mmol). The reaction mixture was stirred at RT for 1 hour. Acetic acid (0.024 mL, 0.416 mmol) was evaporated. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 3% B for 0 min, increase to 3 - 43% B over 25 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation. The substance was further purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; gradient: hold at 0% B for 0 min, increase to 0 - 40% B over 25 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the target compound 249 were combined and dried by centrifugal evaporation.

[0204] Compounds 252 and 253 were prepared similarly. Example 28 - Compound 255

Chem.

[0205] Step 1. A solution of (4 - ((5 - amino - 7 - (butylamino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-3 - methoxyphenyl)methanol 818 (400 mg, 1.122 mmol) in THF (2 mL) was treated with SOCl2 (0.164 mL, 2.244 mmol) and stirred at RT for 1 hour. The solvent was evaporated and the crude chloride 2 was moved to the next step without further purification.

[0206] Step 2. The DMSO solution of chloride 2 was treated with amine 3 (commercially available, CAS: 236406 - 55 - 6), heated at 80 °C for 2 hours and then subjected to LCMS, which showed the completion of the reaction. The reaction mixture was treated with TFA and stirred for 1 hour. TFA was evaporated. The crude substance was further purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 4%B for 0 minutes, increase to 4 - 44%B over 20 minutes, then hold at 100%B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the target compound 255 were combined and dried by centrifugal evaporation.

[0207] The following compounds were prepared similarly: Compound 256, Compound 257, Compound 258, Compound 265, and Compound 266. Example 29 - Compound 259

Chemical Structure

[0208] A solution of compound 255 (18 mg, 0.039 mmol) in DMF (0.5 mL) was treated with K2CO3 (16.06 mg, 0.116 mmol) / 2-bromoethanol (5.49 μl, 0.077 mmol) and heated at 50 °C for 2 h. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.05% TFA; mobile phase B: acetonitrile containing 95:5 0.05% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired compound 259 were combined and dried by centrifugal evaporation.

[0209] The following compounds were prepared similarly: compound 260, compound 261, compound 262, compound 267, and compound 268. Example 30 - Compound 270

Chemical formula

[0210] Step 1. Thionyl chloride (1.235 ml, 16.94 mmol) was added dropwise to a solution of (5-bromo-3-methoxypyridin-2-yl)methanol (Sigma-Aldrich) (2.462 g, 11.29 mmol) in CH2Cl2 (113 ml) at 0 °C. The reaction mixture was stirred at RT for 1 h and concentrated under reduced pressure. The residue was mixed with CH2Cl2 and concentrated under reduced pressure (2x) to give crude 5-bromo-2-(chloromethyl)-3-methoxypyridine. This material was used without further purification. LC-MS m / z 236 / 238 [M+H] +

[0211] Step 2. Cesium carbonate (13.37 g, 41.0 mmol) was added to a suspension of methyl (7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (3.44 g, 10.26 mmol) in DMF (45.6 ml) at RT. The mixture was stirred at 0 °C for 10 min; then a solution of the crude material from Step 1 in DMF (22.80 ml) was added. The reaction mixture was stirred at 0 °C for 1 h. The cooling bath was removed and stirring was continued at RT for 20 h. The reaction mixture was added to H2O (250 mL) and the resulting mixture was allowed to stand at RT. The solid was collected by vacuum filtration and washed with H2O (3 x 15 mL), MeOH (2 x 15 mL), CH2Cl2 (15 mL), and hexane (15 mL) to give methyl (1-((5-bromo-3-methoxypyridin-2-yl)methyl)-7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (4.431 g, 81%). LC-MS m / z 535 / 537[M+H] + 1 H NMR(400MHz,DMSO-d6)δ 13.19-12.96(m,1H),8.95-8.80(m,1H),8.06(s,1H),7.71(s,1H),5.87-5.65(m,2H),3.89(s,3H),3.53(br s,3H)

[0212] Step 3. To a suspension of methyl (1-((5-bromo-3-methoxypyridin-2-yl)methyl)-7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.990 g, 1.850 mmol) in DMSO (12.33 ml) at RT was added (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine, HCl salt (0.870 g, 2.220 mmol) (US 2020 / 0038403 A1, Figure 8, compound 71a), followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (1.245 ml, 8.33 mmol) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (0.982 g, 2.220 mmol). The reaction mixture was stirred at RT for 1 hour, diluted with EtOAc (100 mL), and washed with H2O (100 mL). Liquid separation was performed, and the aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed with saturated NaCl aqueous solution (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude substance was purified by flash chromatography (80 g silica gel; linear gradient 0-100% EtOAc-hexane) to give methyl (S)-(1-((5-bromo-3-methoxypyridin-2-yl)methyl)-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (810 mg, 50%) as a yellow foam. LC-MS m / z 872 / 874[M+H] + 11H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.57 - 7.53 (m, 2H), 7.50 - 7.46 (m, 2H), 7.42 - 7.31 (m, 4H), 7.25 - 7.20 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 5.78 - 5.69 (m, 2H), 4.64 - 4.55 (m, 1H), 3.91 (s, 3H), 3.70 - 3.64 (m, 2H), 3.58 (s, 3H), 1.90 - 1.82 (m, 2H), 1.57 - 1.48 (m, 2H), 1.25 - 1.13 (m, 2H), 0.92 (s, 9H), 0.81 (t, J = 7.3 Hz, 3H)

[0213] Step 4. Zinc (0.607 g, 9.28 mmol) was added to a solution of methyl (S)-(1-((5-bromo-3-methoxypyridin-2-yl)methyl)-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.810 g, 0.928 mmol) in a mixture of MeOH (9.28 mL) and AcOH (9.28 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 minutes and filtered through CELITE® while washing with MeOH (10 mL) and EtOAc (50 mL). The filtrate was diluted with EtOAc (200 mL). While stirring, saturated aqueous NaHCO3 solution (250 mL) was slowly added to this solution (the addition rate was adjusted to control the gas generation rate). Liquid-liquid separation was performed, and the organic layer was washed with saturated aqueous NaCl solution (250 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, methyl (S)-(1-((5-bromo-3-methoxypyridin-2-yl)methyl)-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate. This substance was used without further purification. LC-MS m / z 746 / 748 [M+H] +

