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 TIFF2023512229000155.tif4164 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 / 057,661, filed Jul. 28, 2020, and U.S. Provisional Application Serial No. 62 / 966,092, filed Jan. 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 combat 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 globally stimulating the immune response in the treatment of various pathologies other than actual pathogen infections. Therefore, there is great interest in the use of TLR7 agonists as vaccine adjuvants or as enhancers in cancer immunotherapy. See, for example, Vasilakos and Tomai 2013, Sato-Kaneko et al. 2017, Smits et al. 2008, and Ota et al. 2019.

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

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

Chemical formula

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

Chemical formula

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

Chemical formula

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

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

[0011] There is a disclosure of related molecules in which the 6,5-fused ring system - pyrimidine 6-membered ring and imidazole 5-membered ring of formula (A) are fused and modified. (a) Dellaria et al. 2007, Jones et al. 2010 and 2012, and Pilatte et al. 2017 disclose compounds in which the pyrimidine ring is substituted by 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 by 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 by 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 partner molecules, 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 publication year is set forth 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

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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

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

Mode for Carrying Out the Invention

[0021] Compound In one embodiment, in moiety Ar of formula (I), one X is N and the remaining X's are CH, and one CH has H substituted by W.

[0022] In one embodiment, W is

Chemical formula

Chemical formula

[0023] In one embodiment, the compound of the present disclosure is represented by the following formula (Ia), wherein R 1 , R 5 , and W are as defined for formula (I):

Chemical formula

[0024] In another aspect, the compounds of the present disclosure are represented by the following formula (Ib), wherein R 1 , R 3 , and R 5 are as defined for formula (I):

Chemical Structure

[0025] In one embodiment of the compound represented by formula (Ib), R 3 is NH(C1-C5 alkyl), N(C1-C5 alkyl)2, NH(C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), NH(C1-C4 alkanediyl) 0-1 (C4-C 10 bicycloalkyl), NH(C1-C4 alkanediyl) 0-1 (C5-C 10 spiroalkyl), N(C3-C6 cycloalkyl)2, N[C1-C3 alkyl](C1-C6 alkyl), or the following structure:

Chemical Structure

[0026] In another embodiment of the compound represented by formula (Ib), R 3 is NH[C(=O)](C1-C5 alkyl), NH[C(=O)](C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), NH[C(=O)](C1-C4 alkanediyl) 0-1 (C4-C 10 bicycloalkyl), NH[C(=O)](C1-C4 alkanediyl) 0-1 (C5-C10 (spiroalkyl), or N[C1-C3 alkyl]C(=O)(C1-C6 alkyl) is.

[0027] In another embodiment of the compound represented by formula (Ib), R 3 is O(C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), O(C1-C4 alkanediyl) 0-1 (C4-C8 bicycloalkyl), O(C1-C4 alkanediyl) 0-1 (C5-C 10 (spiroalkyl), or O(C1-C4 alkanediyl) 0-1 (C1-C6 alkyl) is.

[0028] In another aspect, the compounds of the present disclosure are represented by the following formula (Ic), wherein R 1 , R 4 and R 5 are as defined for formula (I): [Chemical formula]

[0029] In one aspect, the present disclosure provides the following formula (Id): [Chemical formula] [wherein, W is [Chemical formula] is] and provides a compound having the structure represented by.

[0030] In one embodiment, W is [Chemical formula] and n is 1, 2, or 3.

[0031] In another embodiment, the compound of the present disclosure has the following formula (Ie): [Chemical formula] [wherein, R 1 is [Chemical formula] ; R 5 is H or Me; R 7 is H, C1-C5 alkyl, or C3-C6 cycloalkyl; where the cycloalkyl group may optionally have a CH2 group substituted with O, NH, or N(C1-C3) alkyl. as shown.

[0032] Examples of the group R 1 are [Chemical formula] .

[0033] Preferably, R 1 is [Chemical formula] selected from the above group (the "preferred R 1 group").

[0034] Examples of the group R 3 include [Chemical formula] [Chemical formula] .

[0035] Preferably, R 3 is [Chemical formula] selected from the above group (the "preferred R 3 group").

[0036] Examples of the group R 4 include: [Chemical formula] etc.

[0037] Preferably, R 4 is [Chemical formula] selected from the above group (the "preferred R 4 group").

[0038] Examples of the group R 5 are H, [Chemical formula] etc.

[0039] Preferably, R 5 is H or Me.

[0040] In one embodiment, the compound represented by formula (Ib) has R 1 selected from the preferred R 1 group, R 3 selected from the preferred R 3 group, and R 5 is H or Me.

[0041] In one embodiment, the compound represented by formula (Ic) has R 1 selected from the preferred R 1 group, R 4 selected from the preferred R 4 group, and R 5 is H or Me.

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

Chemical formula

Chemical formula

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

Chemical formula

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

Chemical formula

Chemical formula

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

Chemical formula

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

Chemical formula

Chemical formula

[0047] In one embodiment, W is:

Chemical formula

[0048] In one aspect, W is [Chem.] 、 particularly [Chem.] and is, a specific and representative embodiment is [Chem.] .

[0049] In one aspect, W is [Chem.] and is, a specific and representative embodiment is [Chem.] .

[0050] In one aspect, W is [Chem.] and is, a specific and representative embodiment is [Chem.] .

[0051] In one aspect, W is [Chem.] and a specific and representative embodiment is [Chemical formula] .

[0052] In one aspect, W is [Chemical formula] and a specific and representative embodiment is [Chemical formula] .

[0053] In one aspect, W is [Chemical formula] , particularly [Chemical formula] and a specific and representative embodiment is [Chemical formula] .

[0054] In one aspect, W is [Chemical formula] , particularly [Chemical formula] and a specific and representative embodiment is [Chemical formula] .

[0055] In one aspect, W is [Chemical formula] and specific and representative embodiments are [Chemical formula] .

[0056] In one aspect, W is [Chemical formula] and specific and representative embodiments are [Chemical formula] .

[0057] In one aspect, W is [Chemical formula] and specific and representative embodiments are [Chemical formula] .

[0058] In one aspect, the compound of the present disclosure has the following formula (Ia): [Chemical formula] [wherein, R 1 is [Chemical formula] ; R 5 is H (preferably) or Me; W is [Chemical formula] [] represented by.

[0059] The above-mentioned representative alkyl, cycloalkyl, spiroalkyl, bicycloalkyl and other groups and the following formula: [Chemical formula] Some of the moieties shown may have any substituent and / or may optionally have one or more CH2 groups substituted with O, SO2, etc., as described in the "Summary of the Invention" above.

[0060] Specific examples of the compounds disclosed herein are shown in Table A below. The table also provides data regarding biological activity: human TLR7 agonism reporter assay and / or induction of the CD69 gene in human whole blood, determined through the procedures provided below. Analytical data (mass spectrum, LC / MS retention time, and NMR) are described in the rightmost column. In one embodiment, the compounds of the present disclosure have (a) an EC 50 value in the human TLR7 (hTLR7) reporter assay of less than 1,000 nM and (b) an EC 50 value for CD69 induction in human whole blood (hWB) of less than 1,000 nM. (When the assay is performed multiple times, the reported value is the average value.) [Chemical formula] [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

Table 17

Table 18

Table 19

Table 20

Table 21

[0061] 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.

[0062] 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).

[0063] 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, subdural, intraspinal, epidural and intrasternal injections and infusions. Alternatively, the pharmaceutical composition may be administered by a non-parenteral route such as a topical, epithelial or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.

[0064] 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 a high drug concentration. The composition may also be provided in the form of a lyophilized product to be reconstituted with water before administration.

[0065] The amount of active ingredient that can combine with the carrier substance to produce a single dosage form will vary depending on the patient being treated and the particular method of administration, and will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 0.01 percent to about 99 percent of the active ingredient, 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.

[0066] 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 the parenteral composition 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.

[0067] The dosage ranges from about 0.0001 to 100 mg / kg, more generally from 0.01 to 5 mg / kg, based on the body weight of the host. For example, the 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 within the range of 1 - 10 mg / kg, or 0.1 to 5 mg / kg. Representative treatment regimens are administrations 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 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 using one of these. 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.

[0068] The "therapeutically effective amount" of the compounds of the present invention preferably results in a reduction in the severity of the symptoms of the disease, an increase in the number and duration of asymptomatic periods of the disease, or the prevention of functional or physical impairment due to the pain of the disease. For example, for the treatment of a patient having cancer, the "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, still more preferably at least about 60%, 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, alternatively, 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, rather than as individual agents.

[0069] The pharmaceutical composition can be a controlled release or sustained release formulation such as an implant, a transdermal patch, and a microencapsulated 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.

[0070] The therapeutic composition can be administered using medical devices such as (1) a needleless subcutaneous injector; (2) a micro-infusion pump; (3) a transdermal device; (4) an infusion device; and (5) an osmotic device.

[0071] 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.

[0072] Industrial Applicability and Use The TLR7 agonist compounds disclosed in this specification can be used for the treatment of diseases or conditions that can be alleviated by the activation of TLR7.

