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

Abstract

The following formula (I): [Case 1] The compound represented by TIFF2023512205000199.tif51101 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 / 057,644, filed Jul. 28, 2020, and U.S. Provisional Application Serial No. 62 / 966,085, 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 Structure

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

Chemical Structure

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

Chemical Structure

[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 of formula (A) - the pyrimidine six-membered ring and the imidazole five-membered ring are fused - is modified. (a) Dellaria et al. 2007, Jones et al. 2010 and 2012, and Pilatte et al. 2017 disclose compounds in which the pyrimidine ring is replaced 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 replaced 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 replaced 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 a partner molecule, which can be, for example, a phospholipid, poly(ethylene glycol) ("PEG"), an antibody, or another TLR (generally TLR2). Representative disclosures include: Carson et al. 2013, 2015, and 2016, Chan et al. 2009 and 2011, Cortez et al. 2017, Gadd et al. 2015, Lioux et al. 2016, Maj et al. 2015, Vernejoul et al. 2014, and Zurawski et al. 2012. The main binding site is the R" group of formula (A).

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

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

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

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

[0018] In one aspect, the following formula (I): CHEMICAL [wherein, W is H, halo, C1-C3 alkyl, CN, (C1-C4 alkanediyl)OH, CHEMICAL and; each X is independently N or CR 2 and; X 1 is O, CH2, NH, S, or N(C1-C3 alkyl); R 1 is (C1-C5 alkyl), (C2-C5 alkenyl), (C1-C8 alkanediyl) 0-1 (C3-C6 cycloalkyl), (C1-C8 alkanediyl) 0-1 (C5-C 10 spiroalkyl), (C2-C8 alkanediyl)OH, (C2-C8 alkanediyl)O(C1-C3 alkyl), (C1-C4 alkanediyl) 0-1 (5-6 membered heteroaryl), (C1-C4 alkanediyl) 0-1 phenyl, (C1-C4 alkanediyl)CF3, (C2-C8 alkanediyl)N[C(=O)](C1-C3 alkyl), or (C2-C8 alkanediyl)NR x R y wherein; each R 2 is independently H, O(C1-C3 alkyl), S(C1-C3 alkyl), SO2(C1-C3 alkyl), C1-C3 alkyl, O(C3-C4 cycloalkyl), S(C3-C4 cycloalkyl), SO2(C3-C4 cycloalkyl), C3-C4 cycloalkyl, Cl, F, CN, or [C(=O)] 0-1 NR x R y wherein; R 3 is H, halo, OH, CN, NH2, NH[C(=O)] 0-1 (C1-C5 alkyl), N(C1-C5 alkyl)2, NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C4-C 10 bicycloalkyl), NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C5-C 10 spiroalkyl), N(C3-C6 cycloalkyl)2, O(C1-C4 alkylene) 0-1 (C3-C8 cycloalkyl), O(C1-C4 alkylene) 0-1 (C4-C8 bicycloalkyl), O(C1-C4 alkylene) 0-1 (C5-C 10 spiroalkyl), O(C1-C4 alkylene) 0-1 (C1-C6 alkyl), N[C1-C3 alkyl]C(=O)(C1-C6 alkyl), NH(SO2)(C1-C5 alkyl), NH(SO2)(C1-C4 alkylene) 0-1 (C3-C8 cycloalkyl), NH(SO2)(C1-C4 alkylene) 0-1 (C4-C 10 bicycloalkyl), NH(SO2)(C1-C4 alkylene) 0-1 (C5-C 10 spiroalkyl), a 6-membered aromatic or heteroaromatic moiety, a 5-membered heteroaromatic moiety, or the following structure: [Chemical formula] and is a moiety having; R 4 is NH2, NH(C1-C5 alkyl), N(C1-C5 alkyl)2, NH(C1-C4 alkylene) 0-1 (C3-C8 cycloalkyl), NH(C1-C4 alkylene) 0-1 (C4-C 10 bicycloalkyl), NH(C1-C4 alkylene) 0-1 (C5-C 10 spiroalkyl), N(C3-C6 cycloalkyl)2, or The following structure: [Chemical formula] which is a part having; R 5 is H, C1-C5 alkyl, C2-C5 alkenyl, C3-C6 cycloalkyl, halo, O(C1-C5 alkyl), (C1-C4 alkanediyl)OH, (C1-C4 alkanediyl)O(C1-C3 alkyl), phenyl, NH(C1-C5 alkyl), a 5- or 6-membered heteroaryl, [Chemical formula] and; R 6 is NH2, (NH) 0-1 (C1-C5 alkyl), N(C1-C5 alkyl)2, (NH) 0-1 (C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), (NH) 0-1 (C1-C4 alkanediyl) 0-1 (C4-C 10 bicycloalkyl), (NH) 0-1 (C1-C4 alkanediyl) 0-1 (C5-C 10 spiroalkyl), N(C3-C6 cycloalkyl)2, or the following structure: [Chemical formula] which is a part having; R x and R y are independently H or C1-C3 alkyl, or R x and R y combine with the nitrogen to which they are attached to form a 3- to 7-membered heterocycle; m is 0 or 1; n is 1, 2, or 3; p is 0, 1, 2, or 3; wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 in each case alkyl, cycloalkyl, alkanediyl, bicycloalkyl, spiroalkyl, cyclic amine, 6-membered aromatic or heteroaromatic moiety, 5-membered heteroaromatic moiety or a moiety of the formula:

Chemical formula

Chemical formula

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

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

Mode for Carrying Out the Invention

[0021] Compound In one embodiment of formula (I), m is 0.

[0022] In one aspect, in formula (I), the following moiety:

Chemical formula

[0023] In another aspect, in formula (I), the following moiety:

Chemical formula

Chemical formula

[0024] In another aspect, in formula (I), the following moiety:

Chemical formula

[0025] In another aspect, in formula (I), the following moiety: [Chemical formula] is [Chemical formula] wherein one X is N and the remaining two are CH.

[0026] In one aspect, W is [Chemical formula] or [Chemical formula] is as follows.

