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 TIFF2023512228000089.tif5166 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. 63 / 058,130, filed July 29, 2020, and U.S. Provisional Application Serial No. 62 / 966,124, filed January 27, 2020, under 35 U.S.C. § 119(e); the disclosures of which are 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] The disclosure of bioactive molecules having a purine-like backbone and their use in the treatment of conditions such as fibrosis, inflammatory diseases, cancer, or pathogenic infections includes: 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 of formula (A) - a pyrimidine 6-membered ring fused with an imidazole 5-membered ring - is 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] A complete citation for the documents cited herein by first author or inventor and year of publication is provided at the end of this specification.

SUMMARY OF THE INVENTION

[0017] This specification relates to compounds having activity as TLR7 agonists and having 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

[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 disclosed herein, or complexes thereof or PEGylated derivatives thereof, can 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 thereof 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 and R 3 are as defined for formula (I):

Chemical formula

[0022] In one aspect, the present disclosure provides a compound having a structure represented by formula (Ia) [wherein R 1 is

Chemical formula

Chemical formula

[0023] Examples of the group R 1 include:

Chemical formula

[0024] R 2 is preferably OMe, O(cyclopropyl), or OCHF 2 , more preferably OMe.

[0025] group R 3 examples include OH,

Chemical formula

[0026] In one embodiment, R 5 is H.

[0027] Specific examples of the compounds disclosed herein are shown in Table A below. The table also provides data on biological activity: 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, HPLC 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) agonist (reporter) assay and (b) an EC 50 value of less than 1,000 nM in the human whole blood (hWB) CD69 induction. (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

Table 16

[0028] 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 drugs. The pharmaceutical composition may be administered in combination therapy with another therapeutic agent, particularly an anti-cancer agent.

[0029] 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, colorants, 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).

[0030] 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, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0031] The pharmaceutical composition may 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.

[0032] 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 an amount of the composition that provides 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.

[0033] The dosing regimen is adjusted to provide a therapeutic response. For example, a single bolus administration may be carried out, the dosage may be divided into several portions and administered over time, or the dosage 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 dosage 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.

[0034] Dosages range from about 0.0001 to 100 mg / kg, more generally 0.01 to 5 mg / kg, based on the body weight of the host. For example, the dosage 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 regimens include one of 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 administration at 1 mg / kg body weight every three weeks, and methods of intravenous administration at 1 mg / kg body weight or 3 mg / kg body weight are mentioned. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / mL, and in some methods about 25-300 μg / mL.

[0035] 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 with cancer, the "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, still more preferably at least about 60%, and still 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 relieve 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.

[0036] The pharmaceutical composition can be a release-controlled 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.

[0037] The therapeutic composition can be administered using medical devices such as (1) a needleless subcutaneous injection device; (2) a microinfusion pump; (3) a transdermal device; (4) an infusion device; and (5) an osmotic device.

[0038] In certain embodiments, the pharmaceutical composition may be formulated to ensure proper 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.

[0039] Industrial Applicability and Uses The TLR7 agonist compounds disclosed in this specification can be used for the treatment of diseases or conditions that can be alleviated by activation of TLR7.

[0040] In one embodiment, the TLR7 agonist is used in combination with an anti-cancer immunotherapeutic agent - also known as an immuno-oncology agent. Anti-cancer immunotherapeutic agents exert their effects, particularly through 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 its inhibition of 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. Binding of an agonist immunotherapeutic agent to a stimulatory checkpoint molecule can result in activation of the latter and enhancement of the immune response against cancer cells. Alternatively, binding of an antagonist immunotherapeutic agent to an inhibitory checkpoint molecule can prevent 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.

[0041] 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, which comprises 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.

[0042] 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 neoplasm, 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.

[0043] 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 17

[0044] 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.

[0045] In another embodiment of the combination therapy with a TLR7 agonist, the anticancer immunotherapeutic agent is an antagonist anti-CTLA-4 antibody, preferably ipilimumab.

[0046] 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.

[0047] The TLR7 agonists disclosed herein are also useful as vaccine adjuvants.

[0048] The implementation of the present invention can be further understood by referring to the following examples provided as illustrations rather than limitations.

Example

[0049] Analysis Procedures NMR The following conditions were used to obtain proton nuclear magnetic resonance (NMR) spectra: DMSO-d6 or CDCl 3 was used as the solvent and internal standard, and NMR spectra were obtained using 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.

[0050] Chemical shifts are reported in parts per million (ppm) on the low magnetic field side, based on the 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 denoted as br. The integration values are approximate. It should be noted that the integration intensity, peak shape, chemical shift, and coupling constant can depend on the solvent, concentration, temperature, pH, and other factors. Furthermore, peaks that overlap with water or solvent peaks in the NMR spectrum or 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.

[0051] Liquid Chromatography The following preparative and analytical (LC / MS) liquid chromatography methods were used:

[0052] LC / MS Method A: 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

[0053] LC / MS Method B: Column: BEH C18 2.1 x 50 mm; Mobile Phase A: 95:5 H 2 O: Acetonitrile containing 0.01M NH 4 OAc; Mobile Phase B: 5:95 H 2 O: Acetonitrile containing 0.01M NH 4 OAc; Temperature: 50 °C; Gradient: 5 - 95% B over 1 minute; Flow Rate: 0.8 mL / min

[0054] LC / MS Method C: 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)

[0055] LC / MS Method D. 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 1.0 minute, then hold at 98% B for 0.50 minutes; Flow Rate: 0.8 mL / min. Detection: MS and UV (220 nm)

[0056] LCMS Method E. Column: Xbridge BEH C18 XP (50 x 2.1 mm), 2.5 μm; Mobile Phase A: 5:95 CH3CN: H containing 10 mM NH4OAc 2 O; Mobile Phase B: 95:5 CH3CN: H containing 10 mM NH4OAc 2 O; Temperature: 50 °C; Gradient: 0 - 100% B over 3 minutes; Flow Rate: 1.1 mL / min)

[0057] Synthesis - General Procedures 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 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 will be separated later, for example, by preparative HPLC. It should be understood that this is the case.

Chemical formula

[0058] The mixture of positional isomers can be separated at the initial stage of the synthesis and the remaining synthesis steps can be carried out using the 1H positional isomer, or alternatively, if necessary, the synthesis can be advanced using the mixture of positional isomers and the separation can be carried out at a later stage.

[0059] The compounds of the present invention can be prepared by a number of methods well known to those skilled in organic synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the technical field of organic synthetic chemistry or variations thereof as recognized by those skilled in the art. Preferred methods include, but are not limited to, the methods described below. All references cited herein are hereby incorporated by reference in their entirety.

[0060] The compounds of the present invention may be prepared using the reactions and techniques described in this section. The reactions are carried out in a solvent appropriate for the reagents and materials used and are suitable for the transformations effected. Similarly, in the description of the synthetic methods described below, it should be understood that all proposed reaction conditions, such as the choice of solvent, reaction atmosphere, reaction temperature, experimental time and work-up procedure, are selected to be standard conditions for that reaction and this should be readily recognized by those skilled in the art. It is understood by those skilled in organic synthesis that the functionality present in the various parts of the molecule must be compatible with the proposed reagents and reactions. Such limitations on substituents compatible with the reaction conditions will be readily apparent to those skilled in the art and alternative methods will have to be used. This may sometimes require a decision to change the order of the synthetic steps or to choose an alternative specific process scheme in order to obtain the desired compounds of the present invention. Another point that should be mainly considered in planning any synthetic route in this field is the wise selection of protecting groups used for the protection of reactive functional groups present in the compounds described in the present invention. A reliable description that lists a number of options for the skilled expert is Greene and Wuts (Protective Groups In Organic Synthesis, 3rd Edition, Wiley and Sons, 1999).

[0061] The compounds of formula (I) may be prepared by reference to the methods described in the following scheme. As shown therein, the final product is a compound having the same structural formula as formula (I). It will be understood that any compound of formula (I) may be prepared by a scheme by appropriate selection of reagents having appropriate substituents. Solvents, temperatures, pressures, and other reaction conditions may be readily selected by those skilled in the art. The starting materials are either commercially available or can be readily prepared by those skilled in the art. The components of the compounds are as defined herein or elsewhere in this specification. Scheme 1 [Chemical]

[0062] A general route to the compounds described in the present invention is illustrated in the scheme, where R 1 , R 5 , L 1 , L 2 , L 3 , Q 1 , Q 2 , the X and W substituents are functional groups that are either defined in the text beforehand or can be converted to the desired final substituents. L is an OH that can be easily converted to a leaving group such as a halide, triflate, thioether, or heterocycle. As shown in Scheme 1, the general procedure for preparing the compounds of the present invention involves starting from a substituted benzyl derivative 1. By substituting 1 with a suitably protected hydrazine using an appropriate reagent, a functional benzyl derivative 2 is obtained. For example, 2 may be obtained by substituting a benzyl halide such as methyl 4-(bromomethyl)-3-methoxybenzoate with a suitably protected hydrazine such as tert-butyl hydrazine carboxylate using one of a number of available base reagents such as DIPEA or K 2 CO 3 in a suitable solvent such as DMF, followed by removal of the protecting group using standard conditions known in the literature. Next, by reacting 2 with a suitably substituted alkenoate 3 using known conditions that bring about cyclization, a suitably substituted nitropyrazole 4 is obtained. For example, by cyclizing benzyl hydrazine 2 with methyl (Z)-4-(dimethylamino)-3-nitro-2-oxobut-3-enoate using an appropriate base, nitropyrazole 4 is obtained. The reduction of nitropyrazole 4 to aminopyrazole 5 is carried out using H 2 (g) and Pd-C or Zn(s) and NH 4It can be achieved using standard conditions known in the literature, such as OAc. Reacting appropriately substituted 5 with appropriately functionalized imidate 6 and cyclizing the resulting guanidino intermediate under basic conditions such as NaOMe - MeOH gives hydroxy pyrimidine 7. Using standard conditions known in the literature, coupling 7 with appropriately substituted amine 8 and then deprotecting if necessary gives compound 9. Scheme 2

Chem.