[0214] Step 5. A solution of Compound 5 (500 mg, 0.670 mmol), Compound 6 (304 mg, 0.870 mmol, CAS 2240187-78-2), and K2CO3 (370 mg, 2.68 mmol) in DMF (2 mL) was purged with nitrogen gas for 2 minutes. PdCl2(dppf)-CH2Cl2 adduct (54.7 mg, 0.067 mmol) was added, and the reaction mixture was purged with N2 again for 1 minute. The reaction flask was sealed and heated at 70 °C for 5 hours. It was purified by eluting with 0 - 50% MeOH / DCM on a 50 g silica gel column to obtain 476 mg of Compound 7. LC / MS [M+H] + =889.5 1 1H NMR (400 MHz, chloroform-d) δ 8.02 (s, 5H), 7.92 (d, J = 19.1 Hz, 1H), 7.63 - 7.50 (m, 3H), 7.37 - 7.23 (m, 2H), 7.22 - 7.14 (m, 2H), 7.04 (s, 1H), 6.62 (d, J = 2.6 Hz, 1H), 5.64 (dd, J = 14.7, 1.4 Hz, 1H), 5.39 (d, J = 14.9 Hz, 1H), 4.63 (s, 1H), 3.95 (d, J = 2.0 Hz, 2H), 3.82 (d, J = 7.5 Hz, 3H), 3.57 (s, 1H), 3.31 - 3.21 (m, 1H), 2.96 (s, 7H), 2.88 (s, 7H), 2.49 (s, 1H), 1.95 (d, J = 17.4 Hz, 1H), 1.47 (s, 5H), 1.52 - 1.36 (m, 2H), 1.26 (d, J = 13.9 Hz, 5H), 1.02 (s, 3H), 1.04 - 0.90 (m, 2H)

[0215] Step 6-7. Solid compound 7 (476 mg, 0.535 mmol) was treated with HCl in dioxane (1.338 mL, 5.35 mmol) with stirring at RT for 2 h and then subjected to LC / MS, which indicated completion of the reaction. HCl was evaporated using a V-10 evaporator. The crude product 8 was dissolved in 1 mL of dioxane and heated with aqueous NaOH solution (1.071 mL, 10.71 mmol) for 2 h and then subjected to LC / MS, which indicated completion of the reaction. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 3% B for 0 min, 3 - 43% over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. Fractions containing compound 272 ((3S)-3-({5-amino-1-[(5-{7-azaspiro[3.5]non-1-en-2-yl}-3-methoxypyridin-2-yl)methyl]-1H-pyrazolo[4,3-d]pyrimidin-7-yl}amino)hexan-1-ol) were combined and dried by centrifugal evaporation.

[0216] Step 8. A solution of compound 272 (40 mg, 0.081 mmol) and tetrahydro-4H-pyran-4-one (37.5 μl, 0.406 mmol) in DMA (1 mL) was treated with acetic acid (46.5 μL, 0.812 mmol), followed by 50 mg of granular 4 Å molecular sieves and sodium triacetoxyborohydride (86 mg, 0.406 mmol). The reaction mixture was stirred overnight at RT and syringe filtered. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 7% B for 0 min, 7 - 47% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 273 ((3S)-3-{[5-amino-1-({3-methoxy-5-[7-(oxan-4-yl)-7-azaspiro[3.5]non-1-en-2-yl]pyridin-2-yl}methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl]amino}hexan-1-ol).

[0217] Step 9. A solution of compound 273 (18 mg, 0.026 mmol) in MeOH (1 mL) and Pd / C (2.73 mg, 0.026 mmol) were purged with hydrogen gas for 1 minute. The reaction mixture was heated at 60 °C under an atmosphere of a hydrogen balloon for 2 hours. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 7% B for 0 min, 7 - 47% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 27.

[0218] The following compounds were prepared similarly: Compound 274, Compound 275, and Compound 278. Example 31 - Compound 271

Chemical formula

[0219] Step 1. A solution of methyl (S)-(1-((5-bromo-3-methoxypyridin-2-yl)methyl)-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (552 mg, 0.739 mmol), tert-butyl 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-azaspiro[3.5]non-6-ene-2-carboxylate 1 (336 mg, 0.961 mmol; CAS 235276-13-4), and K2CO3 (409 mg, 2.96 mmol) in DMF (5 mL) was purged with N2 for 2 minutes. PdCl2(dppf)-CH2Cl2 adduct (60.4 mg, 0.074 mmol) was added and the mixture was purged with N2 again for 1 minute. The reaction vessel was sealed and heated at 70 °C for 5 hours. Purification was performed by eluting with 0 - 50% MeOH / DCM on a 50 g silica gel column to obtain 477 mg of Compound 2. LC / MS [M+H] + =889.5 11H NMR (400 MHz, chloroform-d) δ 8.70 (d, J = 8.1 Hz, 1H), 8.04 - 7.95 (m, 1H), 7.87 (s, 1H), 7.65 - 7.58 (m, 2H), 7.58 - 7.51 (m, 2H), 7.40 - 7.31 (m, 1H), 7.33 - 7.24 (m, 3H), 7.21 - 7.12 (m, 3H), 7.08 (s, 1H), 6.02 (s, 1H), 5.63 (d, J = 14.9 Hz, 1H), 5.40 (d, J = 15.0 Hz, 1H), 4.58 (s, 1H), 3.94 (s, 3H), 3.85 - 3.74 (m, 4H), 3.73 - 3.64 (m, 2H), 3.62 (d, J = 8.3 Hz, 2H), 3.49 (s, 2H), 2.95 (s, 1H), 2.88 (s, 1H), 2.41 (d, J = 4.3 Hz, 4H), 2.02 (dd, J = 13.0, 6.2 Hz, 0H), 1.95 (s, 1H), 1.91 (d, J = 5.7 Hz, 1H), 1.45 (s, 6H), 1.45 - 1.36 (m, 1H), 1.24 (s, 6H), 1.04 (d, J = 8.8 Hz, 0H), 1.03 (s, 6H), 1.02 (s, 1H), 0.94 (t, J = 7.3 Hz, 3H)