[0027] In one aspect, the compound of the present disclosure is represented by the following formula (I'), wherein R 1 , R 5 , X, and W are as defined for formula (I): [Chemical formula]

[0028] In one aspect, 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]

[0029] In another aspect, the compound of the present disclosure is represented by the following formula (Ib), wherein R 1 , R 5, and R 3 is as defined for formula (I):

Chemical formula

[0030] Representative R 2 embodiments include H, OMe, OCHF2, and OCF3, with OMe being a preferred embodiment.

[0031] 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 formula

[0032] 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), or NH[C(=O)](C1-C4alkanediyl) 0-1 (C5-C 10 spiroalkyl) is.

[0033] RepresentativeR 5 embodimentsincludeH,Me,OMe,CH2OH,cyclopropyl,F,Cl,andCF3,withHbeingapreferredembodiment.

[0034] Inanotherembodimentofthecompoundrepresentedbyformula(Ib),R 3 is O(C1-C4alkanediyl) 0-1 (C3-C8cycloalkyl), O(C1-C4alkanediyl) 0-1 (C4-C8bicycloalkyl), O(C1-C4alkanediyl) 0-1 (C5-C 10 spiroalkyl), or O(C1-C4alkanediyl) 0-1 (C1-C6alkyl) is.

[0035] Inanotheraspect,thecompoundsofthepresentdisclosureare representedbythefollowingformula(Ic),whereinR 3 andR 5 areasdefinedforformula(I):

ChemicalFormula

[0036] Inanotheraspect,thecompoundsofthepresentdisclosureare representedbythefollowingformula(Id),whereinR 3 andR 5 areasdefinedforformula(I):

ChemicalFormula

[0037] In another aspect, the compounds of the present disclosure are represented by the following formula (Ie), wherein R 1 , R 4 and R 5 are as defined for formula (I):

Chemical formula

[0038] In another aspect, the present disclosure provides a compound having the structure represented by the following formula (If):

Chemical formula

Chemical formula

Chemical formula

[0039] In another aspect, the present disclosure provides a compound having the structure represented by the following formula (Ig):

Chemical formula

[0040] In another aspect, the present disclosure provides a compound having the structure represented by the following formula (Ih):

Chemical formula

[0041] Group R 1 Examples of [Chemical formula] are as follows.

[0042] Preferably, R 1 is [Chemical formula] selected from the above group (the "preferred R 1 group") consisting of.

[0043] Typical group R 3 includes [Chemical formula] [Chemical formula] are exemplified.

[0044] In another embodiment, R 3 is H, halo, OH, CN, NH2, NH[C(=O)] 0-1 (C1-C5 alkyl), N(C1-C5 alkyl)2, NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C3-C8 cycloalkyl), NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C4-C 10 bicycloalkyl), NH[C(=O)] 0-1 (C1-C4 alkanediyl) 0-1 (C5-C 10 spiroalkyl), N(C3-C6 cycloalkyl)2, N[C1-C3 alkyl]C(=O)(C1-C6 alkyl), A 6-membered aromatic or heteroaromatic moiety, a 5-membered heteroaromatic moiety, or the following structure:

Chem.

[0045] Preferred group R 3 is

Chem.

[0046] Representative group R 4 includes:

Chem.

[0047] Preferred R 4 is

Chem.

[0048] Representative group R 5 is H,

Chem.

[0049] Preferably, R 5 is H or Me.

[0050] By way of example and not limitation, the following formula:

Chem.

Chem.

[0051] By way of illustration and not limitation, the spiroalkyl group includes

Chemical formula

[0052] By way of illustration and not limitation, the following formula:

Chemical formula

Chemical formula

[0053] By way of illustration and not limitation, the bicycloalkyl group includes

Chemical formula

[0054] By way of illustration and not limitation, the following formula:

Chemical formula

Chemical formula

[0055] In one embodiment, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

Chemical formula

[0056] In one embodiment, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

Chemical formula

[0057] In one embodiment, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

[0058] In one embodiment, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

[0059] In one embodiment, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

Chemical formula

[0060] In one aspect, W is preferably in combination with formula (I'), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

[0061] In one aspect, W is preferably in combination with formula (I'), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

[0062] In one aspect, W is preferably in combination with formula (I'), (Ia), (If), or (Ig),

Chemical formula

Chemical formula

[0063] In one aspect, W is preferably in combination with formula (I'), (Ia), (If), or (Ig),

Chemical formula

Chem.

Chem.

[0064] In one aspect, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chem.

Chem.

Chem.

[0065] In one aspect, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chem.

Chem.

Chem.

[0066] In one aspect, W is preferably in combination with formula (I’), (Ia), (If), or (Ig),

Chem.

[0067] In one aspect, the compounds of the present disclosure have the following formula (If): [Chemical formula] [In the formula, R 1 is [Chemical formula] and W is TIFF0007712941000083.tif88164. is represented by

[0068] Some of the above-mentioned representative groups such as alkyl, cycloalkyl, spiroalkyl, bicycloalkyl, etc. and the following formula: [Chemical formula] are optionally substituted and / or may optionally have one or more CH2 groups substituted with O, SO2, etc. as described in the "Summary of the Invention" above.

[0069] Specific examples of the compounds disclosed herein by formula (Ia) are shown in Table A1 below. The table also provides data on biological activity: human TLR7 agonist reporter assay and / or induction of the CD69 gene in human whole blood determined through the procedures provided below. Analytical data (mass spectrum, LC / MS retention time, and NMR) are described in the rightmost column. In one embodiment, the compounds of the present disclosure have (a) an EC 50 value of less than 1,000 nM in the human TLR7 (hTLR7) reporter assay and (b) an EC 50 value of less than 1,000 nM for CD69 induction in human whole blood (hWB). (When the assay is performed multiple times, the reported value is the average value.) [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

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

[0070] Additional compounds of the present disclosure are shown in Table A2 together with their biological properties and analysis data.