[0063] As shown in Scheme 2, the group at R5 may be treated to introduce a substituent before forming the pyrazolopyrimidine ring. A suitable leaving group L4 can be introduced into aminopyrazole 10 in preparation for subsequent chemical reactions. For example, the introduction of a halogen group can be achieved using a suitable halogenating reagent such as NBS or NIS. The subsequent reaction of 11 using a known carbon - carbon bond - forming reaction such as the Suzuki reaction or a known carbon - heteroatom reaction such as the Buchwald reaction under the conditions described in the literature can be used for the introduction of an alkyl, cycloalkyl, aryl or heteroaryl substituent at R 5 Thereby. Scheme 3

Chem.

[0064] Alternative synthetic methods for pyrazolopyrimidine 9 are shown in Schemes 3 and 4. Using the synthetic routes described in Schemes 1 and 2, compound 12 having a placeholder functional group at Q 4 can be prepared. After coupling with amine 8 using standard literature conditions, Q 4 can be converted to W using various means available to those skilled in the art. For example, if Q 4 is an ester, LiAlH 4 or LiBH 4It is reduced to a primary alcohol using standard conditions such as etc., and can be converted to a suitable leaving group such as -Cl, -Br or -OTs, which can be substituted by various nucleophiles. Next, pyrazolopyridimidine 9 is obtained by deprotecting as necessary. In another variation, the placeholder functional group Q as shown in compound 12 of Scheme 4 4 can be converted to W as in compound 14 before coupling with amine 8. Scheme 4

Chemical formula

[0065] Synthesis - Specific Examples To further illustrate the above content, 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 provided with the present disclosure, will recognize that they will be able to prepare and use the compounds disclosed herein even without an exhaustive list of examples.

[0066] The analytical data for compounds numbered 100 or more can be found in Table A. Example 1 - Intermediate A

Chemical formula

[0067] Intermediate A is useful for the synthesis of the compounds of the present disclosure.

[0068] Step 1: A solution of tert-butyl hydrazinecarboxylate (12.75 g, 96 mmol) and DIPEA in DMF (24 mL) was treated by dropwise addition, over 1 hour using a dropping funnel, of methyl 4-(bromomethyl)-3-methoxybenzoate (5 g, 19.30 mmol) in 24 mL of DMF at RT. The reaction mixture was stirred overnight at RT. EtOAc (135 mL) and H 2 O (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 an additional amount of H 2 O (75 mL) and twice with 10% LiCl solution (75 mL), dried over Na 2 SO 4 and concentrated. Column chromatography (Isco, 220 g SiO 2 , 0% CH 2 Cl 2 (5 minutes), then with 15% EtOAc-CH 2 Cl 2 ) gave tert-butyl 2-(2-methoxy-4-(methoxycarbonyl)benzyl)hydrazine-1-carboxylate as a clear oil (3.85 g). 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) LC / MS [M + H] + 311.2; LC RT = 0.80 minutes (Method A)

[0069] 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, 2·HCl (20 g). 1 H 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) LC / MS [M+H] + 211.1; LC RT = 0.51 min (Method A)

[0070] 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 CH 2 Cl 2 (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 h and stirred overnight until it warmed to 25 °C. CH 2 Cl 2 was removed by rotary evaporation and methyl 4-(hydrazinylmethyl)-3-methoxybenzoate dihydrochloride (31.7 g, 112 mmol) was added to the reaction mixture. The solution was stirred for 2 h at RT and the solvent was removed under vacuum. The residue was washed with water, 1N aqueous HCl and extracted with EtOAc (3x). The organic layer was dried over Na 2 SO 4 and concentrated. The residue was taken up in CH 2 Cl 2Dissolve in it, pass through a short silica gel column, and recrystallize from ethanol to obtain ethyl 1-(2-methoxy-4-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate (29.4 g). 1 H 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) LC / MS [M+Na] + 386.0; LC RT = 0.98 min (Method A)

[0071] Step 4: Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (3.04 g, 9.12 mmol, 86% yield) and Pd-C (1.131 g, 0.531 mmol) were suspended in EtOAc / MeOH (1:1) (152 mL). The reaction flask was evacuated under vacuum and purged with H 2 (3X), and stirred under a balloon pressure of H 2 (g). After 5 hours, the reaction mixture was filtered through CELITE™, and unused Pd-C (1.131 g, 0.531 mmol) was added. The reaction flask was evacuated under vacuum and purged with H 2 (3X), and stirred for 16 hours under a balloon pressure of H 2 (g). The reaction mixture was filtered through CELITE™, concentrated, and dried under vacuum to obtain ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (3.04 g) as a cream-colored powder. 11H NMR (400 MHz, DMSO-d6) δ 7.52 - 7.49 (m, 1H), 7.47 (dd, J = 7.9, 1.5 Hz, 1H), 7.19 (s, 1H), 6.40 (d, J = 7.8 Hz, 1H), 5.54 (s, 2H), 5.10 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.91 (s, 3H), 3.84 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H) LC / MS [M+H] + 334.1; LC / RT = 0.85 min (Method B)

[0072] Step 5: Ethyl 4-amino-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (1.65 g, 4.95 mmol) was dissolved in CHCl 3 (49.5 ml) and cooled to 0 °C. NBS (0.925 g, 5.20 mmol) was added. After 15 minutes, the reaction mixture was diluted with CHCl 3 and stirred vigorously with 10% aqueous sodium thiosulfate solution for 10 minutes. The organic phase was separated, washed with H 2 O, dried over MgSO 4 and concentrated. The crude product was purified by column chromatography (80 g SiO 2 , 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. 1 1H 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) LC / MS [M+H] + 412.2; LC RT = 1.02 min (Method A)

[0073] Step 6: Ethyl 4-amino-3-bromo-1-(2-methoxy-4-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (741.2 mg, yield 67.1%), K 2 CO 3 (1.098 g, 7.94 mmol) and TMB (3.5 M in THF) (1.816 ml, 6.36 mmol) were suspended in dioxane (26.5 ml):water (5.30 ml) (5:1). N 2 The reaction mixture was sparged with N 2 gas for 5 minutes, then PdCl 2 (dppf)-CH 2 Cl 2 (dppf)-CH 2 Cl 2 adduct (0.052 g, 0.064 mmol) was added. After stirring for an additional 4 minutes, the reaction flask was sealed and heated to 90 °C. After 3 hours, an additional amount of TMB (3.5 M in THF; 0.908 mL, 3.18 mmoL) and PdCl 2 (dppf)-CH 2 Cl 2 adduct (0.052 g, 0.064 mmol) were added. 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 with additional EtOAc. The crude product was concentrated on 4 g of CELITE®. Column chromatography (80 g SiO 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) LC / MS [M+H] + 348.2; LC / RT = 0.89 minutes (Method A)

[0074] 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 hours. An additional amount of AcOH was added (0.049 mL, 0.854 mmol), and the reaction was stirred at RT for an additional 72 hours. Then, NaOMe (25% wt in MeOH) (5.69 mL, 25.6 mmol) was added. After stirring for 3 hours, the reaction mixture was acidified again with AcOH. The product was collected by filtration, air-dried for 10 minutes, and completely dried in a laboratory dryer to obtain methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (Intermediate A) (722.0 mg) as a cream-colored solid. 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) LC / MS [M+H] + 402.3; LC RT = 0.86 minutes (Method A) Example 2 - Compound 112

Chemical Structure

[0075] Step 1: A suspension of methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (Intermediate A, 200 mg, 0.498 mmol) and BOP (331 mg, 0.747 mmol) in DMF (2491 μl) was treated at RT with (5-methylisoxazol-3-yl)methanamine (72.6 mg, 0.648 mmol) and DBU (3 equiv) (225 μl, 1.495 mmol). The reaction mixture was heated to 40 °C. After 15 minutes, an additional amount of DBU (2 equiv; 150 μL, 0.997 mmol) was added. The reaction mixture was stirred at 40 °C for 16 h. After cooling to RT, the reaction mixture was partitioned between EtOAc and semi-saturated NaHCO 3 aqueous solution. The organic phase was separated and the aqueous phase was extracted with EtOAc (2x). The combined organic layers were washed successively with 10% aqueous LiCl solution and brine, dried over Na 2 SO 4 and concentrated. Column chromatography (12 g SiO 2 , 0 to 10% CH 3 OH-CH 2 Cl 2 gradient elution) gave methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-3-methyl-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (201.1 mg). LC / MS [M+H] + 496.2; LC RT = 0.79 min (Method A)