[0073] 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 the activation of T cells, by stimulating the body's immune system to attack and destroy cancer cells. The immune system has a number of checkpoint (regulatory) molecules that help maintain the balance between its legitimate attack on target cells and 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. The binding of agonist immunotherapeutic agents to stimulatory checkpoint molecules can result in the activation of the latter and an enhanced immune response against cancer cells. Alternatively, the binding of antagonist immunotherapeutic agents to inhibitory checkpoint molecules can prevent the downregulation of the immune system by the latter and help maintain an active response against cancer cells. Examples of stimulatory checkpoint molecules are B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H. Examples of inhibitory checkpoint molecules are CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, CD96 and TIM-4.

[0074] In the mechanism of action of either anti-cancer immunotherapeutic agent, its effectiveness 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.

[0075] 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, heart 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.

[0076] 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, bonvelorizumab. Their alternative names (trade names, former names, research codes, or synonyms) and their respective target checkpoint molecules are shown in Table B below.

Table 22

[0077] 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, renal cancer, head and neck cancer, lymphoma (including Hodgkin lymphoma), skin cancer (including melanoma and Merkel cell carcinoma), urothelial cancer (including bladder cancer), gastric cancer, hepatocellular carcinoma, or colorectal cancer.

[0078] In another embodiment of the combination therapy with a TLR7 agonist, the anticancer immunotherapeutic agent is an antagonist anti-CTLA-4 antibody, preferably ipilimumab.

[0079] 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.

[0080] The TLR7 agonists disclosed herein are also useful as vaccine adjuvants.

[0081] The implementation of the present invention can be further understood by referring to the following examples provided as illustrations rather than limitations.

Example

[0082] Analysis Procedure NMR The following conditions were used to obtain proton nuclear magnetic resonance (NMR) spectra: NMR spectra were obtained using either DMSO-d6 or CDCl3 as the solvent and internal standard, on either a 400 MHz or 500 MHz Bruker instrument. The raw NMR data were analyzed by using either ACD Spectrus version 2015-01 from ADC Labs or MestReNova software.

[0083] Chemical shifts are reported in parts per million (ppm) on the low magnetic field side, relative to the internal tetramethylsilane (TMS) or the position of TMS inferred from 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. Integration values are approximate. It should be noted that integration intensity, peak shape, chemical shift, and coupling constants can depend on the solvent, concentration, temperature, pH, and other factors. Additionally, peaks that overlap with water or solvent peaks in the NMR spectrum, or for which 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.

[0084] Liquid Chromatography The following preparative and / or analytical liquid chromatography methods were used:

[0085] Preparative HPLC / MS Method A: 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: 0 - 47% B over 20 minutes, then hold at 100% B for 0 minutes; Flow Rate: 20 mL / min; Column Temperature: 25 °C

[0086] Preparative HPLC / MS Method B: Column: XBridge C18, 150 mm x 19 mm, 5 μm particles; Mobile Phase A: water containing 0.05% TFA; Mobile Phase B: acetonitrile containing 0.05% TFA; Gradient: hold at 10% B for 2 minutes, 10 - 100% B over 20 minutes, then hold at 100% B for 3 minutes; Flow Rate: 19 mL / min; Column Temperature: 25 °C

[0087] Preparative HPLC / MS Method C: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile Phase A: 5:95 acetonitrile: water containing 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water containing 10 mM ammonium acetate; Gradient: 1 - 65% B over 20 minutes, then hold at 100% B for 0 minutes; Flow Rate: 20 mL / min; Column Temperature: 25 °C

[0088] Analytical LC / MS Method D: 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)

[0089] Analysis of LC / MS Method E: Column: Acquity UPLC BEH C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: Water containing 0.1% formic acid; Mobile Phase B: Acetonitrile containing 0.1% formic acid; Temperature: 40 °C; Gradient: Hold at 5% B for 0.2 minutes; from 5% B to 95% B over 2.3 minutes, then hold at 95% B for 0.20 minutes; Flow rate: 1 mL / min; Detection: UV (254 nm & 220 nm)

[0090] Analysis of LC / MS Method F: Column: Acquity UPLC BEH C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: Water containing 0.1% formic acid; Mobile Phase B: Acetonitrile containing 0.1% formic acid; Temperature: 40 °C; Gradient: Hold at 50% B for 0.2 minutes; from 50% B to 95% B over 2.3 minutes, then hold at 95% B for 0.20 minutes; Flow rate: 1 mL / min; Detection: UV (254 nm & 220 nm)

[0091] Synthesis - General Procedure Generally, the procedures disclosed herein result in a mixture of positional isomers alkylated at the 1H or 2H position of the pyrazolopyrimidine ring system (also referred to as N1 and N2 positional isomers respectively, referring to the alkylated nitrogen). For the sake of simplicity, the N2 positional isomer is not shown, but it is understood to be present in the initially formed mixture and will be separated later, for example, by preparative HPLC.

Chemical Structure

[0092] The mixture of positional isomers may be separated at the initial stage of the synthesis and the remaining synthesis steps may be carried out using the 1H positional isomer, or alternatively, if necessary, the synthesis may be advanced using the mixture of positional isomers and the separation may be carried out at a later stage.

[0093] The compounds of the present disclosure can be prepared by a number of methods well known to those skilled in the art of organic synthetic chemistry. These methods include the methods described below, or variations thereof. Preferred methods include, but are not limited to, the methods described in the following schemes. The schemes are intended to be general, but in some cases, features may be specifically shown for convenience (e.g., methyl esters or specific positional isomers). Scheme 1

Chemical formula

[0094] R a In Scheme 1 and other instances where it appears, for example,

Chemical formula

[0095] Compound 9 can be prepared by the synthetic sequence illustrated in Scheme 1. Convert quinoline 1 (CAS Registry Number 82867-40-6) to hydrazine intermediate 2 with BOC-protected hydrazine. Treating with hydrochloric acid gives intermediate 3. Mix ethyl-2-chloro-2-oxoacetate and (Z)-N,N-dimethyl-2-nitroethene-1-amine, and then add intermediate 3 to obtain intermediate 4. Convert intermediate 4 to intermediate 5 by reducing the nitro group to an amine group with zinc. Treat intermediate 5 with 1,3-bis(methoxycarbonyl)-2-thiourea together with acetic acid, and then with sodium methoxide to obtain intermediate 6. In the presence of BOP and DBU, treat intermediate 6 with R aReacting with NH2 synthesizes intermediate 7. Hydroxylation with NaOH gives intermediate 8. In the last step of Scheme 1, R b NHR c and amide coupling prepares compound 9. Scheme 2

Chemical formula

[0096] The above Scheme 2 shows an alternative method for preparing intermediate 6. Coupling quinoline 1 and methyl 4-nitro-1H-pyrazole-5-carboxylate (CAS registration number 138786-86-9) gives intermediate 10. Reducing the nitro group of intermediate 10 to an amine group with zinc gives intermediate 11. As shown in step 3 of Scheme 2, treating intermediate 11 with 1,3-bis(methoxycarbonyl)-2-thiourea together with acetic acid and then with sodium methoxide gives intermediate 6. Scheme 3

Chemical formula

[0097] The above Scheme 3 shows an alternative method for preparing compound 9. Hydroxylation of intermediate 6 gives acid 12. Amide coupling gives intermediate 13. In the last step, treating intermediate 13 with R a NH2 in the presence of BOP and DBU gives compound 9. Scheme 4

Chemical formula

[0098] R d is, for example, H, F, CO2Me (or Et), or cyano in Scheme 4 and other cases where it appears. R eIn Scheme 4 and other cases where it appears, for example, it is H or CO2Me (or Et) or a protecting group.

[0099] The method of Scheme 4 above can be used to prepare Compound 20. Intermediate 15 is obtained by brominating Compound 14 with NBS (N-bromosuccinimide). Intermediate 15 is coupled with a quinoline compound where R d is a carboxylic acid ester to obtain Intermediate 16. (We have observed that when there is a bromine at the C3 position, the N1 / N2 ratio of the resulting mixture generally becomes higher.) The bromine group of Intermediate 16 is removed by catalytic hydrogenation to obtain Intermediate 17. The carboxylic acid ester in Intermediate 17 is reduced with LiAlH4 or LiBH4 to obtain Intermediate 18. Intermediate 18 is treated with thionyl chloride to obtain Intermediate 19. Treatment with an amine R b NHR c gives Compound 20. When R e contains a carbamate or other protecting group, the latter may be removed at this stage with sodium hydroxide or a suitable deprotecting reagent. Scheme 5

Chemical Structure

[0100] Scheme 5 above shows an alternative method for preparing Compound 20 by reductive amination. (In the case where R d is a cyano group) Intermediate 17 is reduced to Amine 18a. Then, Amine 18a is subjected to reductive amination with the corresponding ketone to obtain Compound 20. Scheme 6

Chemical Structure

[0101] Scheme 6 above shows a method for preparing Compound 23. Intermediate 19 (where R d is a carboxylic acid ester and R eStarting from (where is a carbamate protecting group), intermediate 19 is treated with 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane and PdCl2(dppf)-CH2Cl2 adduct, methylation occurs, and intermediate 21 is obtained. Intermediate 22 is obtained by hydrolysis with sodium hydroxide. In the final step, amide formation of intermediate 22 with R b NHR c gives compound 23. Scheme 7

Chemical Structure

[0102] R f is an amide or amine moiety in Scheme 7 and other cases where it appears, and Hal is a halogen such as Cl or Br.