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

[0071] Pharmaceutical Composition and Administration In another aspect, there is provided a pharmaceutical composition comprising a compound as disclosed herein, or a complex thereof, formulated with a pharmaceutically acceptable carrier or additive. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as biological agents or small molecule agents. The pharmaceutical composition may be administered in combination therapy with another therapeutic agent, particularly an anti-cancer agent.

[0072] 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).

[0073] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epithelial administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated with a substance and protected from the action of acids and other natural conditions that may inactivate the compound. The term "parenteral administration" usually means administration by injection, other than enteral and topical administration, and examples include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the pharmaceutical composition may be administered by a non-parenteral route such as a topical, epithelial or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0074] The pharmaceutical compositions can be in the form of a sterile aqueous solution or a sterile aqueous dispersion. They may also be formulated in microemulsions, liposomes, or other ordered structures suitable for achieving a high drug concentration. The compositions may also be provided in the form of a lyophilized product to be reconstituted with water before administration.

[0075] The amount of the active ingredient that can be combined with the carrier substance to produce a single dosage form will vary depending on the patient being treated and the particular method of administration, and will generally be an amount of the composition that 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.

[0076] The dosing schedule is adjusted to provide a therapeutic response. For example, a single bolus dose may be administered, the dose may be divided into several portions and administered over time, or the dose may be proportionally increased or decreased according to the urgency of the situation. For ease of administration and dose uniformity, it is particularly advantageous to formulate the parenteral composition in unit dosage form. "Unit dosage form" refers to physically discrete units suitable as a single dose for a patient to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic response, together with the necessary pharmaceutical carrier.

[0077] The doses range from about 0.0001 to 100 mg / kg, more typically from 0.01 to 5 mg / kg, based on the body weight of the host. For example, the dose may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight, or may be in the range of 1 - 10 mg / kg, or 0.1 to 5 mg / kg. Representative treatment regimens are administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferred dosing schedules include the following dosing schedules: (i) administer six doses every four weeks, then administer every three months; (ii) administer every three weeks; (iii) administer once at 3 mg / kg body weight, followed by 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 them. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1 - 1000 μg / mL, and in some methods about 25 - 300 μg / mL.

[0078] A "therapeutically effective amount" of a compound of the present invention preferably results in a decrease in the severity of the symptoms of the disease, an increase in the number and duration of asymptomatic periods of the disease, or the prevention of functional or physical impairment due to the pain of the disease. For example, for the treatment of a patient with cancer, a "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, still more preferably at least about 60%, and even more preferably at least about 80% compared to a patient not receiving treatment. A therapeutically effective amount of a therapeutic compound may reduce the size of a tumor or otherwise relieve symptoms in a patient, who is generally a human but may also be another mammal. When two or more therapeutic agents are administered in combination therapy, a "therapeutically effective amount" refers to the effectiveness of the combination as a whole, not as individual agents.

[0079] The pharmaceutical composition can be a controlled-release or sustained-release formulation such as an implant, a transdermal patch, and a microencapsulation delivery system. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by J.R. Robinson, Marcel Dekker, New York, 1978.

[0080] The therapeutic composition can be administered using medical devices such as (1) a needleless subcutaneous injector; (2) a microinfusion pump; (3) a transdermal device; (4) an infusion device; and (5) an osmotic device.

[0081] 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.

[0082] 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 activation of TLR7.

[0083] In one embodiment, the TLR7 agonist is used in combination with an anti-cancer immunotherapeutic agent - also known as an immuno-oncology agent. The anti-cancer immunotherapeutic agent exerts its effect particularly through activation of T cells by stimulating the body's immune system to attack and destroy cancer cells. The immune system has a number of checkpoint (regulatory) molecules that help maintain the balance between its legitimate attack on target cells and the inhibition of its attack on healthy and normal cells. Some are stimulatory (upregulatory) molecules, and their involvement means promoting T cell activation and enhancing the immune response. Others are inhibitory (downregulatory or brake) molecules, and their involvement means inhibiting T cell activation and weakening the immune response. Binding of an agonist immunotherapeutic agent to a stimulatory checkpoint molecule can result in activation of the latter and enhancement of the immune response against cancer cells. Alternatively, binding of an antagonist immunotherapeutic agent to an inhibitory checkpoint molecule can prevent downregulation of the immune system by the latter and help maintain an active response against cancer cells. Examples of stimulatory checkpoint molecules are B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H. Examples of inhibitory checkpoint molecules are CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, CD96 and TIM-4.

[0084] In the mechanism of action of either anti-cancer immunotherapeutic agent, its efficacy can be enhanced by upregulation of the systemic immune system such as activation of TLR7. Therefore, in one embodiment, the present specification provides a method for treating cancer, which comprises administering to a patient suffering from cancer a therapeutically effective combination of an anti-cancer immunotherapeutic agent and a TLR7 agonist as disclosed herein. The timing of administration may be simultaneous, sequential, or alternating. The administration method may be systemic or local. The TLR7 agonist may be delivered using a complex in a targeted manner.

[0085] 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 tumor, colon cancer, colorectal cancer, craniopharyngioma, cholangiocarcinoma, endometrial cancer, epithelioma, esophageal cancer, neuroblastoma, Ewing's sarcoma, eye cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, lip and oral cavity cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0086] Anticancer immunotherapeutic agents that can be used in combination therapies as disclosed herein include AMG 557, AMP-224, atezolizumab, avelumab, BMS 936559, semipramab, CP-870893, dacetuzumab, durvalumab, enoblituzumab, galiximab, IMP321, ipilimumab, lucatumumab, MEDI-570, MEDI-6383, MEDI-6469, muromonab-CD3, nivolumab, pembrolizumab, pidilizumab, spartalizumab, tremelimumab, urelumab, utomilumab, balstilimab, bonlerolizumab. Their alternative names (trade names, former names, research codes, or synonyms) and their respective target checkpoint molecules are shown in Table B below. [Table 36]

[0087] 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.

[0088] In another embodiment of the combination therapy with a TLR7 agonist, the anticancer immunotherapeutic agent is an antagonist anti-CTLA-4 antibody, preferably ipilimumab.

[0089] 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.

[0090] The TLR7 agonists disclosed herein are also useful as vaccine adjuvants.