[0076] Step 2: Methyl 3-methoxy-4-((5-((methoxycarbonyl)amino)-3-methyl-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (200 mg, 0.404 mmol) was suspended in THF at RT and sonicated to assist dissolution. LiAlH 4(In THF, 1 M; 807 μL, 0.807 mmol) was added dropwise over 10 minutes. After 20 minutes, the reaction was quenched with MeOH and partitioned between EtOAc and Rochelle salt. The biphasic mixture was stirred at RT for 2 hours. The aqueous layer was separated and re-extracted with EtOAc (1X). The combined organic layers were washed with brine and concentrated. Column chromatography (12 g SiO 2 , 0 to 10% CH 3 OH-CH 2 Cl 2 gradient elution) gave methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (73 mg). LC / MS [M+H] + 468.4; LC RT = 0.62 min (Method A)

[0077] Step 3: Methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (73 mg, 0.156 mmol) was dissolved in CH 2 Cl 2 (1562 μL) at RT. SOCl 2 (57.0 μl, 0.781 mmol) was added and the reaction was stirred for 20 minutes. Concentration gave methyl (1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (80 mg) in sufficient purity for use without further purification. LC / MS [M+H] + 486.1; LC RT = 0.83 min (Method A)

[0078] Step 4: A stock solution of methyl (1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (20 mg, 0.041 mmol) in acetonitrile (412 μL) was treated with tetrahydro-2H-pyran-4-amine (12.49 mg, 0.123 mmol). The reaction was stirred at 40 °C overnight. After cooling to RT, the reaction mixture was concentrated and redissolved in dioxane (400 μL) and treated with 10 M NaOH (82 μL, 0.823 mmol). The reaction mixture was heated at 80 °C for 5 h. After cooling to RT, the reaction was neutralized with AcOH (42 μL) and concentrated. The crude product was dissolved in DMF, filtered through a PTFE frit, and purified by preparative LC / MS under the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:NH 4 OAc-containing water; Mobile phase B: 95:5 acetonitrile:NH 4 OAc-containing water; 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. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 112 (5.1 mg).

[0079] Compound 113 was prepared similarly: The crude product was purified by preparative LC / MS under the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:NH 4 OAc-containing water; Mobile phase B: 95:5 acetonitrile:NH 4 OAc-containing water; Gradient: Hold at 2% B for 0 min, 2 - 42% over 24 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 113 (8.6 mg). Example 3 - Compound 101

Chem.

[0080] Step 1: A solution of methyl 4 - ((7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-3 - methoxybenzoate (US 2020 / 0038403 A1; 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 H 2 O (3x). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to afford 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, 95% yield). 1 1H NMR (400 MHz, DMSO - d 6 ) 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 RT: 0.67 min. LC / MS [M + H] + 482.3 (Method A)

[0081] 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 (190 mg, 0.395 mmol) in THF (10 mL) was cooled to 0 °C and treated with LiAlH 4 (1 M in THF, 691 μL, 0.691 mmol). The reaction mixture was stirred at 0 °C for 15 minutes, quenched with MeOH and Rochelle salt (saturated aqueous solution), and stirred at RT for 1 hour. The mixture was extracted with EtOAc (3x). The combined organic layers were washed with H 2 O, dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure to give methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (160 mg, 89% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.77 - 9.75 (m, 1H), 7.90 - 7.88 (m, 1H), 7.72 (br t, J = 5.7 Hz, 1H), 6.94 (s, 1H), 6.76 (d, J = 7.5 Hz, 1H), 6.61 - 6.57 (m, 1H), 6.15 (d, J = 0.8 Hz, 1H), 5.68 (s, 2H), 5.16 (t, J = 5.7 Hz, 1H), 4.73 (br d, J = 5.8 Hz, 2H), 4.44 (d, J = 5.6 Hz, 2H), 3.70 (s, 3H), 3.62 (s, 3H), 2.33 (s, 3H) LC RT: 0.58 min LCMS [M+H] + = 454.3 (Method A)

[0082] Step 3: A solution of methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (22 mg, 0.048 mmol) in dioxane (500 μL) was treated with NaOH (10 M aqueous solution, 200 μL, 2.0 mmol) and heated to 75 °C. After 5 h, the reaction mixture was cooled to RT, neutralized with HOAc (114 μL, 2.0 mmol), and concentrated under a nitrogen stream. The residue was dissolved in DMF and filtered through a PTFE frit. The crude material was purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; 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 Compound 101 (3.5 mg, 8% yield). Example 4 - Compound 102

Chemical Structure

[0083] SOCl 2(24 μL, 0.33 mmol) was added to a solution of (4-((5-amino-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxyphenyl)methanol (26.3 mg, 0.067 mmol) in THF (0.7 mL) at RT. After stirring for 30 minutes, the reaction mixture was concentrated under reduced pressure. The residue was redissolved in DCM and concentrated under reduced pressure. The residue was dissolved in DMF (0.7 mL), treated with cyclobutanamine (25.3 mg, 0.355 mmol), and stirred at RT for 3 hours. The temperature was raised to 70 °C. The reaction mixture was stirred for an additional 2 hours and concentrated under reduced pressure. The crude product was dissolved in DMF and filtered through a PTFE frit. The crude material was purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; gradient: hold at 2% B for 0 min, increase to 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 the MS signal. The fractions containing the desired product were combined, dried by centrifugal evaporation to obtain a residue, and further purified by preparative LC / MS under 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 22 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, dried by centrifugal evaporation to obtain compound 102 as the bis TFA salt (4.0 mg, 11%). Example 5 - Compound 103

Chemical Structure

[0084] Step 1: A solution of methyl (1-(4-(hydroxymethyl)-2-methoxybenzyl)-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (159 mg, 0.35 mmol) in DCM (3.5 mL) was treated with SOCl 2 (128 μL, 1.76 mmol). The reaction mixture was stirred at RT for 15 minutes 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-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (182 mg, 100%). LC RT: 0.80 min LCMS [M+H] + = 472.3 (Method A)

[0085] Step 2: A solution of methyl (1-(4-(chloromethyl)-2-methoxybenzyl)-7-(((5-methylisoxazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (25 mg, 0.053 mmol) in DMF (1.1 mL) was treated with tetrahydro-2H-pyran-4-amine (26.8 mg, 0.265 mmol). The reaction mixture was stirred at 70 °C for 2 h and concentrated under reduced pressure. The residue was redissolved in dioxane (0.5 mL) at RT and treated with NaOH (10 M aqueous solution, 27 μL, 0.27 mmol) and heated at 80 °C for 4.5 h. The reaction mixture was neutralized with HOAc (15 μL, 0.27 mmol) at RT and concentrated under reduced pressure. The crude product was dissolved in DMF and filtered through a PTFE frit and purified by preparative LC / MS under 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 - 30% 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 103 as the bis TFA salt (20.2 mg, 54%).

[0086] The following compounds were prepared analogously: Compound 104, Compound 105, Compound 106, Compound 110, and Compound 111. Example 6 - Compound 107

Chemical Structure

[0087] A solution of methyl (1-(4-((cyclobutylamino)methyl)-2-methoxybenzyl)-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (US 2020 / 0038403 A1; 30 mg, 0.073 mmol) in DMF (0.7 mL) was treated with BOP (57.9 mg, 0.131 mmol), (5-methyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (54.4 mg, 0.364 mmol) and DBU (164 μL, 1.091 mmol). The reaction mixture was stirred at RT for 2 h, diluted with EtOAc, and washed with saturated NaHCO 3 solution and H 2 O. The organic layer was concentrated under reduced pressure. The residue was dissolved in dioxane (0.7 mL), treated with NaOH (10 M aqueous solution, 0.20 mL, 2.0 mmol), and heated to 75 °C. After 4 h, the reaction mixture was cooled to RT, neutralized with HOAc (0.12 mL, 2.0 mmol), and concentrated under reduced pressure. The crude product was dissolved in DMF and H 2 O, filtered through a PTFE frit, and purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; 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 the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 107 (8.6 mg, 26% yield). Example 7 - Compound 114

Chemical Structure

[0088] Step 1: A solution of methyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (US 2020 / 0038403 A1, Figure 7, Compound 64; 700 mg, 1.95 mmol) 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 H 2 O. The organic layer was washed with H 2 O (6x), dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was dissolved in DCM / MeOH, 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, 10-50% gradient). The purified product was dissolved in DCM and washed with saturated aqueous NaHCO 3 . The organic layer was dried over Na 2 SO 4 , filtered, and concentrated in vacuo to afford 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). 1 1H NMR (400 MHz, DMSO-d 6)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 A)

[0089] 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 SOCl 2 (179 μL, 2.46 mmol). The reaction mixture was stirred at RT for 10 minutes 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-d 6 )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 LCMS [M + H] + = 473.3 (Method A)

[0090] 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 (34.7 mg, 0.073 mmol) in DMF (1.5 mL) was treated with tetrahydro-2H-pyran-4-amine (37.1 mg, 0.367 mmol). The reaction was stirred at 75 °C for 1 h and concentrated under reduced pressure. The residue was dissolved in dioxane (1.0 mL) and MeOH (0.2 mL), treated with NaOH (10 M aqueous solution, 0.2 mL, 2.0 mmol), and heated at 75 °C for 2 h. After cooling to RT, the reaction mixture was neutralized with HOAc (0.12 mL, 2.0 mmol) and concentrated under reduced pressure. The crude product was dissolved in DMF and H 2 O and filtered through a PTFE frit and purified by preparative LC / MS under the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; Mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; Gradient: hold at 0% B for 0 min, 0 - 40% B over 30 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 114 (7.5 mg, 18%).