[0103] Compound 30 can be prepared by the method of Scheme 7 above by coupling a pyrazolopyrimidine core and a quinoline moiety. The nitro group of starting material 24 is reduced to the amine group of compound 25. Intermediate 25 is treated with 1,3-bis(methoxycarbonyl)-2-thiourea together with acetic acid and then with sodium methoxide to obtain pyrazolopyrimidine 26. The quinoline compound 27 is prepared in the same manner as the reaction described in other schemes above. Pyrazolopyrimidine 26 is coupled with quinoline 27 to obtain intermediate 28. In the presence of BOP and DBU, intermediate 28 is treated with amine R a NH2 to obtain intermediate 29. In the final step, the carbamate protecting group of intermediate 29 is removed with sodium hydroxide to produce compound 30. Scheme 8

Chemical Structure

[0104] The above Scheme 8 shows how, when W is [Chemistry] It shows whether a compound in which n is 0 can be prepared.

[0105] The starting material 31 (CAS registration number 611-32-5) is converted to the intermediate 32 by bromination. The intermediate 33 is obtained by coupling with tert-butyl 4-(3,3,4,4-tetramethylborolan-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate. The intermediate 34 is obtained by hydrogenation. The intermediate 35 is obtained by treatment with NBS and AIBN. The intermediate 37 is obtained by mixing the intermediates 35 and 36 with a base. The intermediate 37 is converted to the intermediate 39 by coupling with the intermediate 38. The iodo group of the intermediate 39 is removed by reduction to obtain the intermediate 40. The compound 41 is obtained by hydrolysis with sodium hydroxide and an acid. The compound 41 is reductively aminated with the ketone R b R c C(=O) to obtain the compound 42.

[0106] Synthesis - Specific Example To further illustrate the above, the following non-limiting representative synthetic schemes are included. Variations of these examples within the scope of the claims are within the scope of those skilled in the art and are considered to be within the scope of the present disclosure. The reader, being a person skilled in the relevant art to whom the present disclosure is provided, will recognize that they will be able to prepare and use the compounds disclosed herein even without an exhaustive list of examples.

[0107] The analytical data for compounds numbered 100 or more can be found in Table A. Example 1 - Compound 111 [Chemistry]

[0108] Step 1. TEA (1.493 mL, 10.71 mmol) was added to a solution of methyl 8-(bromomethyl)quinoline-5-carboxylate (1 g, 3.57 mmol) and tert-butyl hydrazinecarboxylate (2.359 g, 17.85 mmol) in DMF (4 mL). The reaction mixture was stirred at 75 °C for 4 h, diluted with 100 mL of water, and extracted with EtOAc (3 x 75 mL). The organic phases were combined, concentrated, and purified by column chromatography: column: 40 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 min, 0 - 50% B over 14 min, then hold at 100% B for 3 min; flow rate: 40 mL / min; column temperature: 25 °C. The fractions containing the expected product were combined, concentrated, and dried under high vacuum for 1 h to give methyl 8-((2-(tert-butoxycarbonyl)hydrazinyl)methyl)quinoline-5-carboxylate (0.71 g, 60.0% yield). LC-MS m / z 332.2 [M+H] + ; retention time: 1.61 min (method E)

[0109] Step 2. HCl was added to a solution of methyl 8-((2-(tert-butoxycarbonyl)hydrazinyl)methyl)quinoline-5-carboxylate (0.71 g, 2.143 mmol) in MeOH (10 mL) in dioxane (5.36 mL, 21.43 mmol). The reaction mixture was stirred overnight at RT and changed to a slurry. The precipitate was collected by filtration and dried under high vacuum for 1 h to give the HCl salt of methyl 8-(hydrazinylmethyl)quinoline-5-carboxylate (0.58 g, 1.705 mmol, 79.6% yield). LC-MS m / z 232.1 [M+H] + ; retention time: 1.05 min (method E)

[0110] Step 3. A solution of (Z)-N,N-dimethyl-2-nitroethene-1-amine (1.528 g, 13.16 mmol) in DCM (26 mL) and pyridine (17.49 mL, 216 mmol) was cooled to -10 °C. Ethyl 2-chloro-2-oxoacetate (2.226 mL, 19.89 mmol) was added slowly. The reaction mixture was warmed to RT over 2 h and stirred overnight at RT. The reaction mixture was concentrated to 20 mL. Methyl 8-(hydrazinylmethyl)quinoline-5-carboxylate HCl salt (1 g, 4.32 mmol) was added. The resulting mixture was stirred at RT for 2 h. The reaction mixture was concentrated and purified by reverse-phase column chromatography: column: 50 g CombiFlash Aq column; mobile phase A: water containing 0.05 TFA; mobile phase B: acetonitrile containing 0.05% TFA; gradient: hold at 0% B for 1 min, 0 - 50% B over 12 min, then hold at 100% B for 3 min; flow rate: 40 mL / min; column temperature: 25 °C. Fractions containing the expected product were combined and lyophilized to give methyl 8-((5-(ethoxycarbonyl)-4-nitro-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (627 mg, 1.633 mmol, 37.8% yield) as a solid. LC-MS m / z 385.2 [M+H] + ; Retention time: 2.22 min (Method E)

[0111] Step 4. Zinc (358 mg, 5.48 mmol) was added to a solution of methyl 8-((5-(ethoxycarbonyl)-4-nitro-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (421 mg, 1.095 mmol) and ammonium formate (691 mg, 10.95 mmol) in MeOH (3 mL) and THF (5 mL). The reaction mixture was stirred at RT for 1 h. LCMS analysis indicated completion of the reaction. The reaction mixture was filtered, concentrated, and lyophilized with acetonitrile and water to give crude methyl 8-((4-amino-5-(ethoxycarbonyl)-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (285 mg, 0.804 mmol, 73.5%). LC-MS m / z 355.2 [M+H] + ; Retention time: 1.83 min (Method E)

[0112] Step 5. Acetic acid (0.64623 mL, 11.28 mmol) and TFA (0.07 mL) were added to a mixture of 1,3-bis(methoxycarbonyl)-2-methyl-2-thiourea (279 mg, 1.355 mmol) and methyl 8-((4-amino-5-(ethoxycarbonyl)-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (400 mg, 1.129 mmol) in MeOH (20 mL). The reaction mixture was stirred at RT overnight. LCMS analysis indicated conversion to the intermediate (LC-MS m / z 513.3 [M+H] + ). NaOMe (4.2 mL, 33.87 mmol) was added. The reaction mixture was stirred at RT for 1 h. Acetic acid was added to adjust the pH to 5. The product was collected by filtration and dried under high vacuum overnight to give methyl 8-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (313 mg, 0.765 mmol, 67.9% yield). LC-MS m / z 409.2 [M+H] + ; Retention time: 1.67 min (Method E)

[0113] Step 6. ((1H-Benzotriazol-1-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V) (401 mg, 0.906 mmol) was added to a solution of methyl 8-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (185 mg, 0.453 mmol), (S)-3-aminohexan-1-ol (HCl salt, 348 mg, 2.265 mmol), and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (0.305 mL, 2.039 mmol) in DMSO (1.5 mL). The reaction mixture was stirred at 70 °C overnight and worked up with EtOAc, brine, and water. The combined organic phases were concentrated and dried under high vacuum to give the crude intermediate (165 mg, LC-MS m / z 508.2 [M+H] + ). NaOH (10 N, 0.3 mL) was added to a solution of the crude intermediate (165 mg) in dioxane (0.6 mL). The reaction mixture was stirred at 70 °C for 5 h, neutralized with 0.2 mL of acetic acid, and purified by Method B. The fractions containing the expected product were combined and lyophilized to give (S)-8-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylic acid (82 mg, 0.188 mmol, 41.6% yield over 2 steps). LC-MS m / z 436.2 [M+H] + ; retention time: 1.33 min (Method E)

[0114] Step 7. DIPEA (0.032 mL, 0.184 mmol) was added to a solution of (S)-8-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylic acid (20 mg, 0.046 mmol), 2-(piperazin-1-yl)ethan-1-ol (0.023 mL, 0.184 mmol) and HATU (26.2 mg, 0.069 mmol) in DMF (0.5 mL). The reaction mixture was stirred at 20 °C for 3 h, neutralized with 0.05 mL acetic acid and purified by Method C. The fractions containing Compound 111 were combined and dried by centrifugal evaporation (2.74 mg, 0.005 mmol, 14.5%).