[0091] The implementation of the present invention can be further understood by referring to the following examples provided as illustrations rather than limitations.

Example

[0092] 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.

[0093] Chemical shifts are reported in parts per million (ppm) on the low magnetic field side relative to the position of 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 designated 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. Furthermore, peaks that overlap with water or solvent peaks in the NMR spectrum or that undergo exchange 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.

[0094] Liquid Chromatography The following preparative and / or analytical (LC / MS) liquid chromatography methods were used.

[0095] LC / MS Condition A: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile Phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Temperature: 50 °C; Gradient: From 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)

[0096] LC / MS Condition B: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: 5:95 acetonitrile: water containing 0.1% TFA; Mobile Phase B: 95:5 acetonitrile: water containing 0.1% TFA; Temperature: 50 °C; Gradient: From 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)

[0097] LC / MS Condition C: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: acetonitrile containing 0.1% TFA; Mobile Phase B: water containing 0.1% TFA; Temperature: 37 °C; Gradient: From 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 (240 nm)

[0098] LC / MS Condition D: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile Phase A: acetonitrile containing 0.1% formic acid; Mobile Phase B: water containing 0.1% formic acid; Temperature: 37 °C; Gradient: From 0% B to 100% B over 2.5 minutes, then hold at 100% B for 0.50 minutes; Flow Rate: 1 mL / min; Detection: MS and UV (240 nm)

[0099] LC / MS Condition E: Column: Waters X-Bridge BEH C18 XP (50x2.1 mm) 2.5 μm; Mobile Phase A: 5:95 acetonitrile: water containing 10 mM NH4OAc; Mobile Phase B: 95:5 acetonitrile: water containing 10 mM NH4OAc; Temperature: 50 °C; Gradient: 0 - 100% B over 3 minutes; Flow Rate: 1.1 mL / min)

[0100] 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 simplicity, the N2 positional isomer is not shown for convenience, but it is understood to be present in the initially formed mixture and is separated later, for example, by preparative HPLC.

Chemical Structure

[0101] The mixture of positional isomers may be separated at the initial stage of the synthesis and the remaining synthetic 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.

[0102] 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, specific groups (e.g., methyl ester or methoxy) are depicted for convenience. Scheme 1

Chemical Structure

[0103] R aIn Scheme 1 and other cases in which it appears, for example,

Chem.

[0104] Compound 8 can be prepared by the synthetic sequence as illustrated in Scheme 1 above. By reaction with NBS, pyrazolopyrimidine 1 is converted to bromide 2. Alkylation with methyl 3-bromomethyl-4-methoxybenzoate gives compound 3. Compound 3 is hydrogenated under H2 to give compound 4. Compound 4 is reduced to alcohol 5 with LiAlH4. Treatment of alcohol 5 with NaOH gives amine 6. Reaction of amine 6 with SOCl2 gives chloride 7. In the last step of Scheme 1, compound 8 is prepared by alkylating chloride 7 with b NHR c . Scheme 2

Chem.

[0105] The above Scheme 2 shows an alternative method for preparing intermediate 5. Compound 10 is formed by coupling methyl 4-amino-1H-pyrazole-5-carboxylate (CAS Registry Number 923283-54-9) and 1,3-bis(methoxycarbonyl)-2-methyl-2-thioureido (CAS Registry Number 34840-23-8). Compound 11 is obtained by brominating compound 10 with NBS (N-bromosuccinimide). Alkylation with methyl 3-bromomethyl-4-methoxybenzoate gives compound 12. Compound 13 is obtained by hydrogenating compound 12 under H2. Reaction with LiAlH4 reduces compound 13 to alcohol 14. In the presence of BOP and DBU, compound 14 reacts with R a NH2 to synthesize intermediate 5. Scheme 3

Chemical Structure

[0106] The above Scheme 3 shows an alternative method for preparing intermediate 4. Compound 16 is formed by alkylating methyl 4-nitro-1H-pyrazole-5-carboxylate 15 (CAS Registry Number 1345513-95-2) with methyl 3-bromomethyl-4-methoxybenzoate. Compound 17 is obtained by hydrogenating compound 16 under H2. Compound 18 is obtained by reacting compound 17 with 1,3-bis(methoxycarbonyl)-2-methyl-2-thioureido. In the presence of BOP and DBU, compound 18 reacts with R a NH2 to synthesize intermediate 4. Scheme 4

Chemical Structure

[0107] Compound 1 may be directly alkylated with methyl 3-bromomethyl-4-methoxybenzoate to form intermediate 4. However, in this method, the ratio of the N1 isomer to the N2 isomer is generally not very favorable. Scheme 5

Chem.

[0108] The above Scheme 5 shows an alternative method for preparing intermediate 4. Pyrazolopyrimidine 1 is converted to iodide or chloride 19 with NIS (N-iodosuccinimide) or NCS (N-chlorosuccinimide). Alkylation with methyl 3-bromomethyl-4-methoxybenzoate gives compound 20. Compound 20 is hydrogenated under H2 to give compound 4. Scheme 6

Chem.

[0109] The above Scheme 6 shows an alternative method for preparing product 8. Reaction of compound 5 with SOCl2 gives chloride 21. Treatment of chloride 7 with R b NHR c gives compound 22. Deprotection of compound 22 with NaOH gives product 8. Scheme 7

Chem.

[0110] The above Scheme 7 shows an alternative method for preparing product 8. Reaction of compound 14 with SOCl2 gives chloride 23. Treatment of chloride 7 with R b NHR c gives compound 24. Deprotection of compound 24 with NaOH gives compound 25. Product 8 is synthesized by reacting compound 25 with R a NH2 in the presence of BOP and DBU. Scheme 8

Chem.