[0091] The following compounds were prepared analogously: compound 115, compound 117, compound 120, compound 121, compound 122, and compound 123. Example 8 - Compound 116

Chemical Structure

[0092] 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 (19 mg, 0.043 mmol) in dioxane (0.4 mL) and MeOH (0.2 mL) was treated with NaOH (10 M aqueous solution, 50 μL, 0.5 mmol) and heated to 50 °C. After 30 minutes, the reaction mixture was cooled to RT, neutralized with HOAc (30 μL, 0.5 mmol), and concentrated under reduced pressure. The residue was dissolved in DMF and filtered through a PTFE frit. The crude material was purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; gradient: hold at 2% B for 0 minutes, increase to 2 - 42% B over 25 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 116 (3.9 mg, 22% yield). Example 9 - Compound 109a

Chemical Structure

[0093] To a solution of methyl (7-hydroxy-1-(2-methoxy-4-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (75 mg, 0.170 mmol, US 2020 / 0038403 A1) in DMSO (1.5 mL) were added (S)-3-amino-1-cyclopropylpropan-1-ol (39.0 mg, 0.339 mmol), DBU (0.077 mL, 0.509 mmol), and BOP (150 mg, 0.339 mmol); the reaction mixture was heated at 70 °C for 2 h, treated with 5M NaOH (0.136 mL, 0.678 mmol), and heated at 70 °C for 2 h. The reaction mixture was cooled to 25 °C and the crude material was purified by preparative LC / MS under the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH 4 OAc; Mobile phase B: 95:5 acetonitrile: water containing NH 4 OAc; Gradient: Hold at 3% B for 0 min, 3 - 43% over 30 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 material was further purified by preparative LC / MS under 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% 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 material was further purified by preparative LC / MS under the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH 4 OAc; Mobile phase B: 95:5 acetonitrile: water containing NH 4The OAc contained water; gradient: held at 1% B for 0 min, increased from 1 - 41% B over 25 min, then held 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 were combined and dried by centrifugal evaporation to obtain compound 109a (2.3 mg, 4.69 μmol, 2.77% yield).

[0094] Compound 109b was prepared similarly. Example 10 - Compound 108 [Chemical Structure]

[0095] Step 1. To a solution of methyl (7-hydroxy-1-(2-methoxy-4-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (90 mg, 0.203 mmol, US 2020 / 0038403 A1), (S)-2-amino-3-cyclopropylpropan-1-ol hydrochloride (93 mg, 0.610 mmol) and BOP (135 mg, 0.305 mmol) in DMF (2034 μl) was added DBU (153 μl, 1.017 mmol). The reaction mixture was diluted with water (2 mL, 0.2% TFA) at RT overnight and purified on an Accq Prep 20x150 mm Xbridge column (6 injections): 20% acetonitrile / water (0.1% TFA). The fractions collected at 12 min were lyophilized to give methyl (S)-(7-((1-cyclopropyl-3-hydroxypropan-2-yl)amino)-1-(2-methoxy-4-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (65 mg, 59.2% yield) as a white solid. LCMS [M+H] + = 539.3

[0096] Step 2. Methyl (S)-(7-((1-cyclopropyl-3-hydroxypropan-2-yl)amino)-1-(2-methoxy-4-(((tetrahydro-2H-pyran-4-yl)amino)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (167 mg, 0.309 mmol) was dissolved in dioxane (5158 μl), treated with NaOH (619 μl, 3.09 mmol), and heated at 80 °C overnight. The reaction mixture was neutralized with HCl and concentrated. The residue was dissolved in DMF (4 mL) and filtered. The crude material was purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:NH 4 OAc-containing water; mobile phase B: 95:5 acetonitrile:NH 4 OAc-containing water; 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 the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give compound 108 (60 mg, 40% yield).

[0097] Compound 125 was prepared similarly. Example 11 - Compound 126

Chemical Structure

[0098] Step 1. To methyl 4-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (50 mg, 0.129 mmol) in DMF (1 mL) was added NBS (76 mg, 0.427 mmol). The reaction mixture was stirred at 40 °C overnight, cooled to 25 °C, diluted with MeOH, and filtered to give methyl 4-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (40 mg, 0.082 mmol, yield 63.1%). LC-MS m / z 468.2 [M+2H]+ 1 1H NMR (400 MHz, DMSO-d 6 ) δ 11.86 - 11.17 (m, 2H), 7.51 (s, 2H), 7.02 - 6.74 (m, 1H), 5.74 (s, 2H), 3.86 (d, J = 9.7 Hz, 6H), 3.76 (s, 3H)

[0099] Step 2. LiAlH 4 (1 M in THF; 6 mL, 6.00 mmol) was slowly added to a solution of methyl 4-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (1 g, 2.145 mmol) in THF (20 mL) at 0 °C (ice bath). The reaction mixture was stirred at RT for 30 minutes. At 0 °C (ice bath), saturated Na 2 SO 4(5.0 ml) was added slowly to quench the reaction. The mixture was stirred at RT for 30 minutes. The organic solvent was removed by rotary evaporator and the aqueous phase was lyophilized. The lyophilized material was diluted with MeOH (100 ml) and filtered (washed with 3 x 10 mL MeOH). The solvent was removed and the material was purified by silica gel (DCM-MeOH 0 - 30%) to give methyl (3-bromo-7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (330 mg, 0.753 mmol, 30% yield). LC-MS m / z 440.2[M+2H]+ 1 H NMR(400MHz,DMSO-d 6 )δ 7.05 - 6.95(m,1H),6.87 - 6.76(m,2H),5.66(s,2H),5.23 - 5.14(m,1H),4.52 - 4.43(m,2H),3.82 - 3.72(m,6H)

[0100] Step 3. A microwave vial was charged with methyl (3-bromo-7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (200 mg, 0.456 mmol) (about 80% purity and contaminated with N2 isomer), TMB (0.255 ml, 1.825 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (100 mg, 0.137 mmol), K 2 CO 3 (442 mg, 3.19 mmol), dioxane (8 mL) and water (2 mL). The reaction mixture was heated in a microwave at 120 °C for 1 hour, diluted with EtOAc, washed with water, Na 2 SO 4It was dried. The solvent was removed, and the substance was purified by silica gel (dry load) with DCM-MeOH 0 - 50% to obtain 5-amino-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (49 mg, 0.093 mmol, yield 20.43%). LC-MS m / z 316.3[M+H] +

[0101] Step 4. To a 20 mL vial, 5-amino-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (50 mg, 0.159 mmol) and DCM (2 mL) were added, followed by SOCl 2 (0.1 mL, 1.370 mmol) was added. The reaction mixture was stirred at 25 °C and concentrated under reduced pressure to obtain 5-amino-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (52.9 mg, 0.158 mmol, yield 100%), which was used without purification. LC-MS m / z 335.7[M+2H] +

[0102] Step 5. To 5-amino-1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (52 mg, 0.156 mmol) in DMF (2 mL), 2-(piperazin-1-yl)ethan-1-ol (0.1 mL, 0.815 mmol) was added. The reaction mixture was stirred at 25 °C overnight, and the solvent was removed. The substance was purified by silica gel (dry load) with DCM-MeOH 0 - 30% to obtain 5-amino-1-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (53 mg, 0.095 mmol, yield 61.3%). LC-MS m / z 428.3[M+H]+

[0103] A solution of 6.5-amino-1-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (53 mg, 0.124 mmol) and (S)-3-amino-1-cyclopropylpropan-1-ol (30 mg, 0.260 mmol) in DMSO (1.5 mL) was added with DBU (0.075 mL, 0.496 mmol) and BOP (110 mg, 0.248 mmol). The reaction mixture was heated at 70 °C for 1 h. The product was purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile: 0.1% TFA; mobile phase B: acetonitrile containing 95:5 0.1% 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. The fractions containing the target compound were combined and dried by centrifugal evaporation to obtain Compound 126. Example 12 - Compound 118 [Chemical Formula]

[0104] 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; Figure 8, Compound 71) in THF (16 mL) was cooled to 0 °C and treated with LiAlH 4 (1 M in THF, 2.8 mL, 2.8 mmol). The reaction mixture was stirred at 0 °C for 15 min, quenched with H 2 O and Rochelle salt (saturated aqueous solution), and stirred at RT for 3 h. The organic layer was absorbed onto CELITE (trademark), and column chromatography (24 g SiO 2; Purified by elution with a 0 to 20% MeOH-DCM gradient to obtain tert-butyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (460 mg, yield 72%). 1 H (400 MHz, DMSO-d 6 ) 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 D)

[0105] 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 H 2 O (2x). The organic layer was absorbed onto CELITE™ and purified by column chromatography (100 g C18 gold column; mobile phase A: water containing 5:95 acetonitrile: 0.05% TFA; mobile phase B: acetonitrile containing 95:5 0.05% TFA; flow rate: 60 mL / min, 30 - 50% gradient). The purified product was dissolved in DCM and washed with saturated NaHCO 3 aqueous solution. The organic layer was washed with Na 2 SO 4It was dried, filtered, and concentrated under reduced pressure to obtain 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, yield 33%). 1 H NMR(400MHz,DMSO-d 6 )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 D)

[0106] 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 (161 mg, 0.320 mmol) in DCM (0.65 mL) was treated with SOCl 2 (71 μL, 0.97 mmol). The reaction mixture was stirred at RT for 15 minutes and concentrated under reduced pressure. The residue was dissolved in DCM and concentrated under reduced pressure to obtain tert-butyl (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 (166 mg, 100%). LC RT:0.89 min LC / MS[M+H] + =515.2(Method D)

[0107] Step 4. A solution of tert-butyl (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 (33 mg, 0.064 mmol) in DMF (1.3 mL) was treated with DIEA (113 μL, 0.645 mmol) and 3-methoxyazetidine·HCl (23.9 mg, 0.193 mmol). The reaction mixture was stirred at 70 °C for 1 h and dried under a stream of N 2 and then further dried under reduced pressure. The residue was dissolved in dioxane (0.6 mL), treated with HCl (4 M in dioxane, 0.82 mL, 3.3 mmol), stirred at 40 °C for 30 min, and concentrated. The crude product was dissolved in DMF, filtered through a PTFE frit, and purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; gradient: hold at 2% B for 0 min, increase to 2 - 42% B over 30 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 isolated product was further purified by preparative LC / MS under 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 - 30% 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 to give compound 118 (9.4 mg, 21%).