[0115] The following compounds were prepared similarly: Compound 108, Compound 112, Compound 113, Compound 114, Compound 125, Compound 126, Compound 127, Compound 128, Compound 129, Compound 130, Compound 131, Compound 132, Compound 133, Compound 134, Compound 135, Compound 136, and Compound 137. Example 2 - Compound 121

Chemical formula

[0116] Step 1. LiCl (0.908 g, 21.42 mmol) was added to a solution of methyl 8-(bromomethyl)quinoline-5-carboxylate (3 g, 10.71 mmol) in DMF (20 mL). The reaction mixture was stirred at RT for 30 min. LCMS analysis showed that the starting material was the chloro intermediate (LC-MS m / z 236.1 [M+H] +) was shown to be converted. Methyl 4-nitro-1H-pyrazole-5-carboxylate (3 g, 17.53 mmol) and Cs2CO3 (6.98 g, 21.42 mmol) were added. The reaction mixture was stirred overnight at RT. LCMS analysis showed the completion of the reaction and the formation of two isomers (retention times: 1.874 min & 1.992 min at 3 min acidic run, M+H / z 371.1). The reaction mixture was worked up with EtOAc, water and brine. The organic phases were combined, concentrated and purified by column chromatography: column: 80 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 2 min, 0 - 40% B over 24 min, then hold at 100% B for 3 min; flow rate: 60 mL / min; column temperature: 25 °C. The initial fractions with a retention time of 1.992 min were combined, concentrated and dried in vacuo to give methyl 8-((5-(methoxycarbonyl)-4-nitro-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (635 mg, 1.715 mmol, 16.01% yield). LC-MS m / z 371.1 [M+H] + ; retention time: 1.87 min (method E)

[0117] Step 2. Zinc (785 mg, 12.00 mmol) was added portionwise over 1 h to a solution of methyl 8-((5-(methoxycarbonyl)-4-nitro-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (635 mg, 1.715 mmol) in MeOH (7 mL) and THF (15 mL). The reaction mixture was stirred at RT for 2 h, diluted with EtOAc (50 mL) and filtered. The filtrate was concentrated and dried to give methyl 8-((4-amino-5-(methoxycarbonyl)-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate salt (685 mg, 2.013 mmol, 117% yield). LC-MS m / z 341.1 [M+H] + ; retention time: 1.61 min (method E)

[0118] Step 3. Acetic acid (0.530 mL, 9.26 mmol) and TFA (0.4 mL) were added to a solution of 1,3-bis(methoxycarbonyl)-2-methyl-2-thiourea (458 mg, 2.221 mmol) and methyl 8-((4-amino-5-(methoxycarbonyl)-1H-pyrazol-1-yl)methyl)quinoline-5-carboxylate (630 mg, 1.851 mmol) in MeOH (15 mL). The reaction mixture was stirred overnight at RT. LCMS analysis indicated completion of the reaction, and the intermediate (LC-MS m / z 499.2 [M+H] + ) was obtained. Sodium methoxide (5.78 mL, 46.3 mmol) was added. The reaction mixture was stirred at RT for 10 min. LCMS analysis showed completion of the reaction and formation of another intermediate (LC-MS m / z 409.2 [M+H] + ). The reaction mixture was concentrated to dryness. Water, 2 mL of DMF, and 1 mL of NaOH (10 N) were added to the residue. The reaction mixture was stirred at 60 °C for 3 h, neutralized with 1 mL of acetic acid, and concentrated in vacuo. The residue was purified by reverse-phase column chromatography: column: 150 g CombiFlash Aq column; mobile phase A: water containing 0.05 TFA; mobile phase B: acetonitrile containing 0.05% TFA; gradient: hold at 0% B for 2 min, 0 - 40% B over 23 min, then hold at 100% B for 4 min; flow rate: 75 mL / min; column temperature: 25 °C. The fractions containing the expected product were combined and lyophilized to give 8-((5-amino-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylic acid (265 mg, 0.788 mmol, 42.6% yield). LC-MS m / z 337.1 [M+H] + ; retention time: 1.05 min (method E)

[0119] Step 4. DIPEA (50 μL) was added to a solution of 8-((5-amino-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylic acid (28 mg, 0.083 mmol) and HATU (38.0 mg, 0.100 mmol) in DMF (0.5 mL). The reaction mixture was stirred at RT for 1 h, neutralized with 0.1 mL of acetic acid, and purified by Method B. The fractions containing the expected product were combined and lyophilized to give 8-((5-amino-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-N-(1-methylpiperidin-4-yl)quinoline-5-carboxamide (25 mg, 0.058 mmol, 69.7%). LC-MS m / z 433.2 [M+H] + ; Retention time: 0.96 min (Method E)

[0120] Step 5. ((1H-Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (V)) (51.1 mg, 0.116 mmol) was added to a solution of 8-((5-amino-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-N-(1-methylpiperidin-4-yl)quinoline-5-carboxamide (25 mg, 0.058 mmol), 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (0.039 mL, 0.260 mmol), and (S)-3-aminohexan-1-ol (27.1 mg, 0.231 mmol) in DMSO (1.25 mL). The reaction mixture was stirred at 70 °C for 5 h and lyophilized with acetonitrile and water. The residue was purified by Method C. The fractions containing the target compound were combined and dried by centrifugal evaporation to give Compound 121 (9.39 mg, 0.018 mmol, 30.4%).

[0121] The following compounds were prepared analogously: Compound 115, Compound 116, Compound 117, Compound 118, Compound 122, Compound 124, and Compound 138. Example 3 - Compound 110 [Chemical formula]

[0122] Step 1. ((1H-Benzotriazol-1-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate (V) (26.0 mg, 0.059 mmol) was added to a solution of methyl (7-hydroxy-1-(quinolin-8-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (10.3 mg, 0.029 mmol; prepared analogously from 8-(bromomethyl)quinoline according to Example 1), (S)-3-aminohexan-1-ol (17.23 mg, 0.147 mmol), and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (8.79 μl, 0.059 mmol) in DMSO (0.5 mL). The reaction mixture was stirred at 70 °C for 3 hours, neutralized with 0.2 mL of acetic acid, and purified by Method B. The fractions containing the expected product were combined and lyophilized to give methyl (S)-(7-((1-hydroxyhexan-3-yl)amino)-1-(quinolin-8-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (7.3 mg, 0.016 mmol, 55.2% yield). LC-MS m / z 450.1 [M+H] + ; Retention time: 1.64 minutes (Method E)

[0123] Step 2. An aqueous solution of NaOH (0.3 mL, 3.00 mmol) was added to methyl (S)-(7-((1-hydroxyhexan-3-yl)amino)-1-(quinolin-8-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (7.3 mg, 0.016 mmol) in dioxane (0.6 mL). The reaction mixture was stirred at 70 °C for 4 hours, neutralized with HOAc, and purified by Method B to give Compound 110 (0.80 mg, 0.002 mmol, 12.6%). Example 4 - Compound 119 [Chem.]

[0124] Step 1.1 - Bromopyrrolidine-2,5-dione (N-bromosuccinimide (NBS), 2.059 g, 11.57 mmol) was added to a solution of methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (2.2 g, 10.52 mmol) in DMF (20 mL). The reaction mixture was stirred at RT for 1 hour and worked up with EtOAc, water and brine. The organic phases were combined, concentrated and dried under high vacuum for 1 hour to give methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (2.85 g, 9.89 mmol, 94% yield). LC-MS m / z 288.0; 290.0 [M+H] + ; Retention time: 1.07 min (Method E)

[0125] Steps 2&3. LiCl (143 mg, 3.37 mmol) was added to a solution of methyl 8-(bromomethyl)quinoline-5-carboxylate (236 mg, 0.842 mmol) in DMF (3 mL). The reaction mixture was stirred at RT for 30 minutes. LCMS analysis indicated completion of the formation of the chloro intermediate, LC-MS m / z 236.1 [M+H] +was shown. Methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (291 mg, 1.010 mmol) and Cs2CO3 (1098 mg, 3.37 mmol) were added. The reaction mixture was stirred at RT for 120 h and worked up with EtOAc, water and brine. The organic phases were combined, concentrated and purified by column chromatography: column: 24 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 min, 0 - 70% B over 11 min then hold at 100% B for 2 min; flow rate: 35 mL / min; column temperature: 25 °C. The fractions containing the product were combined, concentrated and dried under high vacuum to give methyl 8-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (167 mg, 0.343 mmol, 40.7% yield). LC-MS m / z 487.1; 489.1 [M+H]+; retention time: 1.89 min (method E)

[0126] Step 4. ((1H-Benzotriazol-1-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V) (178 mg, 0.402 mmol) was added to a solution of methyl 8-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (98 mg, 0.201 mmol), (S)-3-aminohexan-1-ol HCl salt (155 mg, 1.006 mmol), and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (92 mg, 0.603 mmol) in DMSO (2.5 mL). The reaction mixture was stirred at 70 °C overnight, neutralized with HOAc, and purified (Method B). The fractions containing the product were combined and lyophilized to give methyl (S)-8-((3-bromo-7-((1-hydroxyhexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (63 mg, 0.107 mmol, 53.4% yield). LC-MS m / z 586.2 [M+H] + ; Retention time: 2.00 min (Method E)

[0127] Step 5. A mixture of methyl (S)-8-((3-bromo-7-((1-hydroxyhexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (50 mg, 0.085 mmol), K2CO3 (41.2 mg, 0.298 mmol), and PdCl2(dppf)-CH2Cl2 adduct (6.24 mg, 8.53 μmol) in dioxane (0.35 mL) and H2O (0.07 mL) was flushed with N2 for 1 minute. 2,4,6-Trimethyl-1,3,5,2,4,6-trioxatriphosphorinane (TMB, 107 mg, 0.853 mmol) was added, flushed with N2 for 1 minute, then sealed and stirred at 110 °C overnight. LCMS analysis showed disappearance of the starting material and formation of a new major peak (LC-MS m / z 464.3 [M+H] + ). Dioxane (0.43 mL) and 0.2 mL of 5N NaOH were added. The reaction mixture was stirred at 60 °C for 2 hours, neutralized with 0.2 mL of acetic acid, and purified by Method B. Fractions containing (S)-8-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylic acid were combined and lyophilized (29 mg, 0.065 mmol, 76% yield). LC-MS m / z 450.3 [M+H] + Retention time: 1.40 min (Method E)

[0128] Step 6. DIPEA (0.019 mL, 0.107 mmol) was added to a solution of (S)-8-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylic acid (12 mg, 0.027 mmol), tetrahydro-2H-pyran-4-amine (10.80 mg, 0.107 mmol) and HATU (15.23 mg, 0.040 mmol) in DMF (0.5 mL). The reaction mixture was stirred at 20 °C for 0.5 h, neutralized with 0.05 mL acetic acid and purified by Method C. The fractions containing compound 119 were combined and dried by centrifugal evaporation (3.49 mg, 0.007 mmol, 24.3%).