[0111] Compound 26 can be prepared by coupling compound 8 with acid R c COOH when R d is H, as illustrated in Scheme 8 above. Scheme 9

Chemical Structure

[0112] Compound 28 can be prepared according to the synthetic sequence illustrated in Scheme 9 above. Compound 4 was hydrolyzed using NaOH to obtain acid 27. Product 28 is obtained by coupling compound 27 with R b NHR c as described above. Scheme 10

Chemical Structure

[0113] Compound 29 can be obtained by the reaction of chloride 7 with alcohol R g OH as illustrated in Scheme 10 above. Scheme 11

Chemical Structure

[0114] Compound 32 can be prepared according to the synthetic sequence illustrated in Scheme 11 above. Compound 30 is obtained by alkylation of compound 2. Deprotection of compound 30 gives compound 31. Product 32 is obtained by hydrolyzing compound 31 with NaOH. Scheme 12

Chemical Structure

[0115] Compound 36 can be prepared according to the synthetic sequence illustrated in Scheme 12 above. Alkylation of compound 2 gives compound 33. Hydrogenation of compound 33 under H2 gives compound 34. Compound 34 is Ri The Grignard reagent R, which is, for example, lower alkyl i is converted to compound 35 by reacting with MgBr. By deprotecting compound 35 using NaOH, the product 36 is obtained.

[0116] Those skilled in the art can prepare the compounds of the present disclosure by referring to the above general procedures and using or modifying the reagents, solvents, and conditions known in the art as appropriate to modify the procedures of the following specific examples.

[0117] 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 provided with the present disclosure, will recognize that they will be able to prepare and use the compounds disclosed herein even without an exhaustive list of examples.

[0118] The analytical data for compounds numbered 101 and above can be found in Table A1 or Table A2. Example A - Compound 105 [Chemical formula]

[0119] Step 1. To a suspension of methyl (7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (4 g, 15.13 mmol) in DMF (7 mL) was added a solution of NBS (2.96 g, 16.65 mmol) in acetonitrile (14 mL). The reaction mixture was stirred at RT for 1 hour. Water (33 mL) was added. The precipitate was collected by filtration. The solid was washed with water (3 x 20 mL) and air-dried overnight to obtain methyl (3-bromo-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate. LC-MS m / z 343.1 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 9.79 (s, 1H), 7.58 (s, 1H), 3.62 (s, 3H), 3.54 (q, J = 6.8 Hz, 2H), 1.68 - 1.56 (m, 2H), 1.47 - 1.33 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H)

[0120] Step 2. Cs2CO3 (5.73 g, 17.59 mmol) was added to a mixture of methyl (3-bromo-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (3.32 g, 9.67 mmol) and methyl 3-(bromomethyl)-4-methoxybenzoate (2.279 g, 8.79 mmol) in DMF (21.72 ml) at RT. The reaction mixture was stirred at RT for 2 h, diluted with EtOAc, washed with water, dried, filtered, and concentrated. The crude material was purified by silica gel column in hexane with 0 - 20% EtOAc to give methyl 3-((3-bromo-7-(butylamino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate as a white solid. LC-MS m / z 521.1 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 7.98 - 7.90 (m, 1H), 7.50 (d, J = 2.2 Hz, 1H), 7.40 (t, J = 5.6 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 5.75 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.63 (s, 3H), 3.55 (q, J = 6.6 Hz, 2H), 1.65 - 1.53 (m, 2H), 1.31 - 1.23 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H)

[0121] Step 3. Pd / C (10 wt%, 30 mg, 0.403 mmol) was added to a solution of methyl 3-((3-bromo-7-(butylamino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.21 g, 0.403 mmol) in MeOH (5 mL) at RT. The reaction mixture was stirred overnight under H2. The catalyst was filtered off and the filtrate was concentrated to give methyl 3-((7-(butylamino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate as a white solid. LC-MS m / z 443.2 [M+H] + 1 1H NMR (400 MHz, chloroform-d) δ 8.77 (t, J = 5.8 Hz, 1H), 8.09 (s, 1H), 7.96 (dd, J = 8.8, 2.2 Hz, 1H), 7.77 (d, J = 2.1 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 6.03 (s, 2H), 3.93 - 3.75 (m, 11H), 1.73 - 1.63 (m, 2H), 1.31 (h, J = 7.6 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H)

[0122] Step 4. In THF (1 M) (1.549 mL, 1.549 mmol), LiAlH4 was added to a mixture of methyl 3-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (60 mg, 0.155 mmol) in THF (8 mL) at 0 °C. The reaction mixture was stirred at RT for 3 h, quenched by slowly adding methanol, and stirred with Rochelle salt (1 M, 3 mL) for 1 h. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in DCM with 0 - 20% MeOH to give methyl (7-(butylamino)-1-(5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate as a white solid. LC-MS m / z 443.2 [M+H] + 1 H NMR (400 MHz, chloroform-d) δ 8.08 (s, 1H), 7.78 (s, 1H), 7.33 - 7.26 (m, 1H), 6.96 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 5.70 (t, J = 5.4 Hz, 1H), 5.57 (s, 2H), 5.29 (s, 2H), 4.47 (s, 2H), 3.90 (s, 3H), 3.73 (s, 3H), 3.44 (td, J = 7.0, 5.3 Hz, 3H), 1.52 - 1.39 (m, 2H), 1.29 - 1.15 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H)

[0123] Step 5. NaOH (10 M, 5.02 mL, 50.2 mmol) was added to a mixture of methyl (7-(butylamino)-1-(5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (1.04 g, 2.509 mmol) in dioxane (25 mL) at RT. The reaction mixture was heated at 54 °C overnight, diluted with water, and extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column chromatography in DCM with 0 - 30% MeOH to give Compound 140 as a white solid. LC-MS m / z 357.2 [M+H] +

[0124] Step 6. SOCl2 (0.410 ml, 5.61 mmol) was added to a solution of (3-((5-amino-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol (0.1 g, 0.281 mmol) in THF (4.60 ml) at RT. The reaction mixture was stirred at RT for 2 h. The solvent was evaporated to give N 7 -butyl-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine as a white solid. LC-MS m / z 375.2 [M+H] +

[0125] Step 7. In DMF (0.5 mL), N 7A mixture of -butyl-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (10 mg, 0.027 mmol) and 3-methoxyazetidine (13.94 mg, 0.160 mmol) was stirred overnight at RT. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; mobile phase B: acetonitrile containing 95:5 acetonitrile:0.1% TFA; gradient: hold at 0%B for 0 min, 0-40%B over 20 min, then hold at 100%B for 0 min; flow rate: 20 mL / min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 105 were combined and dried by centrifugal evaporation.