[0108] Compound 119 was prepared analogously. Example 13 - Compound 127

Chemical formula

[0109] Step 1. A solution of tert-butyl (7-hydroxy-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (200 mg, 0.498 mmol) in DMSO (2.5 mL) was treated with (5-cyclopropyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (175 mg, 0.996 mmol), BOP (331 mg, 0.747 mmol) and DBU (0.30 mL, 2.0 mmol). The reaction mixture was stirred at RT for 2 h, diluted with EtOAc, and washed with H 2 O (2x). The organic layer was concentrated under reduced pressure. The crude product was dissolved in MeOH, filtered through a PTFE frit, and purified by preparative HPLC under the following conditions: Column: Axia C18 100 mm x 30 mm, 5μm particles; Mobile phase A: 10:90 methanol: water containing 0.1% TFA; Mobile phase B: 90:10 MeOH: water containing 0.1% TFA; Gradient: Hold at 40% B for 0 min, 40 - 55% B over 10 min, then hold at 55% B for 5 min; Flow rate: 40 mL / min; UV detection at 220 nm; Column temperature: 25 °C. The purified product was neutralized with saturated NaHCO 3 aqueous solution and washed with DCM. The organic layer was dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure to give tert-butyl (7-(((5-cyclopropyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1-(4-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (93.2 mg, yield 36%). 1 1H NMR (400 MHz, DMSO-d 6)δ 9.25 - 9.17 (m, 1H), 7.88 (s, 1H), 7.71 (t, J = 5.7 Hz, 1H), 6.96 (s, 1H), 6.84 - 6.76 (m, 1H), 6.75 - 6.67 (m, 1H), 5.70 - 5.67 (m, 2H), 5.17 (t, J = 5.7 Hz, 1H), 4.84 (d, J = 4.6 Hz, 2H), 4.45 (d, J = 5.8 Hz, 2H), 3.77 (s, 3H), 2.35 - 2.27 (m, 1H), 1.44 (s, 9H), 1.25 - 1.20 (m, 2H), 1.08 - 1.03 (m, 2H) LC RT: 0.77 min LC / MS [M + H] + = 523.4 (Method D)

[0110] Step 2. A solution of tert-butyl (7 - (((5 - cyclopropyl - 1,2,4 - oxadiazol - 3 - yl)methyl)amino)-1-(4 - (hydroxymethyl)-2 - methoxybenzyl)-1H - pyrazolo[4,3 - d]pyrimidin - 5 - yl)carbamate (93.2 mg, 0.178 mmol) in DCM (3.6 mL) was treated with SOCl 2 (39 μL, 0.54 mmol). The reaction mixture was stirred at RT for 10 minutes and concentrated under reduced pressure. The residue was dissolved in DCM and concentrated under reduced pressure to give tert-butyl (1 - (4 - (chloromethyl)-2 - methoxybenzyl)-7 - (((5 - cyclopropyl - 1,2,4 - oxadiazol - 3 - yl)methyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 5 - yl)carbamate (95.4 mg, yield 99%). 1 1H NMR (400 MHz, DMSO - d 6 )δ 11.70 - 11.19 (m, 1H), 9.46 - 9.20 (m, 1H), 8.10 - 8.06 (m, 1H), 7.10 (s, 1H), 6.97 (s, 2H), 5.79 (s, 2H), 4.97 (br d, J = 5.2 Hz, 2H), 4.73 (s, 2H), 3.74 (s, 3H), 2.40 - 2.30 (m, 1H), 1.53 (s, 9H), 1.30 - 1.22 (m, 2H), 1.10 - 1.04 (m, 2H) LC RT: 0.89 min LC / MS [M + H] +=541.3 (Method D)

[0111] Step 3. A solution of tert-butyl (1-(4-(chloromethyl)-2-methoxybenzyl)-7-(((5-cyclopropyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (30 mg, 0.055 mmol) in DMF (1.1 mL) was treated with DIEA (77 μL, 0.44 mmol) and tetrahydro-2H-pyran-4-amine (22.4 mg, 0.222 mmol). The reaction mixture was stirred at 60 °C for 1 hour, then the temperature was raised to 65 °C and stirring was continued for 1 hour. The reaction mixture was dried under a stream of N 2 and then further dried under reduced pressure. The residue was dissolved in dioxane (1.1 mL), treated with HCl (4 M in dioxane, 0.75 mL, 3 mmol), stirred at 40 °C for 90 minutes, and concentrated under reduced pressure. The crude product was dissolved in DMF, filtered through a PTFE frit, and purified by preparative LC / MS under the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; mobile phase B: 95:5 acetonitrile: 10 mM NH 4The OAc contained water; gradient: held at 2% B for 0 min, increased from 2 - 42% B over 30 min, then held 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 were combined and dried by centrifugal evaporation. The isolated product was further purified by preparative LC / MS under 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: water containing 95:5 acetonitrile: 0.05% TFA; gradient: held at 5% B for 0 min, increased from 5 - 70% 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 the MS signal. The fractions containing the target were combined and dried by centrifugal evaporation to obtain Compound 127 (13.6 mg, 47%). See Table A for analytical data.

[0112] Compound 128 and Compound 129 were prepared similarly. Example 14 - Compound 130

Chemical formula

[0113] Step 1. A solution of ethyl 5-methoxy-6-methylnicotinate (1.32 g, 6.77 mmol) in CCl 4 (19 mL) was treated with NBS (1.44 g, 8.12 mmol) and AIBN (0.22 g, 1.4 mmol). The reaction mixture was stirred at 60 °C for 40 h and washed with saturated Na 2 S 2 O 3 aqueous solution. The organic layer was concentrated under reduced pressure and the crude product was purified by column chromatography (40 g SiO 2 ; eluted with a 0 to 25% EtOAc - hexane gradient) to obtain ethyl 6-(bromomethyl)-5-methoxynicotinate (1.20 g, 4.38 mmol, 65% yield). 11H NMR (400 MHz, chloroform-d) δ 8.83 - 8.75 (m, 1H), 7.78 (d, J = 1.6 Hz, 1H), 4.65 (s, 2H), 4.43 (q, J = 7.1 Hz, 2H), 3.99 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H) LC RT: 0.89 min LC / MS [M+H] + = 274.1 (Method D)

[0114] Step 2. A solution of methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (2.51 g, 12.0 mmol) in DMF (50 mL) was treated with NBS (2.14 g, 12.0 mmol). The reaction mixture was stirred at RT for 15 minutes and filtered. The recovered solid was washed with H 2 2O and diethyl ether to give methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (3.28 g, 95% yield). LC RT: 0.57 min LC / MS [M+H] + = 288.1 (Method D)

[0115] Step 3. A solution of methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (648 mg, 2.25 mmol) in DMF (22.5 mL) was treated with ethyl 6-(bromomethyl)-5-methoxynicotinate (617 mg, 2.25 mmol) and Cs 2 2CO 3 (2199 mg, 6.75 mmol). The reaction mixture was stirred at RT for 2 hours, diluted with EtOAc, and washed with saturated NaHCO 3 3 solution and H 2 2O. The organic layer was concentrated under reduced pressure. The crude product was purified by column chromatography (40 g SiO 2; Purified by gradient elution with 0 to 100% EtOAc - hexane), and ethyl 6 - ((3 - bromo - 7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-5 - methoxynicotinate (653.1 mg, yield 60%) was obtained. 1 H NMR (500 MHz, DMSO - d 6 ) δ 11.61 - 11.41 (m, 1H), 8.49 - 8.47 (m, 1H), 7.81 (d, J = 1.6 Hz, 1H), 5.85 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.96 (s, 3H), 3.74 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H) LC RT: 0.86 min LC / MS [M + H] + = 481.2 (Method D)

[0116] Step 4. A suspension of ethyl 6 - ((3 - bromo - 7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-5 - methoxynicotinate (542 mg, 1.13 mmol) in MeOH (54 mL) was treated with Pd / C (24 mg, 0.23 mmol). The reaction flask was evacuated under vacuum and purged with H 2 (3x). The reaction mixture was stirred under H 2 atmosphere (balloon) for 16 h. The reaction flask was evacuated under vacuum and purged with N 2 (3x). The reaction mixture was diluted with DCM, filtered through CELITE (trademark), and concentrated under reduced pressure to obtain ethyl 6 - ((7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-5 - methoxynicotinate (450 mg, yield 99%). 1 H NMR (400 MHz, DMSO - d 6)δ 8.49 - 8.44 (m, 1H), 7.85 (s, 1H), 7.79 (d, J = 1.6 Hz, 1H), 5.86 (s, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.75 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H) LC RT: 0.78 min LC / MS [M + H] + = 403.0 (Method D)