[0129] Compounds 120 and 123 were prepared analogously. Example 5 - Compound 109 [Chemical formula]

[0130] Step 1. LiCl (236 mg, 5.57 mmol) was added to a solution of methyl 8-(bromomethyl)quinoline-5-carboxylate (236 mg, 0.842 mmol) in DMF (3 mL). The reaction mixture was stirred at RT for 2 h. LCMS analysis indicated completion of the reaction (chloro intermediate, LC-MS m / z 236.1 [M+H] + ). 3-Bromo-N7-butyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (200 mg, 0.701 mmol) and Cs2CO3 (914 mg, 2.81 mmol) were added. The reaction mixture was stirred at RT over the weekend. LCMS analysis indicated the completion of the reaction along with two isomers corresponding to the desired mass (LC-MS m / z 484.2; 486.2 [M+H] +) was shown. The reaction mixture was washed with EtOAc, water and brine. The organic phases were combined, concentrated, and purified by column chromatography: column: 40 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 minute, 0 - 100% over 14 minutes, then hold at 100% B for 3 minutes; flow rate: 40 mL / min; column temperature: 25 °C. The fractions containing the product were combined, concentrated, and dried under high vacuum to give methyl 8-((5-amino-3-bromo-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (161 mg, 0.332 mmol, 47.5%). LC-MS m / z 484.2; 486.2 [M+H] + ; retention time: 1.85 minutes (Method E)

[0131] To a solution of methyl 8-((5-amino-3-bromo-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (161 mg, 0.332 mmol) in MeOH (10 mL) was added Pd-C (10%, 53 mg). The reaction mixture was stirred overnight under a hydrogen balloon and filtered. The filtrate was concentrated and dried under high vacuum to give methyl 8-((5-amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-4-carboxylate (128 mg, 0.316 mmol, 95.2%). LC-MS m / z 406.3」 [M+H] + Retention time: 1.67 minutes (Method E)

[0132] Step 3. To a solution of methyl 8-((5-amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (60 mg, 0.148 mmol) in THF (1 mL) and MeOH (0.1 mL) was added LiBH4 in THF (0.740 mL, 0.740 mmol). The reaction mixture was stirred at 40 °C for 1 h, neutralized with 0.07 mL of HOAc and purified by Method B. The fractions containing the expected product were combined and lyophilized to give (8-((5-amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)methanol (25 mg, 0.066 mmol, 44.8%). LC-MS m / z 378.3 [M+H] + Retention time: 1.45 min (Method E)

[0133] Step 4. To a solution of (8-((5-amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)methanol (25 mg, 0.066 mmol) in THF (1 mL) was added thionyl chloride (0.024 mL, 0.331 mmol). The reaction mixture was stirred at RT for 5 min. LCMS analysis showed completion of the reaction (LC-MS m / z 396.3 [M+H] + ). The reaction mixture was concentrated in vacuo and co-evaporated with dry DCM (2 x 5 mL). The residue was dried under high vacuum for 10 min and dissolved in DMF (1 mL). Tetrahydro-2H-pyran-4-amine (67.0 mg, 0.662 mmol) was added. The reaction mixture was stirred at 25 °C for 4 h and purified by Method A. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 109 (14.49 mg, 0.021 mmol, 31.9%).

[0134] Compound 101 and Compound 107 were prepared similarly. Example 6 - Compound 102

Chemical Structure

[0135] Step 1. Imidazole (1.452 g, 21.33 mmol) was added to a solution of (S)-3-aminohexan-1-ol (1 g, 8.53 mmol) and tert-butyldichlorophenylsilane (TBPDSCl 3.28 mL, 12.80 mmol) in DMF (6 mL). The reaction mixture was stirred overnight at RT and worked up with EtOAc, water and brine. The organic phases were combined, concentrated and purified by column chromatography: column: 40 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 min, 0-100% over 14 min then hold at 100% B for 3 min; flow rate: 40 mL / min; column temperature: 25 °C. The fractions containing the expected product were combined, concentrated and dried under high vacuum to give (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (2.09 g, 5.88 mmol, 68.9% yield). LC-MS m / z 356.2 [M+H]+; retention time: 2.51 min (method E)

[0136] Step 2. BOP (433 mg, 0.979 mmol) was added to a solution of methyl 8-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (200 mg, 0.490 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (871 mg, 2.449 mmol) and DBU (0.148 mL, 0.979 mmol) in DMSO (3 mL). The reaction mixture was stirred at 70 °C for 3 h, neutralized with 0.2 mL acetic acid, and purified by reverse-phase column chromatography: column: 50 g CombiFlash Aq column; mobile phase A: water containing 0.05% TFA; mobile phase B: acetonitrile containing 0.05% TFA; gradient: hold at 0% B for 0.75 min, 0 - 50% B over 8.75 min, then hold at 100% B for 1.5 min; flow rate: 35 mL / min; column temperature: 25 °C. Fractions containing methyl (S)-8-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate were combined and lyophilized (258 mg, 0.346 mmol, 70.6% yield). LC-MS m / z 746.3 [M+H] + Retention time: 2.57 min (Method E)

[0137] Step 3. LiBH4 (2N, 0.4 mL) was added to a solution of methyl (S)-8-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinoline-5-carboxylate (121 mg, 0.162 mmol) in THF (1.8 mL) and MeOH (0.2 mL). The reaction mixture was stirred at 40 °C for 1 h, neutralized with 0.2 mL acetic acid and purified by method B. Fractions containing methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-(hydroxymethyl)quinolin-8-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate were combined and lyophilized (43 mg, 0.060 mmol, 36.9%). LC-MS m / z 718.3 [M+H] + Retention time: 2.51 min (Method E)

[0138] Step 4. SOCl2 (0.024 mL, 0.334 mmol) was added to a solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-(hydroxymethyl)quinolin-8-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (48 mg, 0.067 mmol) in THF (1 mL). The reaction mixture was stirred at RT for 5 min. LCMS analysis showed that the starting material was completely converted to the chloro intermediate (LC-MS m / z 736.3 [M+H] +) was shown to be converted. The reaction mixture was concentrated in vacuo and co-evaporated with dry DCM (2 x 5 mL). The residue was dried under high vacuum for 10 minutes to give a residue. The residue was dissolved in DMF (1 mL), and DIEA (0.070 mL, 0.401 mmol) and 3-methoxyazetidine (34.9 mg, 0.401 mmol) were added. The reaction mixture was stirred at 70 °C for 30 minutes and lyophilized with acetonitrile and water to give crude methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-((3-methoxyazetidin-1-yl)methyl)quinolin-8-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (52.1 mg, 0.066 mmol, 99%). LC-MS m / z 787.3 [M+H] + Retention time: 2.60 minutes (Method E)

[0139] Step 5. NaOH (0.3 mL, 3.00 mmol) in water was added to a solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-((3-methoxyazetidin-1-yl)methyl)quinolin-8-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (52.1 mg, 0.066 mmol) in 1,4-dioxane (0.6 mL). The reaction mixture was stirred at 70 °C for 3 hours, neutralized with 0.3 mL of HCl (12 M), and lyophilized with acetonitrile and water to give a crude intermediate. HCl (12 M, 0.3 mL) was added to a mixture of the intermediate (143 mg crude) in MeOH (0.8 mL). The slurry was stirred at RT for 1 hour, diluted with acetonitrile (10 mL) and water (10 mL), and lyophilized to give a crude product. The crude product was dissolved in 1 mL of DMSO and filtered. The filtrate was purified by Method C. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 102 (7.62 mg, 0.016 mmol, 24.3%).