[0220] Step 2. Compound 2 (90 mg, 0.101 mmol) was treated with TFA (0.078 mL, 1.012 mmol). The reaction mixture was stirred at RT for 30 minutes. TFA was evaporated in a V - 10 evaporator. The residue was dissolved in DMA (0.5 mL) and treated with tetrahydro - 4H - pyran - 4 - one (0.028 mL, 0.506 mmol), acetic acid (0.029 mL, 0.506 mmol), 50 mg of 4 Å molecular sieves, and finally sodium triacetoxyborohydride (107 mg, 0.506 mmol). After stirring at RT for 1 hour, the reaction mixture was treated with triethylamine trihydrofluoride (0.165 mL, 1.012 mmol) and stirred at RT for 2 hours. The reaction mixture was directly purified by 50 g reverse - phase ISCO, eluting with 0 - 50% MeCN / water (0.05% TFA) to afford Compound 3 as a white solid. LC / MS [M + H] + = 635.3

[0221] Step 3, Part 1. A solution of the compound (58 mg, 0.091 mmol) in DMSO (0.5 mL) was treated with NaOH (0.091 mL, 0.914 mmol) and heated at 80 °C for 2 hours to obtain the decarbamoylated compound 3. LC / MS [M+H] + =577.3

[0222] Step 3, Part 2. A solution of the decarbamoylated compound 3 (12 mg, 0.021 mmol) in MeOH (1 mL) containing Pd-C (2.214 mg, 0.021 mmol) was bubbled with H2 for 1 minute. The reaction mixture was heated at 60 °C for 2 hours under a hydrogen balloon atmosphere. The crude product was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 9% B for 0 minutes, increase from 9 - 49% B over 20 minutes, then hold at 100% B for 0 minutes; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the target product were combined and dried by centrifugal evaporation to obtain 4.7 mg of compound 271.

[0223] Compound 277 was prepared similarly. Example 32 - Compound 250

Chemical Structure

[0224] Step 1. A solution of benzyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate 1 (CAS#1363383-32-7; 3 g, 12.13 mmol) in DCM (20 mL) was treated with triethylamine (2.029 mL, 14.56 mmol), DMAP (0.296 g, 2.426 mmol) and tosyl-Cl (2.54 g, 13.34 mmol) at 0 °C. The reaction was allowed to proceed for 2 hours. The reaction was quenched with 50 mL of water, washed with 50 mL of 1M aqueous HCl, brine (50 mL), dried over Na2SO4, filtered, and concentrated to give the crude tosylated intermediate as a yellowish residue. This was dissolved in DMSO (20 mL) and treated with sodium iodide (5.46 g, 36.4 mmol). After heating at 120 °C for 2 hours, the reaction mixture was dissolved in 50 mL of EtOAc, washed with saturated aqueous Na2S2O3 (50 mL), water (50 mL), brine (50 mL), and dried over Na2SO4. Filtered and concentrated, and purified by elution with 0 - 50% EtOAc / hexane on an 80 g silica gel column to give compound 2 as a white solid. LC / MS [M+H] + =357.9 1 H NMR (400 MHz, DMSO-d6) δ 7.42 - 7.27 (m, 5H), 5.01 (s, 2H), 4.43 (p, J = 7.9 Hz, 1H), 3.96 (s, 4H), 2.92 (ddd, J = 10.4, 7.5, 3.1 Hz, 2H), 2.74 - 2.61 (m, 2H)

[0225] Step 2. A 4 mL THF solution of compound 2 (1649 mg, 4.62 mmol) was added to Rieke zinc in THF (12.08 mL, 9.23 mmol) in a round bottom flask dried in a dryer under N2. The temperature of the flask rose, indicating the formation of zinc reagent 3. The reaction mixture was stirred at RT for 1 hour and kept under N2 for future use.

[0226] Step 3.5-Bromo-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-methoxypyridine (1.4 g, 4.21 mmol), 1,1'-bis(diphenylphosphino)ferrocenedichloropalladium(II) dichloromethane complex (0.308 g, 0.421 mmol), and copper(I) iodide (0.160 g, 0.843 mmol) in DMF (10 mL) were bubbled with N2 for 1 minute. (2-((Benzyloxy)carbonyl)-2-azaspiro[3.3]heptan-6-yl)zinc(II) iodide 3 (17.49 mL, 5.06 mmol) was added. The reaction mixture was heated at 70 °C for hours. The solution was treated with triethylamine trihydrofluoride (1.372 mL, 8.43 mmol) and stirred overnight. LCMS indicated the formation of compound 5 (167 mg, 0.453 mmol, 10.76% yield). The reaction mixture was directly purified on a 150 g reverse-phase C-18 column, eluting with 0-50% MeCN / water (0.05% TFA) to collect the desired fraction, and compound 5 was obtained as a pale yellow solid. LC / MS[M+H] + =369.2 1 H NMR(400MHz,DMSO-d6)δ 8.08(s,1H),7.81(s,1H),7.43-7.28(m,5H),5.04(s,2H),4.71(s,2H),4.11(s,2H),3.66-3.56(m,3H),3.61-3.48(m,1H),2.58(ddt,J = 10.6,8.4,2.5 Hz,2H),2.41(td,J = 9.5,2.9 Hz,2H),1.84-1.70(m,3H)

[0227] Step 4-5. A solution of compound 5 (167 mg, 0.453 mmol) in THF (1 mL) was treated with SOCl2 (0.066 mL, 0.907 mmol) and stirred at RT for 30 minutes. The solvent was evaporated using a V-10 evaporator. The crude product 6 in 1 mL of DMF was added to a 1 mL DMF solution of methyl (7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate 7 (152 mg, 0.453 mmol) and Cs2CO3 (295 mg, 0.907 mmol). After heating at 60 °C for 2 hours, the reaction mixture was filtered and purified directly by elution with 0-50% MeCN / water (0.05% TFA) on a 50 g reverse-phase C-18 column. The desired fraction was lyophilized to give compound 8 as a pale yellow solid. LC / MS[M+H] + =886.1

[0228] Step 6-7. A solution of compound 8 (140 mg, 0.204 mmol) and (S)-3-aminohexan-1-ol 9 (47.9 mg, 0.408 mmol) in DMSO (1 mL) was treated with DBU (0.092 mL, 0.613 mmol), followed by BOP (135 mg, 0.306 mmol). After heating at 40 °C for 1 hour, it was subjected to LCMS which indicated completion of the reaction, and intermediate 10 was obtained. The reaction mixture was treated with NaOH (0.204 mL, 2.042 mmol) and heated at 80 °C for 2 hours. The reaction mixture was purified directly by elution with 0-50% MeCN / water (0.05% TFA) on a 50 g C-18 reverse-phase column. The desired fraction was lyophilized to give compound 11 as a pale yellow solid. LC / MS[M+H] + =593.1