[0117] Step 5. A solution of ethyl 6 - ((7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-5 - methoxynicotinate (543 mg, 1.35 mmol) in THF (28 mL) was cooled to 0 °C and treated with LiAlH 4 (1 M in THF, 2.4 mL, 2.4 mmol). The reaction mixture was stirred at 0 °C for 15 minutes and quenched with H 2 O and Rochelle salt (saturated aqueous solution), and stirred at RT for 2 hours. The organic layer was absorbed onto CELITE™ and purified by column chromatography (40 g SiO 2 ; elution with a 0 to 10% MeOH - DCM gradient) to give methyl (7 - hydroxy - 1 - ((5 - (hydroxymethyl)-3 - methoxypyridin - 2 - yl)methyl)-1H - pyrazolo[4,3 - d]pyrimidin - 5 - yl)carbamate (191 mg, 39% yield). 1 1H NMR (400 MHz, DMSO - d 6 )δ 7.89 - 7.84 (m, 1H), 7.80 (s, 1H), 7.37 (d, J = 1.5 Hz, 1H), 5.80 - 5.72 (m, 2H), 5.28 (t, J = 5.7 Hz, 1H), 4.48 (d, J = 5.4 Hz, 2H), 3.87 - 3.81 (m, 3H), 3.74 (s, 3H) LC RT: 0.56 min LC / MS [M + H] + = 361.0 (Method D)

[0118] Step 6. A solution of methyl (7-hydroxy-1-((5-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (190 mg, 0.527 mmol) in DMSO (2.6 mL) was treated with (5-methyl-1,2,4-oxadiazol-3-yl)methanamine·HCl (103 mg, 0.685 mmol), BOP (303 mg, 0.685 mmol) and DBU (0.28 mL, 1.8 mmol). The reaction mixture was stirred at RT for 1 h, diluted with DCM, and washed with H 2 O(6x). The organic layer was concentrated under reduced pressure. The crude product was dissolved in MeOH, filtered through a PTFE frit, and purified by preparative HPLC under the following conditions: column: Axia C18 100 mm x 30 mm, 5μm particles; mobile phase A: 10:90 methanol: water containing 0.1% TFA; mobile phase B: 90:10 methanol: water containing 0.1% TFA; gradient: hold at 5% B for 0 min, 5 - 30% B over 10 min, then hold at 30% B for 2 min; flow rate: 40 mL / min; UV detection at 220 nm; column temperature: 25 °C. The purified product was neutralized with saturated NaHCO 3 aqueous solution and washed with DCM. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give methyl (1-((5-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (102.4 mg, yield 43%). 1 1H NMR (400 MHz, DMSO-d 6 ) δ 9.68 (s, 1H), 8.99 (br s, 1H), 7.98 - 7.92 (m, 1H), 7.84 (s, 1H), 7.45 (d, J = 1.1 Hz, 1H), 5.77 (s, 2H), 5.35 (br s, 1H), 4.92 (br s, 2H), 4.51 (br s, 2H), 3.88 (s, 3H), 3.61 (s, 3H), 2.57 (s, 3H) LC RT: 0.61 min LC / MS [M+H]+ =456.1 (Method D)

[0119] Step 7. A solution of methyl (1-((5-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (102 mg, 0.225 mmol) in DCM (4.5 mL) was treated with SOCl 2 (49 μL, 0.68 mmol). The reaction mixture was stirred at RT for 30 minutes and concentrated under reduced pressure. The residue was dissolved in DCM and concentrated under reduced pressure to give methyl (1-((5-(chloromethyl)-3-methoxypyridin-2-yl)methyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (107 mg, yield 100%). LC RT: 0.67 min LC / MS [M+H] + =474.3 (Method D)

[0120] Step 8. A solution of methyl (1-((5-(chloromethyl)-3-methoxypyridin-2-yl)methyl)-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (35 mg, 0.074 mmol) in DMF (0.7 mL) was treated with DIEA (103 μL, 0.591 mmol) and tetrahydro-2H-pyran-4-amine (29.9 mg, 0.295 mmol). The reaction mixture was stirred at 70 °C for 2 hours, N 2It was dried under air flow and then further dried under reduced pressure. The residue was dissolved in dioxane (0.8 mL) and treated with NaOH (10 M aqueous solution, 37 μL, 0.37 mmol). The reaction mixture was heated to 60 °C. An additional amount of NaOH (10 M aqueous solution, 120 μL, 1.2 mmol) was added to the reaction mixture over 8 hours. The reaction mixture was neutralized with HOAc at RT and concentrated under reduced pressure. The crude product was dissolved in DMF, filtered through a PTFE frit, and purified by preparative LC / MS under the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 10 mM NH 4 OAc; Mobile phase B: 95:5 acetonitrile: water containing 10 mM NH 4 OAc; Gradient: Hold at 1% B for 0 min, increase from 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 target compound were combined and dried by centrifugal evaporation. The isolated product was further purified by preparative LC / MS under 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 from 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 were combined and dried by centrifugal evaporation to obtain compound 130 as the bis-TFA salt (11 mg, 20%).

[0121] Compound 131 was prepared similarly. Example 15 - Compound 134

Chemical Structure

[0122] Step 1. To a stirred solution of methyl (7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (5.0 g, 14.92 mmol) in DMF (50.0 mL) at 0 °C was added Cs 2 CO 3 (9.72 g, 29.8 mmol) and methyl 4-(bromomethyl)-3-methoxybenzoate (3.87 g, 14.92 mmol). The reaction mixture was stirred at 0 °C for 1 h and water was added. The precipitated solid was filtered and washed with an excess of water followed by petroleum ether. The solid was dried in vacuo. The crude compound was purified by ISCO combiflash chromatography eluting with 0 - 100% ethyl acetate in chloroform to give methyl 4-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (3.88 g, 6.20 mmol, 41.5% yield) as an off-white solid. 1 1H NMR (400 MHz, DMSO-d 6 ) δ ppm: 11.69 (br s, 1H), 11.38 (s, 1H), 7.56 - 7.45 (m, 2H), 6.87 - 6.78 (m, 1H), 5.75 (s, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.75 (s, 3H) LC-MS m / z 514.0 [M+H] +

[0123] Step 2. To a stirred solution of methyl 4-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (3.5 g, 6.82 mmol) in 1,4-dioxane (35.0 mL) were added K 2 CO 3 (1.885 g, 13.64 mmol), TMB (1.907 mL, 13.64 mmol) and PdCl 2 (dppf).CH 2 Cl 2 adduct (0.557 g, 0.682 mmol) under N2 It was added under purge. The reaction mixture was stirred at 100 °C for 6 hours. The reaction mixture was filtered through a CELITE™ bed and washed with an excess amount of ethyl acetate. The filtrate was concentrated under reduced pressure to obtain a residue. The crude compound was purified by ISCO Combiflash chromatography (0 - 20% methanol in chloroform) to give methyl 4-((5-amino-7-hydroxy-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (2.1 g, 4.10 mmol, 60.1% yield) as a brown solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ = 10.90 (s, 1H), 7.51 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 6.63 - 6.50 (m, 1H), 6.18 - 6.01 (m, 2H), 5.71 - 5.54 (m, 2H), 3.91 (s, 3H), 3.87 - 3.78 (s, 3H), 2.23 (s, 3H) LC-MS m / z 344.0 [M+H] +

[0124] To a stirred solution of methyl 4-((5-amino-7-hydroxy-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxybenzoate (0.5 g, 1.456 mmol) in THF (5.0 mL) at 0 °C was added LiAlH 4 (1.214 mL, 2.91 mmol). The reaction mixture was warmed to RT, stirred for 1 hour, quenched with ice-cold water, filtered through a CELITE™ bed, and washed with an excess amount of ethyl acetate. The organic layer was dried over Na 2 SO 4 and filtered, and concentrated under reduced pressure to give 5-amino-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (0.31 g, 0.551 mmol, 37.8% yield) as a brown semi-solid. 1 H NMR (400 MHz, DMSO-d 6)δ=6.99 - 6.95(m, 1H), 6.73(br d, J = 7.5 Hz, 1H), 6.44 - 6.38(m, 1H), 5.75 - 5.49(m, 2H), 5.26 - 4.99(m, 1H), 4.44(s, 2H), 3.87 - 3.80(m, 3H), 2.23(s, 3H) LC-MS m / z 316.3 [M + H] +