[0140] The following compounds were prepared similarly: Compound 103, Compound 104, Compound 105, Compound 106, Compound 139, and Compound 140. Example 7 - Compound 141

Chemical formula

[0141] Step 1. Bromine (3.60 ml, 69.8 mmol) was added to a solution of 8-methylquinoline (9.51 ml, 69.8 mmol) and silver sulfate (32.7 g, 105 mmol) in concentrated H2SO4 (98%, 100 mL) cooled to 0 °C in an ice bath. The reaction mixture was stirred at 25 °C for 4 hours and diluted with ice. NH4OH solution (14.8 M) was added slowly to raise the pH above 7. The reaction mixture was extracted with EtOAc (4 x 250 mL). The organic phases were combined, concentrated, and purified by column chromatography: Column: 80 g CombiFlash column; Mobile phase A: hexane; Mobile phase B: ethyl acetate; Gradient: held at 0% B for 3 minutes, 0 - 10% over 45 minutes, then held at 10% B for 3 minutes; Flow rate: 85 mL / min; hexane containing 0.05% TEA; Column temperature: 25 °C to give 5-bromo-8-methylquinoline (13.1 g, 59.0 mmol, 84% yield). LC-MS m / z 222.1&224.1 [M+H]+; Retention time: 2.05 minutes (Method E)

[0142] Step 2. In DMF (15 mL), a mixture of 5-bromo-8-methylquinoline (1 g, 4.50 mmol), tert-butyl 4-(3,3,4,4-tetramethylborolan-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.787 g, 5.85 mmol) and 5-bromo-8-methylquinoline (1 g, 4.50 mmol), tert-butyl 4-(3,3,4,4-tetramethylborolan-1-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.787 g, 5.85 mmol) was purged with N2 for 3 minutes. PdCl2(dppf) (0.329 g, 0.450 mmol) was added. N2 was purged for an additional 2 minutes. The reaction vessel was sealed. The reaction mixture was stirred at 80 °C for 5 hours, diluted with EtOAc, and filtered through CELITE. The filtrate was concentrated and purified by column chromatography: column: 80 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: held at 0% B for 3 minutes, 0 - 10% over 45 minutes, then held at 10% B for 3 minutes; flow rate: 85 mL / min; hexane containing 0.05% TEA; column temperature: 25 °C. The desired fraction was concentrated to obtain tert-butyl 4-(8-methylquinolin-5-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.28 g, 3.95 mmol, 88% yield). LC-MS m / z 324.9 [M+H] + ; Retention time: 1.98 minutes (Method E) 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (dd, J = 4.1, 1.8 Hz, 1H), 8.35 (dd, J = 8.5, 1.8 Hz, 1H), 7.64 - 7.49 (m, 2H), 7.32 (d, J = 7.2 Hz, 1H), 5.74 (s, 1H), 4.06 (q, J = 2.8 Hz, 2H), 3.64 (t, J = 5.6 Hz, 2H), 2.71 (d, J = 0.9 Hz, 3H), 2.44 (ddt, J = 7.9, 5.6, 2.7 Hz, 2H), 1.46 (s, 9H)

[0143] Step 3. A mixture of tert-butyl 4-(8-methylquinolin-5-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.35 g, 4.16 mmol) and Pd-C (0.222 g, 0.21 mmol) in MeOH (15 mL) was stirred under a hydrogen balloon. The reaction was monitored by LCMS. The reaction was 40% complete in 8 hours. The reaction mixture was filtered and the filtrate was concentrated and purified by column chromatography: column: 40 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 minute, 0 - 10% over 14 minutes, then hold at 10% B for 1 minute; flow rate: 40 mL / min; hexane containing 0.05% TEA; column temperature: 25 °C. The fractions containing the expected product were combined, concentrated, and dried under high vacuum to give tert-butyl 4-(8-methylquinolin-5-yl)piperidine-1-carboxylate (0.412 g, 1.262 mmol, 30.3% yield). LC-MS m / z 324.9 [M+H]+; retention time: 1.98 min (method E) 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (dd, J = 4.1, 1.6 Hz, 1H), 8.64 (dd, J = 8.7, 1.6 Hz, 1H), 7.61 - 7.53 (m, 2H), 7.38 (dd, J = 7.5, 1.5 Hz, 1H), 4.13 (d, J = 12.9 Hz, 2H), 3.64 - 3.46 (m, 1H), 3.31 (s, 1H), 2.69 (s, 3H), 1.87 - 1.78 (m, 2H), 1.60 (qd, J = 12.5, 4.1 Hz, 2H), 1.44 (s, 9H)

[0144] Step 4. AIBN (14.59 mg, 0.089 mmol) was added to a solution of tert-butyl 4-(8-methylquinolin-5-yl)piperidine-1-carboxylate (290 mg, 0.888 mmol) and NBS (190 mg, 1.066 mmol) in CCl4 (4 mL). The reaction mixture was stirred overnight at RT. LCMS analysis showed 30% conversion to the intermediate (2.393 min by method E, M+H / z = 405.2; 407.2). An additional amount of AIBN (14.59 mg, 0.089 mmol) was added. The reaction mixture was stirred overnight at RT. LCMS analysis showed 50% conversion. The reaction mixture was worked up with EtOAc, water and brine. The organic phase was concentrated and dried in vacuo to give the crude intermediate tert-butyl 4-(8-(bromomethyl)quinolin-5-yl)piperidine-1-carboxylate (349 mg).

[0145] Cs2CO3 (868 mg, 2.67 mmol) was added to a solution of methyl (7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (298 mg, 0.888 mmol) and the crude intermediate tert-butyl 4-(8-(bromomethyl)quinolin-5-yl)piperidine-1-carboxylate (349 mg) in DMF (3 mL). The reaction mixture was stirred at 25 °C for 30 min. LCMS indicated completion of the reaction. The reaction mixture was worked up with EtOAc, water and brine. The organics were combined, concentrated and purified by column chromatography: column: 24 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 min, 0 - 100% over 14 min, then hold at 100% B for 1 min; flow rate: 25 mL / min; column temperature: 25 °C to give tert-butyl 4-(8-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (135 mg, 0.205 mmol, 23.04% yield). LC-MS m / z [M+H]+; Retention time: minutes (Method E)

[0146] Step 5. DBU (0.371 mL, 2.464 mmol) was added to a solution of tert-butyl 4-(8-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (325 mg, 0.493 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (350 mg, 0.986 mmol) and BOP (436 mg, 0.986 mmol) in DMSO (4.5 mL). The reaction mixture was stirred at 45 °C for 4 h and worked up with EtOAc, water and brine. The organic phases were combined, concentrated and purified by column chromatography: column: 12 g CombiFlash column; mobile phase A: hexane; mobile phase B: ethyl acetate; gradient: hold at 0% B for 1 min, 0 - 10% over 15 min then hold at 10% B for 1 min; flow rate: 20 mL / min; column temperature: 25 °C. The fractions containing the desired product were concentrated and dried under reduced pressure to give tert-butyl (S)-4-(8-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (315 mg, 0.316 mmol, 64.1% yield). LC-MS m / z 997.6 [M+H]+; Retention time: 2.56 min (Method F) 11H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.98 (dd, J = 4.2, 1.6 Hz, 1H), 8.78 - 8.70 (m, 1H), 7.66 (dd, J = 8.7, 4.2 Hz, 1H), 7.53 - 7.46 (m, 2H), 7.42 - 7.13 (m, 10H), 6.82 (s, 1H), 6.21 (s, 2H), 4.56 (s, 2H), 3.58 (s, 4H), 3.48 (s, 2H), 2.91 (s, 5H), 2.68 (s, 1H), 2.53 (s, 1H), 1.74 (d, J = 12.8 Hz, 2H), 1.58 (s, 1H), 1.53 - 1.47 (m, 1H), 1.43 (s, 9H), 0.99 (s, 1H), 0.86 (s, 9H), 0.72 (t, J = 7.3 Hz, 3H)

[0147] Step 6. Zinc (168 mg, 2.57 mmol) was added to a solution of tert-butyl (S)-4-(8-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (256 mg, 0.257 mmol) in MeOH (4 mL) and AcOH (2 mL). The reaction mixture was stirred at 25 °C for 1 h and worked up with EtOAc, water and brine. The organic phases were combined, concentrated to afford the intermediate tert-butyl (S)-4-(8-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (186 mg). LC-MS m / z 871.7 [M+H]+; retention time: 1.82 min (method F)

[0148] NaOH (10 N, 1 mL) was added to a solution of the intermediate (186 mg) in dioxane (4 mL). The reaction mixture was stirred at 78 °C overnight and worked up with EtOAc, water, and brine. The organic phase was concentrated and dried in vacuo to give tert-butyl (S)-4-(8-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (151 mg, 0.186 mmol, 72.3% yield). LC-MS m / z 813.7 [M+H]+; retention time: 2.48 min (Method E)

[0149] Step 7. TFA (0.5 mL) was added to a solution of tert-butyl (S)-4-(8-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)quinolin-5-yl)piperidine-1-carboxylate (32 mg, 0.039 mmol) in DCM (0.5 mL), and the reaction mixture was stirred at 25 °C for 30 min. LCMS indicated removal of the Boc protecting group. The reaction mixture was concentrated and dissolved in dioxane (0.5 ml). HCl (12 N, 0.5 mL) was added thereto. The reaction mixture was stirred at RT for 15 min, concentrated, and purified by Method C. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 141 (2.4 mg, 0.005 mmol, 13.0%). Example 8 - Compound 142

Chemical Structure

[0150] A solution of (S)-3-((5-amino-1-((5-(piperidin-4-yl)quinolin-8-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)hexan-1-ol (20 mg, 0.042 mmol) in DMF (0.6 mL) was treated with Molecular Sieves, tetrahydro-4H-pyran-4-one (21.09 mg, 0.211 mmol) and 1 drop of HOAc, followed by sodium triacetoxyborohydride (35.7 mg, 0.169 mmol). The reaction mixture was stirred overnight at RT and purified by Method C to give Compound 142 (5.0 mg, 8.55 μmol, 20.28% yield).