[0126] The following compounds were prepared similarly: Compound 101, Compound 102, Compound 103, Compound 104, Compound 106, Compound 107, Compound 108, Compound 109, Compound 110, Compound 111, Compound 112, Compound 113, Compound 114, Compound 115, Compound 116, Compound 117, Compound 118, Compound 119, Compound 120, Compound 121, Compound 122, Compound 123, Compound 124, Compound 125, Compound 126, Compound 127, Compound 129, Compound 130, Compound 131, Compound 132, Compound 133, Compound 134, Compound 135, Compound 137, Compound 138, Compound 142, and Compound 151. Example B - Compound 128

Chemical formula

[0127] DIEA (6.07 μl, 0.035 mmol) was added to a mixture of N7-butyl-1-(2-methoxy-5-((methylamino)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (Compound 132, 9.9 mg, 0.027 mmol), 3-(dimethylamino)propanoic acid (3.45 mg, 0.029 mmol), and HATU (12.22 mg, 0.032 mmol) in DMF (1 mL) at RT. The reaction mixture was stirred at RT overnight. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; mobile phase B: acetonitrile containing 95:5 acetonitrile:0.1% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. Fractions containing Compound 128 were combined and dried by centrifugal evaporation.

[0128] The following compounds were prepared similarly: Compound 136, Compound 146, Compound 147, and Compound 148. Example C - Compound 139

Chemical formula

[0129] NaH (60%) (6.40 mg, 0.160 mmol) was added to a solution of oxetan-3-ol (11.86 mg, 0.160 mmol) in DMF (0.5 mL) at RT. The mixture was stirred at RT for 10 minutes, and N 7A solution of 1-butyl-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (10 mg, 0.027 mmol) in DMF (0.5 mL) was added at RT. The reaction mixture was stirred at RT for 2 h. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; mobile phase B: acetonitrile containing 95:5 acetonitrile:0.1% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fractions containing compound 139 (collection triggered by MS and UV signals) were combined and dried by centrifugal evaporation.

[0130] The following compounds were prepared analogously: Compound 141, Compound 143, and Compound 144. Example D - Compound 145

Chemical Structure

[0131] Step 1. Cs2CO3 (0.380 g, 1.166 mmol) was added at RT to a mixture of methyl (3-bromo-7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.2 g, 0.583 mmol) and 3-(bromomethyl)-4-methoxybenzonitrile (0.132 g, 0.583 mmol) in DMF (2 mL). The reaction mixture was stirred at RT over the weekend. The reaction mixture was diluted with EtOAc, washed with water, dried, filtered, and concentrated. The crude material was purified by silica gel column in hexane with 0 - 70% EtOAc to give methyl (3-bromo-7-(butylamino)-1-(4-cyano-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate as a white solid. LC-MS m / z 488.1 [M+H] +

[0132] Step 2. A mixture of methyl (3-bromo-7-(butylamino)-1-(5-cyano-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (81 mg, 0.166 mmol) and 10 wt% Pd / C (20 mg, 0.166 mmol) in methanol (2 mL) was stirred overnight under H2. After filtering the catalyst, the filtrate was concentrated to give methyl (7-(butylamino)-1-(4-cyano-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate as a white solid. LC-MS m / z 410.2 [M+H] +

[0133] Step 3. A mixture of methyl (7-(butylamino)-1-(5-cyano-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (42.6 mg, 0.104 mmol) and 10N NaOH (0.208 mL, 2.081 mmol) in dioxane (1.5 mL) was stirred overnight at 54 °C. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile: 0.1% TFA; mobile phase B: acetonitrile containing 95:5 0.1% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 145 were combined and dried by centrifugal evaporation. Example E - Compound 149

Chemical formula

[0134] Step 1. A mixture of methyl 3-(bromomethyl)-4-methoxybenzoate (3.6 g, 13.89 mmol), methyl 4-nitro-1H-pyrazole-5-carboxylate (2.377 g, 13.89 mmol) and K2CO3 (2.496 g, 18.06 mmol) in DMF (30 mL) was stirred at RT for 3 h. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were dried, filtered and concentrated. The crude material was purified by silica gel column with 0 - 50% EtOAc in hexane to give methyl 1-(2-methoxy-5-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate as a white solid. LC-MS m / z 350.1 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.98 (dd, J = 8.6, 2.2 Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 5.52 (s, 2H), 3.98 (s, 3H), 3.82 (d, J = 5.1 Hz, 6H)

[0135] Step 2. Zinc (0.599 g, 9.16 mmol) was added to a mixture of methyl 1-(2-methoxy-5-(methoxycarbonyl)benzyl)-4-nitro-1H-pyrazole-5-carboxylate (1 g, 2.86 mmol) and ammonium formate (0.903 g, 14.31 mmol) in THF (9 mL) and MeOH (9 mL) at RT. The reaction mixture was stirred at RT for 1 h. The solid was filtered off. The filtrate was concentrated to give methyl 4-amino-1-(2-methoxy-5-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate as a white solid. LC-MS m / z 320.1 [M+H] +

[0136] Step 3. A mixture of 3-bis(methoxycarbonyl)-2-methyl-2-thiourea (0.452 g, 2.192 mmol) and methyl 4-amino-1-(2-methoxy-5-(methoxycarbonyl)benzyl)-1H-pyrazole-5-carboxylate (0.7 g, 2.192 mmol) was placed in MeOH (18 mL), treated with acetic acid (0.627 mL, 10.96 mmol) at RT. The reaction mixture was stirred overnight. Next, sodium methoxide in methanol (4.37 M) (5.02 mL, 21.92 mmol) was added to the reaction mixture, and then stirred overnight at RT. The pH was adjusted to 5 by slowly adding acetic acid. The precipitate was collected by filtration, washed with water and acetonitrile, and dried to obtain methyl 3-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate as a white solid. LC-MS m / z 388.1 [M+H] +