[0229] Step 8. A solution of compound 11 (30 mg, 0.051 mmol) in MeOH (1 mL) was purged with H2 (10.21 mg, 5.06 mmol) for 1 minute together with Pd-C (5.39 mg, 0.051 mmol). The reaction mixture was heated at 50 °C under a H2 balloon for 2 hours. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 6% B for 0 min, 6 - 46% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation to give 7.6 mg of compound 12. LC / MS [M+H] + =466.9 1 H NMR (500 MHz, DMSO-d6) δ 7.93 (s, 2H), 7.90 (d, J = 1.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 5.96 (s, 1H), 5.71 - 5.59 (m, 3H), 4.43 (s, 2H), 4.11 (s, 1H), 3.90 (d, J = 6.9 Hz, 3H), 2.68 (d, J = 9.9 Hz, 0H), 2.57 (d, J = 22.7 Hz, 2H), 2.35 (d, J = 14.2 Hz, 2H), 2.24 (s, 1H), 1.92 (s, 1H), 1.78 (d, J = 6.3 Hz, 2H), 1.77 - 1.69 (m, 2H), 1.58 (s, 4H), 1.29 (s, 2H), 0.91 - 0.84 (m, 3H)

[0230] Step 9. A solution of compound 12 (30 mg, 0.064 mmol) and tetrahydro-4H-pyran-4-one (0.012 mL, 0.129 mmol) in DMF (0.5 mL) was treated with 2 drops of acetic acid and 50 mg of 4 Å molecular sieves and sodium triacetoxyborohydride (54.5 mg, 0.257 mmol). After stirring at RT for 1 hour, the crude material was purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: Hold at 13% B for 0 min, 13 - 53% B over 20 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation. The material was further purified by preparative LC / MS using the following conditions: Column: XBridge Phenyl, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: Hold at 9% B for 0 min, 9 - 49% B over 20 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give 1.9 mg of compound 250.

[0231] Compound 251 was prepared similarly. Example 33 - Starting Materials and Intermediates

[0232] The following chart shows a scheme for creating compounds that may be useful as starting materials or intermediates for the preparation of the TLR7 agonists disclosed herein. The scheme may be applied to the creation of other similar compounds that may be used as starting materials or intermediates. The reagents used are well known in the art and in many cases their use is shown in the foregoing examples. Chart 1 [Chemistry] Chart 2 [Chemistry] Chart 3 [Chemistry]

[0233] Biological Activity The biological activity of the compounds disclosed herein as TLR7 agonists may be quantified by the following procedure.

[0234] Human TLR7 Agonist Activity Assay This procedure describes a method for quantifying the human TLR7 (hTLR7) agonist activity of the compounds disclosed herein.

[0235] Modified human embryonic kidney HEK-Blue™ TLR cells (Invivogen) having a human TLR7 secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in non-selective medium (DMEM high glucose (Invitrogen) supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to each well of a 384-well tissue culture plate (15,000 cells per well) and incubated at 37 °C, 5% CO2 for 16 - 18 hours. Compounds (100 nl) were added to the wells containing HEK-Blue™ TLR cells and the treated cells were incubated at 37 °C, 5% CO2. Eighteen hours after treatment, 10 microliters of freshly prepared Quanti-Blue™ reagent (Invivogen) was added to each well and incubated for 30 minutes (37 °C, 5% CO2), and SEAP levels were measured using an Envision plate reader (OD = 620 nm). The half maximal effective concentration value (EC 50 ; the concentration of the compound that elicits a response midway between the assay reference value and the maximum value) was calculated.

[0236] Induction of Type I Interferon Gene (MX-1) and CD69 in Human Blood The induction of type I interferon (IFN) MX-1 gene and B cell activation marker CD69 are downstream events that occur upon activation of the TLR7 pathway. The following is a human whole blood assay that measures their induction in response to TLR7 agonists.

[0237] Heparinized human whole blood was collected from human patients and treated with a TLR7 agonist test compound at 1 mM. The blood was diluted with RPMI 1640 medium and predotted at 10 nL per well using an Echo to a final concentration of 1 μM (10 nL in 10 μL of blood). After mixing on a shaker for 30 seconds, the plate was covered and placed in a 37°C chamber overnight = 17 hours. Fixation / Lysis buffer was prepared (5x in H20 → 1x, warmed at 37°C; Cat# BD 558049), and the Perm buffer was maintained (on ice) for later use.

[0238] Surface antibodies were prepared for surface marker staining (CD69): 0.045 μl hCD14-FITC (ThermoFisher Cat # MHCD1401) + 0.6 μl hCD19-ef450 (ThermoFisher Cat # 48-0198-42) + 1.5 μl hCD69-PE (cat# BD555531) + 0.855 μl FACS buffer. Added at 3 μl / well, centrifuged at 1000 rpm for 1 minute, mixed on a shaker for 30 seconds, and placed on ice for 30 minutes. After 30 minutes, the stimulation was stopped with 70 μL of pre-warmed 1x Fixation / Lysis buffer and resuspended using a Felix mate (15 times, changing the tip for each plate) and incubated at 37°C for 10 minutes.

[0239] Centrifuge at 2000 rpm for 5 minutes, aspirate with an HCS plate washer, mix on a shaker for 30 seconds, then wash with 70 μL of dPBS and pellet twice (2000 rpm, 5 minutes), wash with 50 μL of FACS buffer and pellet once (2000 rpm, 5 minutes). Mix on a shaker for 30 seconds. For intracellular marker staining (MX-1): Add 50 μl of BD Perm buffer III, mix on a shaker for 30 seconds. Incubate on ice for 30 minutes (in the dark). Wash twice with 50 μL of FACS buffer (centrifuge at 2300 rpm for 5 minutes after permeabilization), then mix on a shaker for 30 seconds. Resuspend in 20 μL of FACS buffer containing MX1 antibody ((4812)-Alexa 647: Novus Biologicals #NBP2-43704AF647) (20 μl FACS buffer + 0.8 ul hIgG + 0.04 μl MX-1). Centrifuge at 1000 rpm for 1 minute, mix on a shaker for 30 seconds, incubate the sample at RT in the dark for 45 minutes, then wash with 2x FACS buffer (centrifuge at 2300 rpm for 5 minutes after permeabilization). Resuspend in 20 μl of FACS buffer (total 35 μL per well), cover with foil, place at 4°C, and read the next day. Read the plate with iQuePlus. Load the results into the toolset and create an IC50 curve with CurveMaster. 100% on the y-axis is set to 1 μM of resiquimod.