[0151] Compound 143 was prepared similarly. Example 9 - Starting Materials and Intermediates

[0152] The following chart shows a scheme for creating compounds that may be useful as starting materials or intermediates for the preparation of the TLR7 agonists disclosed herein. The scheme may be applied to the creation of other similar compounds that may be used as starting materials or intermediates. The reagents used are well known in the art and in many cases their use is shown in the foregoing examples. Chart 1

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[0153] Biological Activity The biological activity of the compounds disclosed herein as TLR7 agonists may be quantified by the following procedure.

[0154] Human TLR7 Agonist Activity Assay This procedure describes a method for quantifying the human TLR7 (hTLR7) agonist activity of the compounds disclosed in this specification.

[0155] Modified human embryonic kidney HEK-Blue™ TLR cells (Invivogen) having a human TLR7 secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in non-selective medium (DMEM high glucose (Invitrogen) supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue™ TLR7 cells were added to each well of a 384-well tissue culture plate (15,000 cells per well) and incubated at 37 °C, 5% CO2 for 16 - 18 hours. A compound (100 nl) was added to the wells containing HEK-Blue™ TLR cells, and the treated cells were incubated at 37 °C, 5% CO2. Eighteen hours after treatment, 10 microliters of freshly prepared Quanti-Blue™ reagent (Invivogen) was added to each well and incubated for 30 minutes (37 °C, 5% CO2), and the SEAP levels were measured using an Envision plate reader (OD = 620 nm). The half maximal effective concentration value (EC 50 ; the compound concentration that elicits a response midway between the assay reference value and the maximum value) was calculated.

[0156] 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 a TLR7 agonist.

[0157] Heparin-treated human whole blood was collected from human patients and treated with a TLR7 agonist test compound at 1 mM. The blood was diluted with RPMI 1640 medium, predotted at 10 nL per well using Echo, and the final concentration was set to 1 μM (10 nL in 10 μL of blood). After mixing on a shaker for 30 seconds, the plate was covered and placed in a 37 °C chamber overnight = 17 hours. Fixation / Lysis Buffer was prepared (5x in H20 → 1x, warmed at 37 °C; Cat# BD 558049), and the Perm Buffer was maintained (on ice) for later use.

[0158] Surface antibodies were prepared for surface marker staining (CD69): 0.045 μl hCD14-FITC (ThermoFisher Cat # MHCD1401) + 0.6 μl hCD19-ef450 (ThermoFisher Cat # 48-0198-42) + 1.5 μl hCD69-PE (cat# BD555531) + 0.855 μl FACS Buffer. Added at 3 μl / well, centrifuged at 1000 rpm for 1 minute, mixed on a shaker for 30 seconds, and placed on ice for 30 minutes. After 30 minutes, the stimulation was stopped with 70 μL of pre-warmed 1x Fixation / Lysis Buffer, resuspended using Felix mate (15 times, changing the tip for each plate), and incubated at 37 °C for 10 minutes.

[0159] Centrifuge at 2000 rpm for 5 minutes, aspirate with an HCS plate washer, mix on a shaker for 30 seconds, then wash with 70 μL of dPBS and pellet twice (2000 rpm, 5 minutes), wash with 50 μL of FACS buffer and pellet once (2000 rpm, 5 minutes). Mix on a shaker for 30 seconds. For intracellular marker staining (MX-1): Add 50 μl of BD Perm buffer III, mix on a shaker for 30 seconds. Incubate on ice for 30 minutes (in the dark). Wash twice with 50 μL of FACS buffer (centrifuge at 2300 rpm for 5 minutes after permeabilization), then mix on a shaker for 30 seconds. Resuspend in 20 μL of FACS buffer containing MX1 antibody ((4812)-Alexa 647: Novus Biologicals #NBP2-43704AF647) (20 μl FACS buffer + 0.8 ul hIgG + 0.04 μl MX-1). Centrifuge at 1000 rpm for 1 minute, mix on a shaker for 30 seconds, incubate the sample at RT in the dark for 45 minutes, then wash with 2x FACS buffer (centrifuge at 2300 rpm for 5 minutes after permeabilization). Resuspend in 20 μl of FACS buffer (total 35 μL per well), cover with foil, place at 4°C, and read the next day. Read the plate on an iQuePlus. Load the results into the toolset and create an IC50 curve with CurveMaster. 100% on the y-axis is set to 1 μM of resiquimod.

[0160] Induction of TNF-alpha and Type I IFN Response Genes in Mouse Blood The induction of TNF-alpha and type I IFN response genes are downstream events that occur upon activation of the TLR7 pathway. The following is an assay to measure their induction in mouse whole blood in response to a TLR7 agonist.

[0161] Heparin-treated mouse whole blood was diluted in RPMI 1640 medium containing Pen-Strep at a ratio of 5:4 (50 μL of whole blood and 40 μL of medium). 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% CO2 incubator for 17 hours. After incubation, 100 μL of medium was added to each well. The plate was centrifuged and 130 μL of the supernatant was removed and used for the assay of TNFα production by ELISA (from Invitrogen, catalog number 88-7324 Thermo-Fisher Scientific). 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 sample in the well and mixed 5 times by pipetting. The plate was then shaken at room temperature 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.

[0162] The frozen samples were thawed, and mRNA was extracted using the Invitrogen mRNA Catcher Plus Kit (Cat# K1570-02) according to the manufacturer's instructions. Using half of the mRNA obtained from the RNA extraction, cDNA was synthesized in a 20 μL reverse transcriptase reaction using the Invitrogen SuperScript IV VILO Master Mix (Cat# 11756500). TaqMan (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 for mouse IFIT1, IFIT3, MX1, and PPIA gene expression and TaqMan Master Mix. PPIA was used as a housekeeping gene. The recommendations from the manufacturer were followed. All raw data (Ct) were normalized by the average housekeeping gene (Ct), and then the comparative Ct (ΔΔCt) method was utilized to quantify the relative gene expression level (RQ) for experimental analysis.

[0163] Definition "Aliphatic" means a straight or branched chain saturated or unsaturated non-aromatic hydrocarbon moiety having a specific number of carbon atoms (e.g., "C3 aliphatic", "C 1-5 aliphatic", "C1-C5 aliphatic", or "C1 to C5 aliphatic". The latter three expressions are synonymous with an aliphatic moiety having 1 to 5 carbon atoms), and when the number of carbon atoms is not specifically identified, it is 1 to 4 carbon atoms (2 to 4 carbons in the case of an unsaturated aliphatic moiety). A similar understanding applies to the number of carbons in other types, i.e., C 2-4 alkene, C4-C7 cycloaliphatic, etc. Similarly, terms such as "(CH2) 1-3 " should be understood as an abbreviated representation where the subscript is 1, 2, or 3, and thus such terms would represent CH2, CH2CH2, and CH2CH2CH2.

[0164] "Alkyl" means a saturated aliphatic moiety following the same convention for specifying the number of applicable carbon atoms. Examples of C1-C4 alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl, and the like. "Alkandiyl" (sometimes also called "alkylene") means the divalent counterpart of an alkyl group, for example, [Chemical formula] and the like.

[0165] "Alkenyl" means an aliphatic moiety having at least one carbon-carbon double bond following the same convention for specifying the number of applicable carbon atoms. Examples of C2-C4 alkenyl moieties include, but are not limited to, ethenyl (vinyl), 2-propenyl (allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E-(or Z)-2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl), and the like.

[0166] "Alkynyl" means an aliphatic moiety having at least one carbon-carbon triple bond following the same convention for specifying the number of applicable carbon atoms. Examples of C2-C4 alkynyl groups include ethynyl (ethynyl), propargyl (prop-2-ynyl), 1-propynyl, but-2-ynyl, and the like.

[0167] "Cycloaliphatic" means a saturated or unsaturated non-aromatic hydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to 8 (preferably 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, particularly cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. "Cycloalkanediyl" (sometimes also called "cycloalkylene") means the divalent counterpart of a cycloalkyl group. Similarly, "bicycloalkanediyl" (or "bicycloalkylene") and "spiroalkanediyl" (or "spiroalkylene") refer to the divalent counterparts of bicycloalkyl and spiroalkyl (or "spirocycloalkyl") groups. Examples of

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[0168] "Heterocycloaliphatic" means a cycloaliphatic moiety in which, in at least one of its rings, up to 3 (preferably 1 to 2) carbons are replaced by heteroatoms independently selected from N, O or S, where N and S may be optionally oxidized and N may be optionally quaternized. Preferred cycloaliphatic moieties consist of one ring with a size of 5 to 6 members. 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.

[0169] "Alkoxy", "aryloxy", "alkylthio", and "arylthio" each mean -O(alkyl), -O(aryl), -S(alkyl), and -S(aryl), respectively. Examples are methoxy, phenoxy, methylthio, and phenylthio, respectively.