[0137] Step 4. A solution of spiro[2.3]hexan-5-ylmethanamine (0.201 g, 1.808 mmol) and methyl 3-((7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.35 g, 0.904 mmol) in DMSO (5 mL) was treated with DBU (0.545 mL, 3.61 mmol) and BOP (0.799 g, 1.807 mmol). The reaction mixture was heated at 40 °C for 1 h. Water was added to quench the reaction. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in DCM with 0 - 20% MeOH to obtain methyl 4-methoxy-3-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexan-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate as a white solid. LC-MS m / z 481.2 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.96 - 7.87 (m, 2H), 7.25 (d, J = 2.2 Hz, 1H), 7.23 - 7.03 (m, 2H), 5.76 (s, 2H), 3.88 (d, J = 6.6 Hz, 3H), 3.74 (s, 3H), 3.69 - 3.52 (m, 5H), 2.84 - 2.68 (m, 1H), 2.08 - 1.90 (m, 2H), 1.86 - 1.77 (m, 2H), 0.34 (s, 4H)

[0138] Step 5. A solution of methyl 4-methoxy-3-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (0.122 g, 0.254 mmol) in THF (3 mL) was cooled to 0 °C and treated with dropwise addition of LiAlH4 (0.127 mL, 0.254 mmol). After 20 minutes, the reaction was quenched by slowly adding methanol and stirred with Rochelle salt (1 M, 3 mL) for 1 hour. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in DCM with 0 - 30% MeOH to give methyl (1-(5-(hydroxymethyl)-2-methoxybenzyl)-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate as a white solid. LC-MS m / z 453.2 [M+H] +

[0139] Step 6. NaOH (10 N, 0.350 mL, 3.50 mmol) was added to a mixture of methyl (1-(5-(hydroxymethyl)-2-methoxybenzyl)-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (79.1 mg, 0.175 mmol) in dioxane (2 mL) and DMSO (1 mL) at RT. The reaction mixture was heated at 54 °C overnight. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column chromatography in DCM with 0 - 30% MeOH to give (3-((5-amino-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol as a white solid. LC-MS m / z 395.2 [M+H] +

[0140] Step 7. SOCl2 (0.221 mL, 3.04 mmol) was added to a solution of (3-((5-amino-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol (60 mg, 0.152 mmol) in THF (1.5 mL) at RT. The reaction mixture was stirred at RT for 2 h. The solvent was evaporated to give 1-(5-(chloromethyl)-2-methoxybenzyl)-N7-(spiro[2.3]hexane-5-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine as a white solid. LC-MS m / z 413.2 [M+H] +

[0141] Step 8. A mixture of 1-(5-(chloromethyl)-2-methoxybenzyl)-N7-(spiro[2.3]hexane-5-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (10 mg, 0.024 mmol) and 3-methoxyazetidine (2.110 mg, 0.024 mmol) in DMF (0.5 mL) was stirred at RT for 2 h. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; mobile phase B: acetonitrile containing 95:5 0.1% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 149 were combined and dried by centrifugal evaporation.

[0142] Compound 150 was prepared analogously according to this example. Example F - Compound 152

Chemical Structure

[0143] Step 1. NaOH (10N, 0.237 mL, 2.372 mmol) was added to methyl 4-methoxy-3-((5-((methoxycarbonyl)amino)-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)benzoate (57 mg, 0.119 mmol) in DMSO (1 mL) at RT. The reaction mixture was heated at 54 °C overnight and neutralized by adding 6N HCl. The solvent was evaporated to give 3-((5-amino-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoic acid as a white solid. LC-MS m / z 409.2 [M+H] +

[0144] Step 2. DIEA (8.53 μl, 0.049 mmol) was added to a mixture of 1-methylpiperidin-4-amine (16.77 mg, 0.147 mmol), 3-((5-amino-7-((spiro[2.3]hexane-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoic acid (10 mg, 0.024 mmol) and HATU (12.10 mg, 0.032 mmol) in DMF (0.5 mL) at RT. The reaction mixture was stirred at RT for 2 h. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; mobile phase B: acetonitrile containing 95:5 acetonitrile:0.1% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 152 were combined and dried by centrifugal evaporation.

[0145] Compound 153 was prepared similarly according to this example. Example G - Compound 154

Chemical formula

[0146] Step 1. A mixture of methyl 3-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.5 g, 1.072 mmol) in DMSO (5 mL) was treated with (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (0.763 g, 2.145 mmol), 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (0.5 mL, 3.32 mmol), and then ((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate (V) (0.949 g, 2.145 mmol). The reaction mixture was heated at 70 °C for 2 h, diluted with EtOAc, and washed with water. The solvent mixture was dried over Na2SO4 and the solvent was removed. The residue was diluted with MeOH and filtered to remove the starting material. The solvent was removed and the substance was purified by silica gel (dry load) hexane-EtOAc 0-100% to give methyl (S)-3-((3-bromo-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.59 g, 0.734 mmol, 68.4% yield). LC-MS m / z 803.3 [M+H] + 1 H NMR(400MHz,DMSO-d6)δ 9.84(s,1H),7.98-7.81(m,1H),7.66-7.31(m,10H),7.30-7.19(m,2H),7.14-7.06(m,1H),6.71-6.58(m,1H),5.87-5.59(m,2H),4.77-4.53(m,1H),3.80-3.75(m,3H),3.75-3.71(m,3H),3.67-3.61(m,2H),3.60-3.56(m,3H),1.95-1.77(m,2H),1.64-1.42(m,2H),1.29-1.10(m,2H),0.92(s,9H),0.78(br t,J=7.3 Hz,3H)