[0125] Step 4. To a stirred solution of 5-amino-1-(4-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (1.1 g, 3.49 mmol) in DMSO (10.0 mL) were added DBU (1.577 mL, 10.47 mmol), BOP (2.314 g, 5.23 mmol) and (5-methyl-1,2,4-oxadiazol-3-yl)methanamine hydrochloride (0.522 g, 3.49 mmol). The reaction mixture was stirred at RT for 2 h. (5-Methyl-1,2,4-oxadiazol-3-yl)methanamine, HCl (0.3 g, 2.0 mmol) was added. The reaction mixture was stirred at RT for 16 h and partitioned between EtOAc and water. The organic layer was washed with brine and dried over Na 2 SO 4 and filtered, concentrated in vacuo to give a residue. The crude compound was purified by ISCO Combiflash chromatography eluting with 0 - 20% methanol in chloroform to give (4-((5-amino-3-methyl-7-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-3-methoxyphenyl)methanol (0.81 g, 1.243 mmol, 35.6% yield) as a brown solid. 1 1H NMR (400 MHz, DMSO-d 6)δ = 7.60 - 7.55 (m, 1H), 7.26 (br t, J = 5.8 Hz, 1H), 6.98 - 6.93 (m, 1H), 6.77 (br d, J = 7.5 Hz, 1H), 6.68 - 6.60 (m, 1H), 5.68 (s, 2H), 5.55 - 5.48 (m, 1H), 5.20 - 5.13 (m, 1H), 4.78 (br d, J = 5.5 Hz, 2H), 4.49 - 4.42 (m, 2H), 3.82 - 3.77 (m, 3H), 2.56 (d, J = 2.0 Hz, 4H), 2.55 - 2.50 (m, 6H) LC-MS m / z 411.2 [M + H] +

[0126] Step 5. To a stirred solution of (4 - ((5 - amino - 3 - methyl - 7 - (((5 - methyl - 1,2,4 - oxadiazol - 3 - yl)methyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-3 - methoxyphenyl)methanol (0.45 g, 1.096 mmol) in THF (10.0 mL) at 0 °C was added SOCl 2 (1.0 ml, 13.70 mmol). The reaction mixture was stirred at 0 °C for 1 h, warmed to RT, concentrated in vacuo, and crude 1 - (4 - (chloromethyl)-2 - methoxybenzyl)-3 - methyl - N7 - ((5 - methyl - 1,2,4 - oxadiazol - 3 - yl)methyl)-1H - pyrazolo[4,3 - d]pyrimidine - 5,7 - diamine (0.51 g, estimated yield 100%) was obtained as a brown solid and used as such in the next step. LC-MS m / z 429.4 [M + H] +

[0127] Step 6. To a stirred solution of 1 - (4 - (chloromethyl)-2 - methoxybenzyl)-3 - methyl - N7 - ((5 - methyl - 1,2,4 - oxadiazol - 3 - yl)methyl)-1H - pyrazolo[4,3 - d]pyrimidine - 5,7 - diamine (0.15 g, 0.350 mmol) in DMF (3.0 mL) were added 1 - methylpiperazine (0.053 g, 0.525 mmol) and K 2 CO 3(0.145 g, 1.049 mmol) was added. The reaction mixture was stirred at 50 °C for 90 minutes, filtered through a CELITE™ bed, and washed with excess ethyl acetate. The filtrate was concentrated under reduced pressure to give a residue. The crude compound was purified by reverse-phase preparative LC / MS (column: TRIART-YMC-EXRS (250 mm x 19 mm); mobile phase A: in water, 10 mM NH 4 OAc pH-4.5, mobile phase B: CH 3 CN; flow rate: 20 mL / min; gradient: 0 / 0, 10 / 15, 20 / 15, 22 / 100, 24 / 0). Fraction collection was triggered by MS and UV signals. The fractions containing the desired product were combined and dried by centrifugal evaporation using a Genevac apparatus to give Compound 134 (12.6 mg, 0.025 mmol, 7.15% yield). Example 16 - Compound 132

Chemical Structure

[0128] To a stirred solution of 1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-N7-((5-methyl-1,2,4-oxadiazol-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (0.15 g, 0.350 mmol) in DMF (3.0 mL), 2-(piperazin-1-yl)ethan-1-ol (0.068 g, 0.525 mmol), 2-(piperazin-1-yl)ethan-1-ol (0.068 g, 0.525 mmol) and K 2 CO 3 (0.097 g, 0.699 mmol) were added. The reaction mixture was stirred at 50 °C for 90 minutes, filtered through a CELITE™ bed, and washed with excess ethyl acetate. The filtrate was concentrated under reduced pressure to give a residue, which was purified by reverse-phase preparative LC / MS (column: Gemini NX (250 x 21.2 mm) 5μm, mobile phase A: in water, 10 mM ammonium bicarbonate 9.5pH, mobile phase B: CH 3Purified by CN, flow rate: 20 mL / min, gradient T / %B: 0 / 10, 7 / 35, 12 / 35, 12.01 / 100). Fraction collection was triggered by MS and UV signals. The fractions containing the target were combined and dried by centrifugal evaporation using a Genevac apparatus to obtain Compound 132 (51.2 mg, 0.095 mmol, yield 27.2%). Example 17 - Compound 133 [Chemical formula]

[0129] To a stirred solution of 1-(4-(chloromethyl)-2-methoxybenzyl)-3-methyl-N7-((5-methyl-1,2,4-oxadiazol-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (0.15 g, 0.350 mmol) in acetonitrile (3.0 mL) were added tetrahydro-2H-pyran-4-amine hydrochloride (0.072 g, 0.525 mmol), Na 2 CO 3 (0.111 g, 1.049 mmol) and KI (0.058 g, 0.350 mmol). The reaction mixture was stirred at 50 °C for 3 h. The reaction mixture was filtered through a CELITE™ bed and washed with an excess of ethyl acetate. The filtrate was concentrated under reduced pressure to give a residue. The crude compound was purified by reverse-phase preparative LC / MS (column: Gemini NX (250 x 21 mm) x 5 micron; mobile phase A: in water, 10 mM NH 4 OAc, mobile phase B: CH 3 CN:MeOH (1:1), flow rate: 19 mL / min, gradient: 0 / 35, 12 / 45). Fraction collection was triggered by MS and UV signals. The fractions containing the target were combined and dried by centrifugal evaporation using a Genevac to obtain Compound 133 (17.4 mg, 0.035 mmol, yield 10.08%). Example 18 - Starting Materials and Intermediates

[0130] The following charts show schemes for creating compounds that may be useful as starting materials or intermediates for the preparation of TLR7 agonists disclosed herein. The schemes 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 [Chemical formula] Chart 2 [Chemical formula] Chart 3 [Chemical formula]

[0131] Biological Activity The biological activity of the compounds disclosed herein as TLR7 agonists may be quantified by the following procedure.

[0132] Human TLR7 Agonist Activity Assay This procedure describes a method for quantifying the human TLR7 (hTLR7) agonist activity of the compounds disclosed herein.

[0133] 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% CO 2 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% CO 2Incubated at [temperature]. 18 hours after the treatment, 10 microliters of freshly prepared Quanti-Blue™ reagent (Invivogen) was added to each well and incubated for 30 minutes (37 °C, 5% CO 2 ), and the SEAP level was measured using an Envision plate reader (OD = 620 nm). The half-maximal effective concentration value (EC 50 ; the compound concentration that causes a response midway between the assay reference value and the maximum value) was calculated.

[0134] 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.

[0135] Heparinized human whole blood was collected from human patients and treated with TLR7 agonist test compounds 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 (H 2 0 medium 5x→1x, warm at 37 °C; Cat# BD 558049), and the palm buffer was maintained (on ice) for later use.

[0136] 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 / permeabilization buffer, resuspended using a Felix mate (changing the tip for each plate 15 times), and incubated at 37 °C for 10 minutes.

[0137] Centrifuged at 2000 rpm for 5 minutes, aspirated with an HCS plate washer, mixed on a shaker for 30 seconds, then washed with 70 μL of dPBS and pelleted twice (2000 rpm, 5 minutes), washed with 50 μL of FACS buffer and pelleted once (2000 rpm, 5 minutes). Mixed on a shaker for 30 seconds. For intracellular marker staining (MX-1): 50 μl of BD Perm buffer III was added and mixed on a shaker for 30 seconds. Incubated on ice for 30 minutes (in the dark). Washed twice with 50 μL of FACS buffer (centrifuged at 2300 rpm for 5 minutes after permeabilization), then mixed on a shaker for 30 seconds. Resuspended in 20 μL of FACS buffer containing MX1 antibody ((4812)-Alexa 647: Novus Biologicals #NBP2-43704AF647) (20 μl FACS buffer + 0.8 μl hIgG + 0.04 μl MX-1). Centrifuged at 1000 rpm for 1 minute, mixed on a shaker for 30 seconds, incubated the sample at RT in the dark for 45 minutes, and then washed with 2x FACS buffer (centrifuged at 2300 rpm for 5 minutes after permeabilization). Resuspended in 20 μl of FACS buffer (35 μL in total per well), covered with foil, placed at 4 °C, and read the next day. The plate was read with an iQuePlus. The results were loaded into the toolset and an IC50 curve was created with CurveMaster. 100% on the y-axis was set to 1 μM of resiquimod.

[0138] Induction of TNF-alpha and Type I IFN Response Genes in Mouse Blood The induction of TNF-alpha and type I IFN response genes is a downstream event that occurs upon activation of the TLR7 pathway. The following is an assay for measuring their induction in mouse whole blood in response to a TLR7 agonist.

[0139] Heparinized 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). Ninety microliters 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% CO 2 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). Seventy microliters 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 of sample in the wells 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.

[0140] 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 RNA extraction, cDNA was synthesized in a 20 μL reverse transcriptase reaction using the Invitrogen SuperScript IV VILO Master Mix (Cat# 11756500). TaqMan (registered trademark) 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 and TaqMan Master Mix for mouse IFIT1, IFIT3, MX1, and PPIA gene expression. PPIA was used as a housekeeping gene. According to the manufacturer's recommendations. 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.