[0170] "Halogen" or "halo" means fluorine, chlorine, bromine or iodine, unless a narrower meaning is indicated.

[0171] "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.

[0172] "Heteroaryl" means a moiety having a mono-, bi- or tricyclic ring system (preferably a 5- to 7-membered monocyclic) in which each ring has from 3 to 7 carbon atoms and at least one ring is an aromatic ring containing from 1 to 4 heteroatoms independently selected from N, O, or S, where N and S may be optionally oxidized and N may be optionally quaternized. Such an aromatic ring containing at least one heteroatom may be fused to other types of rings (such as benzofuranyl or tetrahydroisoquinolyl) or directly bonded to other types of rings (such as phenylpyridyl or 2-cyclopentylpyridyl). Further examples of the heteroaryl moiety include, but are not limited to, pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, quinoxalinyl, naphthyridinyl, benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl, phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl, carbazolyl, dibenzothiophenyl, acridinyl, and the like. "Heteroarylene" means the divalent counterpart of a heteroaryl group.

[0173] For example, when using "unsubstituted or substituted" or "optionally substituted", that is, expressing as "unsubstituted or substituted C1-C5 alkyl" or "optionally substituted heteroaryl", etc., indicating that a moiety may be substituted, such a moiety may have one or more independently selected substituents, preferably 1 to 5 in number, more preferably 1 to 2 in number. The substituents and substitution patterns may be selected by those skilled in the art considering the moiety to which the substituent is attached, and provide compounds that are chemically stable and can be synthesized by techniques known in the art and by the methods described herein. When a moiety is specified as "unsubstituted or substituted" or "optionally substituted", in a preferred embodiment, such a moiety is unsubstituted.

[0174] "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, moieties having an open (unsatisfied) valence with an alkyl, alkenyl, or alkynyl moiety, such as benzyl, phenethyl, N-imidazolylethyl, N-morpholinoethyl, and the like. Conversely, "alkylaryl", "alkenylcycloalkyl", and the like, in some cases, mean an aryl, cycloalkyl, or other moiety substituted with alkyl, alkenyl, etc., and in some cases, moieties such as methylphenyl (tolyl) or allylcyclohexyl. "Hydroxyalkyl", "haloalkyl", "alkylaryl", "cyanoaryl", and the like, in some cases, mean an alkyl, aryl, or other moiety substituted with one or more specific substituents (in some cases, hydroxyl, halo, etc.).

[0175] For example, acceptable substituents include alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl, aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especially fluoro), haloalkyl (especially trifluoromethyl), hydroxyl, hydroxyalkyl (especially hydroxyethyl), cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl) (especially -OCF3), -O(cycloalkyl), -O(heterocycloalkyl), -O(aryl), alkylthio, arylthio, =O, =NH, =N(alkyl), =NOH, =NO(alkyl), -C(=O)(alkyl), -C(=O)H, -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(cycloalkyl), -S(=O)alkyl, -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), -SO2N(alkyl)2, and the like, but are not limited thereto.

[0176] When the moiety to be replaced is an aliphatic moiety, preferred substituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo, hydroxyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl), -O(cycloalkyl), -O(heterocycloalkyl), -O(aryl), alkylthio, arylthio, =O, =NH, =N(alkyl), =NOH, =NO(alkyl), -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(=O)alkyl, -S(cycloalkyl), -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), and -SO2N(alkyl)2. More preferred substituents are halo, hydroxyl, cyano, nitro, alkoxy, -O(aryl), =O, =NOH, =NO(alkyl), -OC(=O)(alkyl), -OC(=O)O(alkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, and -NHC(=NH)NH2. Particularly preferred substituents are phenyl, cyano, halo, hydroxyl, nitro, C1-C4 alkoxy, O(C2-C4 alkanediyl)OH, and O(C2-C4 alkanediyl)halo.

[0177] When the moiety to be replaced is a cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituents are alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl), -O(aryl), -O(cycloalkyl), -O(heterocycloalkyl), alkylthio, arylthio, -C(=O)(alkyl), -C(=O)H, -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azido, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(cycloalkyl), -S(=O)alkyl, -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), and -SO2N(alkyl)2.More preferred substituents are alkyl, alkenyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -C(=O)(alkyl), -C(=O)H, -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, and -NHC(=NH)NH2. Particularly preferred substituents are C1-C4 alkyl, cyano, nitro, halo, and C1-C4 alkoxy.

[0178] When ranges are recited, such as "C1-C5 alkyl" or "5 to 10%", the range includes the endpoints of the range, i.e., C1 and C5 in the first example, and 5% and 10% in the second example.

[0179] Unless a particular stereoisomer is explicitly indicated (e.g., by making the relevant valence bond at the associated stereocenter in the structural formula bold or dashed, or by depicting the double bond in the structural formula as having an E or Z configuration, or by using a nomenclature or symbol that specifies stereochemistry), all stereoisomers are included within the scope of the invention as pure compounds as well as mixtures thereof. Unless otherwise indicated, racemates, individual enantiomers (whether optically pure or partially resolved), diastereomers, geometric isomers, and combinations thereof, and mixtures thereof are all encompassed by the present invention.

[0180] 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, and zwitterionic forms.

[0181] "Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (e.g., within the human body) to produce the parent compound or a salt thereof, or that itself has activity similar to that of the parent compound. Suitable esters include C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl esters, particularly methyl, ethyl or n-propyl esters.

[0182] "Pharmaceutically acceptable salt" means a salt of a compound suitable for use in pharmaceutical formulations. When the compound has one or more basic groups, the salt can be an acid addition salt, e.g., 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 salt can be a calcium, potassium, magnesium, meglumine, ammonium, zinc, piperazine, tromethamine, lithium, choline, diethylamine, 4-phenylcyclohexylamine, benzathine, sodium, tetramethylammonium, and the like salts. Polymorphic crystalline forms and solvates are also included within the scope of the present invention.

[0183] "Subject" refers to an animal, including but not limited to primates (e.g., humans), monkeys, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice. The terms "subject" and "patient" are used interchangeably herein with respect to mammalian patients, e.g., humans.

[0184] 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 disorders, diseases, or pathologies, or symptoms associated with the disorder, disease, or pathology; or delaying the progression, spread, or worsening of the disease, disorder, or pathology, 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) decrease in the number of tumor cells; (3) maintenance of tumor size; (4) decrease in tumor size; (5) inhibition of tumor cell infiltration into peripheral organs, including (i) decrease, (ii) delay, or (iii) complete prevention; (6) inhibition of metastasis, including (i) decrease, (ii) delay, or (iii) complete prevention; (7) enhancement of the anti-tumor immune response, which may result in (i) maintenance of tumor size, (ii) decrease in tumor size, (iii) delay in tumor growth, (iv) decrease, 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.

[0185] In the formulas herein, a wavy line ([

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[0186] 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 vacant by the removal of a hydrogen that is implicitly there (or, if fully written, explicitly there). As an example:

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[0187] 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. Common 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 using isotopically labeled appropriate reagents, by conventional techniques known to those skilled in the art, or by procedures similar to those described herein, instead of the unlabeled reagents used in other cases. As an example, a C1-C3 alkyl group may be non-deuterated, partially deuterated, or fully deuterated, and "CH3" may include CH3, 13 CH3, 14It includes CH3, CH2T, CH2D, CHD2, CD3, etc. In one embodiment, various elements in the compound are present in their natural isotopic abundances.

[0188] 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.

[0189] Acronyms and Abbreviations Table C provides a list of the acronyms and abbreviations used herein, along with their meanings. [Table 23] [Table 24]

[0190] References At the beginning of 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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[0263] The foregoing detailed description of the present invention includes sections that relate primarily or exclusively to particular parts or aspects of the present invention. This is for purposes of clarity and convenience, and it is understood that particular features may be relevant not only in the sections in which they are disclosed but also in other sections, and that the disclosure herein includes all appropriate combinations of the information described in the different sections. Similarly, although the various figures and descriptions herein relate to particular embodiments of the present invention, if a specific feature is disclosed in the context of a particular figure or embodiment, it is understood that such feature may be used in combination with other features in the context of another figure or embodiment, or in the context of the invention generally, to the extent appropriate.

[0264] Furthermore, although the present invention has been particularly described with respect to certain preferred embodiments, the present invention is not limited to such preferred embodiments. On the contrary, the scope of the present invention is defined by the appended claims.

Claims

1. A compound having a structure represented by the following formula (Id): 【Chemical 1】 [wherein, W is 【Chemical 2】 as follows]

2. A compound having a structure represented by the following formula (Ie): [wherein, [Chemical Formula 3] is; R １ is 【Chemical Formula 4】

3. R ５ is H or Me; R ７ is H, C １ -C ５ alkyl, or C ３ -C ６ cycloalkyl; wherein the cycloalkyl group may optionally have a CH １ -C ３ group substituted with O, NH, or N(C ２ )alkyl as appropriate] A pharmaceutical composition for treating cancer, comprising an anti-cancer immunotherapeutic agent and the compound according to claim 1 or 2.

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

5. The pharmaceutical composition according to claim 3, wherein the anti-cancer immunotherapeutic agent is ipilimumab, nivolumab, or pembrolizumab.

6. The pharmaceutical composition according to any one of claims 3 to 5, 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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