[0240] Induction of TNF-alpha and Type I IFN Response Genes in Mouse Blood The induction of TNF-alpha and type I IFN response genes are downstream events that occur upon activation of the TLR7 pathway. The following is an assay to measure their induction in mouse whole blood in response to a TLR7 agonist.

[0241] Heparin-treated mouse whole blood was diluted in RPMI 1640 medium containing Pen-Strep at a ratio of 5:4 (50 μL of whole blood and 40 μL of medium). 90 μL of the diluted blood was transferred to the wells of a Falcon flat-bottom 96-well tissue culture plate, and the plate was incubated at 4 °C for 1 hour. The test compound in 100% DMSO stock was diluted 20-fold in the same medium for the concentration-response assay and then 10 μL of the diluted test compound was added to the wells to give a final DMSO concentration of 0.5%. To the control wells, 10 μL of medium containing 5% DMSO was added. The plate was then incubated at 37 °C in a 5% CO2 incubator for 17 hours. After incubation, 100 μL of medium was added to each well. The plate was centrifuged and 130 μL of the supernatant was removed and used for the assay of TNFα production by ELISA (from Invitrogen, catalog number 88-7324 Thermo-Fisher Scientific). 70 μL of mRNA catcher lysis buffer (1x) containing DTT, derived from the Invitrogen mRNA Catcher Plus kit (Cat# K1570-02), was added to the remaining 70 μL sample in the well and mixed 5 times by pipetting. The plate was then shaken at RT for 5 - 10 minutes, followed by the addition of 2 μL of proteinase K (20 mg / mL) to each well. The plate was then shaken at RT for 15 - 20 minutes. The plate was then stored at -80 °C until further processing.

[0242] The frozen samples were thawed, and mRNA was extracted using the Invitrogen mRNA Catcher Plus Kit (Cat# K1570-02) according to the manufacturer's instructions. Using half of the mRNA obtained from the RNA extraction, cDNA was synthesized in a 20 μL reverse transcriptase reaction using the Invitrogen SuperScript IV VILO Master Mix (Cat# 11756500). TaqMan® real-time PCR was performed using the ThermoFisher (Applied Biosystems) QuantStudio Real-Time PCR system. All real-time PCR reactions were repeated twice using commercially available pre-designed TaqMan assays for mouse IFIT1, IFIT3, MX1, and PPIA gene expression and TaqMan Master Mix. PPIA was used as a housekeeping gene. The recommendations from the manufacturer were followed. All raw data (Ct) were normalized by the mean housekeeping gene (Ct), and then the comparative Ct (ΔΔCt) method was utilized to quantify the relative gene expression level (RQ) for experimental analysis.

[0243] Definition "Aliphatic" means a straight or branched chain saturated or unsaturated non-aromatic hydrocarbon moiety having a specific number of carbon atoms (e.g., "C3 aliphatic", "C 1-5 aliphatic", "C1-C5 aliphatic", or "C1 to C5 aliphatic". The latter three expressions are synonymous with an aliphatic moiety having 1 to 5 carbon atoms), and when the number of carbon atoms is not specifically identified, it is 1 to 4 carbon atoms (2 to 4 carbons in the case of an unsaturated aliphatic moiety). A similar understanding applies to the number of carbons in other types, i.e., C 2-4 alkene, C4-C7 cycloaliphatic, etc. Similarly, terms such as "(CH2) 1-3 " should be understood as an abbreviated representation where the subscript is 1, 2, or 3, and thus such terms would represent CH2, CH2CH2, and CH2CH2CH2.

[0244] "Alkyl" means a saturated aliphatic moiety following the same convention for specifying the number of applicable carbon atoms. Examples of C1-C4 alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl, and the like. "Alkandiyl" (sometimes also called "alkylene") means the divalent counterpart of an alkyl group, for example,

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[0245] "Alkenyl" means an aliphatic moiety having at least one carbon-carbon double bond following the same convention for specifying the number of applicable carbon atoms. Examples of C2-C4 alkenyl moieties include, but are not limited to, ethenyl (vinyl), 2-propenyl (allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E-(or Z)-2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl), and the like.

[0246] "Alkynyl" means an aliphatic moiety having at least one carbon-carbon triple bond following the same convention for specifying the number of applicable carbon atoms. Examples of C2-C4 alkynyl groups include, but are not limited to, ethynyl (acetylenyl), propargyl (prop-2-ynyl), 1-propynyl, but-2-ynyl, and the like.

[0247] "Cycloaliphatic" means a saturated or unsaturated non-aromatic hydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to 8 (preferably from 3 to 6) carbon atoms. "Cycloalkyl" means a cycloaliphatic moiety in which each ring is saturated. "Cycloalkenyl" means a cycloaliphatic moiety in which at least one ring has at least one carbon-carbon double bond. "Cycloalkynyl" means a cycloaliphatic moiety in which at least one ring has at least one carbon-carbon triple bond. Examples of cycloaliphatic moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl. Preferred cycloaliphatic moieties are cycloalkyl moieties, especially cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. "Cycloalkanediyl" (sometimes also called "cycloalkylene") means the divalent counterpart of a cycloalkyl group. Similarly, "bicycloalkanediyl" (or "bicycloalkylene") and "spiroalkanediyl" (or "spiroalkylene") refer to the divalent counterparts of bicycloalkyl and spiroalkyl (or "spirocycloalkyl") groups. Examples of

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[0248] "Heterocycloaliphatic" means a cycloaliphatic moiety in which, in at least one of its rings, up to 3 (preferably 1 to 2) carbons are replaced by heteroatoms independently selected from N, O or S, where N and S may be optionally oxidized and N may be optionally quaternized. Preferred cycloaliphatic moieties consist of one ring of 5 to 6 members in size. Similarly, "heterocycloalkyl", "heterocycloalkenyl", and "heterocycloalkynyl" each mean a cycloalkyl, cycloalkenyl, or cycloalkynyl moiety in which at least one of its rings is so modified. Representative heterocycloaliphatic moieties include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl, tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl, thietanyl, and the like. "Heterocycloalkylene" means the divalent counterpart of a heterocycloalkyl group.

[0249] "Alkoxy", "aryloxy", "alkylthio", and "arylthio" mean -O(alkyl), -O(aryl), -S(alkyl), and -S(aryl), respectively. Examples are methoxy, phenoxy, methylthio, and phenylthio, respectively.