[0141] Definitions "Aliphatic" means a straight-chain or branched-chain saturated or unsaturated non-aromatic hydrocarbon moiety having a specific number of carbon atoms (e.g., "C 3 aliphatic", "C 1-5 aliphatic", "C 1 -C 5 aliphatic", or "C 1 to C 5 aliphatic" as such. 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 specified, 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, namely C 2-4 alkene, C 4 -C 7 cycloaliphatic, etc. Similarly, "(CH 2 ) 1-3Terms such as "」" should be understood as an abbreviated expression where the subscript is 1, 2, or 3. Therefore, such terms represent CH 2 CH 2 CH 2 and CH 2 CH 2 CH 2 will represent.

[0142] "Alkyl" means a saturated aliphatic moiety following the same convention for specifying the number of applicable carbon atoms. As examples, C 1 -C 4 The alkyl moiety includes, but is 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,

Chemical formula

[0143] "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. As examples, C 2 -C 4 The alkenyl moiety includes, but is 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.

[0144] "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. As examples, C 2 -C 4 The alkynyl group includes, but is not limited to, ethynyl (acetylenyl), propargyl (prop-2-ynyl), 1-propynyl, but-2-ynyl, and the like.

[0145] "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.

[0146] "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 optionally be oxidized and N may optionally be 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.

[0147] "Alkoxy", "aryloxy", "alkylthio", and "arylthio" mean -O(alkyl), -O(aryl), -S(alkyl), and -S(aryl), respectively. Examples are methoxy, phenoxy, methylthio, and phenylthio, respectively.

[0148] "Halogen" or "halo" means fluorine, chlorine, bromine or iodine, unless a more narrow meaning is indicated.

[0149] "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, for example 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

[0150] "Heteroaryl" means a moiety having a mono-, bi- or tricyclic ring system (preferably a 5- to 7-membered monocyclic ring system) 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.

[0151] For example, when using "unsubstituted or substituted" or "optionally substituted", that is, "unsubstituted or substituted C 1 -C 5 alkyl" or "optionally substituted heteroaryl", etc., indicating that the moiety may be substituted, such 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 substituent is attached, and provide compounds that are chemically stable, known in the art, and can be synthesized by the methods described herein. When a moiety is specified as "unsubstituted or substituted" or "optionally substituted", in a preferred embodiment, such moiety is unsubstituted.

[0152] "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, for example, an alkyl, alkenyl, or alkynyl moiety having an open (unsatisfied) valence like 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, for example, a moiety like 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.).

[0153] 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 -OCF 3 ), -O(cycloalkyl), -O(heterocycloalkyl), -O(aryl), alkylthio, arylthio, =O, =NH, =N(alkyl), =NOH, =NO(alkyl), -C(=O)(alkyl), -C(=O)H, -CO 2 H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH 2 , -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)NH 2 , -OC(=O)NH(alkyl), -OC(=O)N(alkyl) 2 , azide, -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH 2 , -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl) 2 , -NHC(=NH)NH 2 , -OSO 2 (alkyl), -SH, -S(alkyl), -S(aryl), -S(cycloalkyl), -S(=O)alkyl, -SO 2 (alkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), -SO 2 N(alkyl) 2 and the like, but are not limited thereto.

[0154] 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), -CO 2 H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH 2 、-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)NH 2 、-OC(=O)NH(alkyl), -OC(=O)N(alkyl) 2 、azide, -NH 2 、-NH(alkyl), -N(alkyl) 2 、-NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH 2 、-NHC(=O)NH(alkyl), -NHC(=O)N(alkyl) 2 、-NHC(=NH)NH 2 、-OSO 2 (alkyl), -SH, -S(alkyl), -S(aryl), -S(=O)alkyl, -S(cycloalkyl), -SO 2 (alkyl), -SO 2 NH 2 、-SO 2 NH(alkyl), and -SO 2 N(alkyl) 2 are. More preferred substituents are halo, hydroxyl, cyano, nitro, alkoxy, -O(aryl), =O, =NOH, =NO(alkyl), -OC(=O)(alkyl), -OC(=O)O(alkyl), -OC(=O)NH 2 、-OC(=O)NH(alkyl), -OC(=O)N(alkyl)2 , azide, -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH 2 , -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl) 2 , and -NHC(=NH)NH 2 are. Particularly preferred substituents are phenyl, cyano, halo, hydroxyl, nitro, C 1 -C 4 alkoxy, O(C 2 -C 4 alkanediyl)OH, and O(C 2 -C 4 alkanediyl)halo.

[0155] When the substituted moiety 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, -CO 2 H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH 2 , -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)NH 2 , -OC(=O)NH(alkyl), -OC(=O)N(alkyl) 2 , azide, -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH 2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl) 2 , -NHC(=NH)NH 2 , -OSO 2 (alkyl), -SH, -S(alkyl), -S(aryl), -S(cycloalkyl), -S(=O)alkyl, -SO 2 (alkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), and -SO 2 N(alkyl) 2 is. More preferred substituents are alkyl, alkenyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -C(=O)(alkyl), -C(=O)H, -CO 2 H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH 2 , -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)NH 2 , -OC(=O)NH(alkyl), -OC(=O)N(alkyl) 2 , -NH 2 , -NH(alkyl), -N(alkyl) 2 , -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH 2 , -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl) 2 , and -NHC(=NH)NH 2 is. Particularly preferred substituents are C 1 -C 4 alkyl, cyano, nitro, halo, and C 1 -C 4 alkoxy.

[0156] "C 1 -C 5When a range is recited, such as "alkyl" or "5 to 10%", such range includes the endpoints of the range, i.e., in the first example, C 1 and C 5 , and in the second example, 5% and 10%.

[0157] (For example, by making the valence marks at the relevant stereocenters in the structural formula thick or dashed lines, or by depicting the double bond in the structural formula as having an E or Z configuration, or by using a nomenclature or symbol that specifies stereochemistry) Unless a particular stereoisomer is clearly indicated, all stereoisomers are included within the scope of the invention as pure compounds as well as mixtures thereof. Unless otherwise specified, 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.

[0158] 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 equivalent to those depicted by the structural formulas used herein, and that the structural formulas include such tautomers, resonance structures, zwitterionic forms.

[0159] "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 C 1 -C 5 alkyl, C 2 -C 5 alkenyl or C 2 -C 5 alkynyl esters, particularly methyl, ethyl or n-propyl esters.

[0160] "Pharmaceutically acceptable salts" means salts of compounds suitable for pharmaceutical formulations. When the compound has one or more basic groups, the salts can be acid addition salts such as 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 the compound has one or more acidic groups, the salts can be salts such as 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.

[0161] "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 such as humans.

[0162] 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 tumor cell infiltration 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 anti-tumor immune response that 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.

[0163] In the formulas herein, a wavy line (

Chem.

Chem.

Chem.

Chem.

Chem.

[0164] In the formulas of this specification, a valence bond that crosses an aromatic ring between two carbons means that the group attached to that valence bond can be at any of the positions of the aromatic ring that become available by the removal of hydrogen that is implicitly there (or explicitly there if fully written). As an example:

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0165] 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 isotopically labeled appropriate reagents instead of the unlabeled reagents used in other cases. As an example, a C 1 -C 3 alkyl group may be not deuterated, partially deuterated, or fully deuterated, and "CH 3 " includes CH 3, 13 CH 3 , 14 CH 3 , CH 2 T, CH 2 D, CHD 2 , CD 3 etc. are included. In one embodiment, the various elements in the compound are present at their natural isotopic abundances.

[0166] 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.

[0167] Acronyms and Abbreviations Table C provides a list of the acronyms and abbreviations used herein, along with their meanings.

Table 18

Table 19

[0168] References Earlier in this specification, the complete citations for the following references, which are cited in abbreviated form by the first author (or inventors) and date, are provided below. Each of these references is hereby incorporated by reference into this specification for all purposes.

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[0241] The foregoing detailed description of the invention includes sections that relate primarily or exclusively to particular parts or aspects of the invention. This is for purposes of clarification and convenience, and it is to be understood that a particular feature may be relevant not only in the section in which it is disclosed but also in other sections, and that the disclosure herein includes all appropriate combinations of the information described in the different sections. Similarly, although the various figures and descriptions herein relate to particular embodiments of the invention, if a specific feature is disclosed in the context of a particular figure or embodiment, it is to 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 invention, as appropriate.

[0242] Furthermore, although the invention has been particularly described with respect to certain preferred embodiments, the invention is not limited to such preferred embodiments. On the contrary, the scope of the invention is defined by the appended claims.

Claims

1. A compound having a structure represented by the following formula (Ia): 【Chemical 1】 [wherein, R １ is 【Chemical 2】 is; R ３ is OH, 【Chemical Formula 3】 is]

2. A pharmaceutical composition for treating cancer, comprising an anti-cancer immunotherapeutic agent and the compound according to Claim 1.

3. The pharmaceutical composition according to Claim 2, wherein the anti-cancer immunotherapeutic agent is an antagonist anti-CTLA-4, anti-PD-1, or anti-PD-L1 antibody.

4. The pharmaceutical composition according to Claim 2, wherein the anti-cancer immunotherapeutic agent is ipilimumab, nivolumab, or pembrolizumab.

5. The pharmaceutical composition according to any one of Claims 2 to 4, wherein the cancer is 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. ​

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