[0250] "Halogen" or "halo" means fluorine, chlorine, bromine or iodine, unless a narrower meaning is indicated.

[0251] "Aryl" means a hydrocarbon moiety having a mono-, bi- or tricyclic ring system (preferably monocyclic) in which each ring has from 3 to 7 carbon atoms and at least one ring is aromatic. The rings in the ring system may be fused to each other (such as naphthyl), bonded to each other (such as biphenyl), or fused or bonded to a non-aromatic ring (such as indanyl or cyclohexylphenyl). Further examples of the aryl moiety include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracenyl, and acenaphthyl. "Arylene" means the divalent counterpart of an aryl group, such as 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

[0252] "Heteroaryl" means a moiety having a mono-, bi- or tricyclic ring system (preferably a 5- to 7-membered monocyclic) in which each ring has from 3 to 7 carbon atoms and at least one ring is an aromatic ring containing from 1 to 4 heteroatoms independently selected from N, O, or S, where N and S may be optionally oxidized and N may be optionally quaternized. Such an aromatic ring containing at least one heteroatom may be fused to other types of rings (such as benzofuranyl or tetrahydroisoquinolyl) or directly bonded to other types of rings (such as phenylpyridyl or 2-cyclopentylpyridyl). Further examples of the heteroaryl moiety include, but are not limited to, pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, quinoxalinyl, naphthyridinyl, benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl, phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl, carbazolyl, dibenzothiophenyl, acridinyl, and the like. "Heteroarylene" means the divalent counterpart of a heteroaryl group.

[0253] For example, when using "unsubstituted or substituted" or "optionally substituted", that is, expressing as "unsubstituted or substituted C1-C5 alkyl" or "optionally substituted heteroaryl", etc., it indicates that the moiety may be substituted. Such a moiety may have one or more independently selected substituents, preferably 1 to 5 in number, more preferably 1 to 2 in number. The substituents and substitution patterns may be selected by those skilled in the art considering the moiety to which the substituents are attached, and provide compounds that are chemically stable and can be synthesized by techniques known in the art and the methods described herein. When a moiety is specified as "unsubstituted or substituted" or "optionally substituted", in a preferred embodiment, such a moiety is unsubstituted.

[0254] "Arylalkyl", "(heterocycloaliphatic)alkyl", "arylalkenyl", "arylalkynyl", "biarylalkyl", and the like, in some cases, mean an alkyl, alkenyl, or alkynyl moiety substituted with aryl, heterocycloaliphatic, biaryl, etc., and in some cases, a moiety having an open (unsatisfied) valence with an alkyl, alkenyl, or alkynyl moiety, such as benzyl, phenethyl, N-imidazolylethyl, N-morpholinoethyl, and the like. Conversely, "alkylaryl", "alkenylcycloalkyl", and the like, in some cases, mean an aryl, cycloalkyl, or other moiety substituted with alkyl, alkenyl, etc., and in some cases, a moiety such as methylphenyl (tolyl) or allylcyclohexyl. "Hydroxyalkyl", "haloalkyl", "alkylaryl", "cyanoaryl", and the like, in some cases, mean an alkyl, aryl, or other moiety substituted with one or more specific substituents (in some cases, hydroxyl, halo, etc.).

[0255] For example, acceptable substituents include alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl, aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especially fluoro), haloalkyl (especially trifluoromethyl), hydroxyl, hydroxyalkyl (especially hydroxyethyl), cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl) (especially -OCF3), -O(cycloalkyl), -O(heterocycloalkyl), -O(aryl), alkylthio, arylthio, =O, =NH, =N(alkyl), =NOH, =NO(alkyl), -C(=O)(alkyl), -C(=O)H, -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(cycloalkyl), -S(=O)alkyl, -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), -SO2N(alkyl)2, and the like, but are not limited thereto.

[0256] When the moiety to be replaced is an aliphatic moiety, preferred substituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo, hydroxyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl), -O(cycloalkyl), -O(heterocycloalkyl), -O(aryl), alkylthio, arylthio, =O, =NH, =N(alkyl), =NOH, =NO(alkyl), -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(=O)alkyl, -S(cycloalkyl), -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), and -SO2N(alkyl)2. More preferred substituents are halo, hydroxyl, cyano, nitro, alkoxy, -O(aryl), =O, =NOH, =NO(alkyl), -OC(=O)(alkyl), -OC(=O)O(alkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, and -NHC(=NH)NH2. Particularly preferred substituents are phenyl, cyano, halo, hydroxyl, nitro, C1-C4 alkoxy, O(C2-C4alkanediyl)OH, and O(C2-C4alkanediyl)halo.

[0257] When the moiety to be replaced is a cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituents are alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl), -O(aryl), -O(cycloalkyl), -O(heterocycloalkyl), alkylthio, arylthio, -C(=O)(alkyl), -C(=O)H, -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azido, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(cycloalkyl), -S(=O)alkyl, -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), and -SO2N(alkyl)2.More preferred substituents are alkyl, alkenyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -C(=O)(alkyl), -C(=O)H, -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, and -NHC(=NH)NH2. Particularly preferred substituents are C1-C4 alkyl, cyano, nitro, halo, and C1-C4 alkoxy.

[0258] When ranges are recited, such as "C1-C5 alkyl" or "5 to 10%", the range includes the endpoints of the range, i.e., C1 and C5 in the first example, and 5% and 10% in the second example.

[0259] Unless a specific stereoisomer is clearly indicated (e.g., by making the valence bond at the relevant stereocenter in the structural formula bold or dashed, or by depicting the double bond in the structural formula as having an E or Z configuration, or by using nomenclature or symbols that specify stereochemistry), all stereoisomers are included within the scope of the invention as pure compounds as well as mixtures thereof. Unless otherwise indicated, racemates, individual enantiomers (whether optically pure or partially resolved), diastereomers, geometric isomers, and combinations thereof, and mixtures thereof are all encompassed by the present invention.

[0260] One of ordinary skill in the art will recognize that a compound may have tautomers (e.g., keto and enol forms), resonance structures, and zwitterionic forms that are equivalent to those depicted by the structural formulas used herein, and that the structural formulas include such tautomers, resonance structures, and zwitterionic forms.

[0261] "Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (e.g., in the human body) to produce the parent compound or a salt thereof, or that itself has activity similar to that of the parent compound. Suitable esters include C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl esters, especially methyl, ethyl or n-propyl esters.

[0262] "Pharmaceutically acceptable salt" means a salt of a compound suitable for use in a pharmaceutical formulation. When a compound has one or more basic groups, the salt can be an acid addition salt, e.g., a sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate, methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like. When a compound has one or more acidic groups, the salt can be a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like. Polymorphic forms and solvates are also included within the scope of the present invention.

[0263] "Subject" refers to an animal, including but not limited to primates (e.g., humans), monkeys, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice. The terms "subject" and "patient" are used interchangeably herein with respect to mammalian patients, e.g., humans.

[0264] The terms "treat", "treating", and "treatment" are intended to include, in the context of treating a disease or disorder, reducing or suppressing one or more of the disorder, disease, or condition, or symptoms associated with the disorder, disease, or condition; or delaying the progression, spread, or worsening of the disease, disorder, or condition, or one or more of their symptoms. "Treatment of cancer" refers to one or more of the following effects: (1) inhibition of tumor growth to some extent, including (i) delay and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintenance of tumor size; (4) reduction in tumor size; (5) inhibition of infiltration of tumor cells into peripheral organs, including (i) reduction, (ii) delay, or (iii) complete prevention; (6) inhibition of metastasis, including (i) reduction, (ii) delay, or (iii) complete prevention; (7) enhancement of the anti-tumor immune response, which may result in (i) maintenance of tumor size, (ii) reduction in tumor size, (iii) delay in tumor growth, (iv) reduction, delay, or prevention of infiltration; and / or (8) reduction in the severity or number of one or more symptoms associated with the disorder to some extent.

[0265] In the formulas herein, a wavy line (

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[0266] In the formulas of this specification, a valence bond that crosses an aromatic ring between two of its carbons means that the group attached to that valence bond may be located at any of the positions of the aromatic ring that are made available by the removal of a hydrogen that is implicitly there (or, if fully written, explicitly there). As an example:

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[0267] This disclosure includes all isotopes of atoms that occur in the compounds described herein. Isotopes include atoms that have the same atomic number but different mass numbers. General examples, but not limited to, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include 13 C and 14 C. Isotopically labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by procedures similar to those described herein, using an appropriate isotopically labeled reagent in place of the unlabeled reagent otherwise employed. As an example, a C1-C3 alkyl group may be non-deuterated, partially deuterated, or fully deuterated, and "CH3" may include CH3, 13 CH3, 14It includes CH3, CH2T, CH2D, CHD2, CD3, etc. In one embodiment, various elements in the compound are present at their natural isotopic abundances.

[0268] Those skilled in the art will recognize that a particular structure may be depicted in either tautomeric form - for example, keto or enol - and that the two forms are equivalent.

[0269] Acronyms and Abbreviations Table C provides a list of the acronyms and abbreviations used herein, along with their meanings.

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Table 111

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[0343] The foregoing detailed description of the present invention includes sections that relate primarily or exclusively to particular parts or aspects of the present invention. This is for purposes of clarification and convenience, and it should be understood that particular features may be relevant not only in the sections in which they are disclosed but also in other sections, and that the disclosure herein includes all appropriate combinations of the information described in different sections. Similarly, although the various figures and descriptions herein relate to particular embodiments of the present invention, if a specific feature is disclosed in the context of a particular figure or embodiment, it should be understood that such feature may be used in combination with other features in the context of another figure or embodiment, or generally in the present invention, to the extent appropriate.

[0344] Furthermore, although the present invention has been particularly described with reference to certain preferred embodiments, the present invention is not limited to such preferred embodiments. On the contrary, the scope of the present invention is defined by the appended claims.

Claims

1. The following formula (I): 【Chemical 1】 [Wherein, W is H, halo, C １ -C ３ -alkyl, CN, (C １ -C ４ -alkanediyl)OH, [Chemical 2] is; Each X is independently N or CR ２ and; R １ is (C １ -C ５ alkyl), (C ２ -C ５ alkenyl), (C １ -C ８ alkanediyl) ０－１ (C ３ -C ６ cycloalkyl), (C １ -C ８ alkanediyl) ０－１ (C ５ -C １０ spirocycloalkyl), (C ２ -C ８ alkanediyl)OH, (C ２ -C ８ alkanediyl)O(C １ -C ３ alkyl), (C １ -C ４ alkanediyl) ０－１ (5-6 membered heteroaryl), (C １ -C ４ alkanediyl) ０－１ phenyl, (C １ -C ４ alkanediyl)CF ３ , (C ２ -C ８ alkanediyl)N[C(=O)](C １ -C ３ alkyl), (C ２ -C ８ alkanediyl) ０－１ (C ３ -C ６ cycloalkanediyl)(C ３ -C ６ cycloalkyl), or (C ２ -C ８ alkanediyl)NR ｘ R ｙ is; Each R ２ is independently H, O(C １ -C ３ alkyl), S(C １ -C ３ alkyl), SO ２ (C １ -C ３ alkyl), C １ -C ３ alkyl, O(C ３ -C ４ cycloalkyl), S(C ３ -C ４ cycloalkyl), SO ２ (C ３ -C ４ cycloalkyl), C ３ -C ４ cycloalkyl, Cl, F, CN, or [C(=O)] ０－１ NR ｘ R ｙ wherein; R ３ is H, halo, OH, CN, NH ２ , NH[C(=O)] ０－１ (C １ -C ５ (alkyl), N(C １ -C ５ alkyl) ２ , NH[C(=O)] ０－１ (C １ -C ４ alkanediyl) ０－１ (C ３ -C ８ cycloalkyl), NH[C(=O)] ０－１ (C １ -C ４ alkanediyl) ０－１ (C ４ -C １０ bicycloalkyl), NH[C(=O)] ０－１ (C １ -C ４ alkanediyl) ０－１ (C ５ -C １０ spirocycloalkyl), N(C ３ -C ６ cycloalkyl) ２ , O(C １ -C ４ alkanediyl) ０－１ (C ３ -C ８ cycloalkyl), O(C １ -C ４ alkanediyl) ０－１ (C ４ -C ８ bicycloalkyl), O(C １ -C ４ alkanediyl) ０－１ (C ５ -C １０ spirocycloalkyl), O(C １ -C ４ alkanediyl) ０－１ (C １ -C ６ (alkyl), N[C １ -C ３ alkyl]C(=O)(C １ -C ６ alkyl), NH(SO ２ )(C １ -C ５ -alkyl), NH(SO ２ )(C １ -C ４ alkanediyl) ０－１ (C ３ -C ８ cycloalkyl), NH(SO ２ )(C １ -C ４ alkanediyl) ０－１ (C ４ -C １０ bicycloalkyl), NH(SO ２ )(C １ -C ４ alkanediyl) ０－１ (C ５ -C １０ spirocycloalkyl), a 6-membered aromatic or heteroaromatic moiety, a 5-membered heteroaromatic moiety, or the following structure: 【Chemical Formula 3】 is a moiety having; R ４ is NH ２ , NH(C １ -C ５ alkyl), N(C １ -C ５ alkyl) ２ , NH(C １ -C ４ alkanediyl) ０－１ (C ３ -C ８ cycloalkyl), NH(C １ -C ４ alkanediyl) ０－１ (C ４ -C １０ bicycloalkyl), NH(C １ -C ４ alkanediyl) ０－１ (C ５ -C １０ spirocycloalkyl), N(C ３ -C ６ cycloalkyl) ２ , or the following structure: 【Chemical Formula 4】 is a moiety having; R ５ is H, C １ -C ５ alkyl, or halo; R ６ is NH ２ , (NH) ０－１ (C １ -C ５ alkyl), N(C １ -C ５ alkyl) ２ , (NH) ０－１ (C １ -C ４ alkanediyl) ０－１ (C ３ -C ８ cycloalkyl), (NH) ０－１ (C １ -C ４ alkanediyl) ０－１ (C ４ -C １０ bicycloalkyl), (NH) ０－１ (C １ -C ４ alkanediyl) ０－１ (C ５ -C １０ spirocycloalkyl), N(C ３ -C ６ -cycloalkyl) ２ , or the following structure: [Chemical Formula 6] is a moiety having; R ｘ and R ｙ are independently H or C １ -C ３ alkyl, or R ｘ and R ｙ combine with the nitrogen to which they are attached to form a 3- to 7-membered heterocycle; n is 1, 2, or 3; p is 0, 1, 2, or 3; Here, R １ , R ２ , R ３ , R ４ , R ５ , and R ６ in, alkyl, alkenyl, cycloalkyl, alkanediyl, bicycloalkyl, spirocycloalkyl, cyclic amine, 6-membered aromatic or heteroaromatic moiety, 5-membered heteroaromatic moiety or the following formula: 【Chemical Formula 7】 the moiety represented by OH, halo, CN, (C １ -C ３ -alkyl), O(C １ -C ３ -alkyl), C(=O)(C １ -C ３ alkyl), SO ２ (C １ -C ３ alkyl), NR ｘ R ｙ , (C １ -C ４ alkanediyl)OH, (C １ -C ４ alkanediyl)O(C １ -C ３ alkyl) may be optionally substituted with one or more substituents selected from; alkyl, alkenyl, alkanediyl, cycloalkyl, bicycloalkyl, spirocycloalkyl, or the following formula: 【Chemical Formula 8】 the moiety represented by O, SO ２ , CF ２ , C(=O), NH, N[C(=O)] ０－１ (C １ -C ５ (alkyl), N[C(=O)] ０－１ (C １ -C ４ alkanediyl)CF ３ , N[C(=O)] ０－１ (C ２ -C ４ alkanediyl)OH, N(SO ２ )(C １ -C ３ alkyl), N(C １ -C ３ alkanediyl) ０－１ [C(=O)]NR ｘ R ｙ 、 or N[C(=O)] ０－１ (C １ -C ４ (alkanediyl) ０－１ (C ３ -C ５ (cycloalkyl) CH to be replaced ２ optionally having a ２ group; However, at least one of R １ and W contains a spirocycloalkyl or spirocycloalkanediyl moiety, and the compound of formula (I) is 【Chemical Formula 9】 is other than] A compound having the structure represented by.

2. W is, 【Chemical 10】 The compound according to claim 1, which is.

3. W is, 【Chemical 11】 The compound according to claim 1, which is.

4. R １ The compound according to claim 1, wherein each of R and W comprises a spirocycloalkyl or spirocycloalkanediyl moiety.

5. R １ The compound according to claim 1, wherein R contains a spirocycloalkyl moiety and W contains a bicycloalkyl or bicycloalkanediyl moiety.

6. R １ is 【Chemical Formula 12】 The compound according to claim 1, which is selected from the group consisting of.

7. R ２ is OMe or OCHF ２ The compound according to claim 1.

8. R ５ The compound according to claim 1, wherein R is H or Me.

9. The following formula (Ia): 【Chemical Formula 13】 The compound according to claim 1, which has the structure represented by.

10. The following formula (Ib): 【Chemical 14】 The compound according to claim 1, which has the structure represented by.

11. 【Fig. 15】 is, 【Chemical 16】 The compound according to claim 10, which is selected from the group consisting of.

12. The following formula (Ic): 【Chemical 17】 The compound according to claim 1, which has the structure represented by.

13. 【Fig. 18】 is, 【Chemical 19】 The compound according to claim 12, which is selected from the group consisting of.

14. The following formula (Id): 【Chemical 20】 [Wherein, R １ is 【Chemical 21】 is; W is, 【Chemical 22】 is] A compound having the structure represented by.

15. A pharmaceutical composition for treating cancer, comprising an anti-cancer immunotherapeutic agent and the compound according to any one of claims 1 to 14.

16. The pharmaceutical composition according to claim 15, wherein the anti-cancer immunotherapeutic agent is an antagonist anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody.

17. The pharmaceutical composition according to claim 15, wherein the anti-cancer immunotherapeutic agent is ipilimumab, nivolumab, or pembrolizumab.

18. The pharmaceutical composition according to any one of claims 15 to 17, wherein the cancer is lung cancer (including non-small cell lung cancer), pancreatic cancer, renal cancer, head and neck cancer, lymphoma (including Hodgkin lymphoma), skin cancer (including melanoma and Merkel cell carcinoma), urothelial cancer (including bladder cancer), gastric cancer, hepatocellular carcinoma, or colorectal cancer.

19. The following formula (Ie): 【Chemical 23】 [wherein, W' is 【Chemical 24】 and; R ９ is H, C １ -C ５ alkyl, (CH ２ ) １ - ２ (C ３ -C ５ cycloalkyl), or 【Chemical 25】 ] A compound having a structure represented by

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