[0147] Step 2. Methyl (S)-3-((3-bromo-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.59 g, 0.734 mmol), methanol (10 mL), and Pd / C (20 mg, 0.188 mmol) were added to a Parr bottle. The hydrogenation reaction was carried out at 25 °C for 2 hours under 50 psi. The substance was filtered and the solvent was removed to obtain methyl (S)-3-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.466 g, 0.624 mmol, 85% yield). LC-MS m / z 725.4 [M+H]+ 1 H NMR (400 MHz, DMSO-d6) δ 7.76 - 7.66 (m, 3H), 7.62 (s, 5H), 7.55 - 7.42 (m, 8H), 7.42 - 7.33 (m, 2H), 7.31 - 7.06 (m, 1H), 5.93 - 5.68 (m, 1H), 3.85 - 3.68 (m, 5H), 1.98 - 1.80 (m, 2H), 1.79 - 1.68 (m, 1H), 1.62 - 1.39 (m, 3H), 1.38 - 1.23 (m, 2H), 1.01 (s, 9H), 0.92 (s, 3H), 0.87 (t, J = 7.3 Hz, 3H), 0.83 - 0.77 (m, 1H)

[0148] Step 3. 460 mg (0.635 mmol) of methyl (S)-3-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate, 4 mL of dioxane, and 1.3 mL (7.98 mmol) of triethylamine trihydrofluoride (TREAT-HF™) were placed in a 20 mL scintillation vial. The reaction mixture was stirred at 50 °C for 2 hours. 6 mL (30.0 mmol) of NaOH was added, and then the mixture was stirred at 80 °C for 1 hour. After cooling, the reaction mixture was neutralized with 5N HCl and evaporated to dryness using a V10 evaporator. (S)-3-((5-Amino-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoic acid (100 mg, 0.241 mmol, 38.0% yield) was obtained as a white lyophilized solid by flash chromatography (EZ Prep., 50 g column, DMSO / water packed, 0 to 60% MeCN in water containing 0.05% TFA over 14 minutes). LC-MS m / z 451.2 [M+H]+ 1 H NMR (400 MHz, DMSO-d6) δ 12.74 - 12.22 (m, 2H), 7.81 - 7.62 (m, 3H), 7.57 (s, 1H), 7.47 - 7.35 (m, 1H), 7.16 - 7.06 (m, 1H), 6.96 - 6.84 (m, 1H), 5.56 (br d, J = 14.7 Hz, 2H), 4.53 - 4.17 (m, 2H), 3.61 (s, 3H), 1.51 (br d, J = 6.4 Hz, 2H), 1.36 - 1.17 (m, 2H), 1.01 - 0.80 (m, 2H), 0.56 (t, J = 7.4 Hz, 3H)

[0149] Step 4. In a 20 mL scintillation vial, (S)-3-((5-amino-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoic acid (30 mg, 0.072 mmol), HATU (33.0 mg, 0.087 mmol), (R)-1-methylpyrrolidin-3-amine (14.50 mg, 0.145 mmol) and DMF (1.5 mL) were added. DIPEA (0.038 mL, 0.217 mmol) was added and the reaction mixture was stirred at RT for 1 h. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; mobile phase B: acetonitrile containing 95:5 0.1% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 30 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 154 were combined and dried by centrifugal evaporation. Example H - Compound 157 [Chemical formula]

[0150] Step 1a. A mixture of methyl 3-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (1.1 g, 2.359 mmol) and Pd / C (0.500 g, 2.359 mmol, prepared in a prior patent) in DMSO (30 mL) and EtOH (10 mL) was stirred under H2 at 80 °C for 3 days. The catalyst was filtered off and the filtrate was concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 10% MeOH to give methyl 3-((5-amino-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate as a white solid. LC-MS m / z 330.1 [M+H]+ 1 1H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 7.90 (dd, J = 8.6, 2.2 Hz, 1H), 7.61 (s, 1H), 7.21 - 7.12 (m, 2H), 6.10 (s, 2H), 5.65 (s, 2H), 3.91 (s, 3H), 3.75 (s, 3H)

[0151] Step 1b. A mixture of methyl 3 - ((7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-4 - methoxybenzoate (0.1 g, 0.258 mmol, prepared by BBRC) and K2CO3 (0.107 g, 0.774 mmol) in DMSO (2 mL) was stirred at 80 °C for 90 minutes. After cooling, water was added to quench the reaction mixture. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 10% MeOH to give methyl 3 - ((5 - amino - 7 - hydroxy - 1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-4 - methoxybenzoate as a white solid. LC - MS m / z 330.1 [M + H] +

[0152] Step 2. A solution of methyl 3-((5-amino-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.274 g, 0.832 mmol) in THF (20 mL) was cooled to 0 °C, and then treated with a dropwise addition of LiAlH4 (2 M in THF) (0.416 mL, 0.832 mmol). LCMS indicated the completion of the reaction after 1 hour. Methanol was slowly added to quench the reaction, and then the reaction mixture was stirred with Rochelle salt (1 M, 10 mL) for 1 hour. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 10% MeOH to obtain 5-amino-1-(5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol as a white solid. LC-MS m / z 302.1 [M+H] + 1 H NMR(400MHz,DMSO-d6)δ 7.55(s,1H),7.17(dd,J = 8.3,2.1 Hz,1H),6.96(d,J = 8.4 Hz,1H),6.51(d,J = 2.1 Hz,1H),6.10(s,2H),5.62(s,2H),4.96(t,J = 5.8 Hz,1H),4.28(d,J = 5.4 Hz,2H),3.81(s,3H)

[0153] 3.5-Amino-1-(5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ol (0.13 g, 0.431 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (0.307 g, 0.863 mmol) in DMSO (5 mL) was treated with BOP (0.382 g, 0.863 mmol) and DBU (0.260 mL, 1.726 mmol). The reaction mixture was heated at 60 °C overnight. Water was added to quench the reaction. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 10% MeOH to give (S)-(3-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol as a white solid. LC-MS m / z 639.3 [M+H] +

[0154] Step 4. A mixture of (S)-(3-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol (0.24 g, 0.376 mmol) and SOCl2 (0.545 mL, 7.51 mmol) in THF (2 mL) was stirred at RT for 30 minutes. The solvent was evaporated to give (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine as a white solid. LC-MS m / z 657.3 [M+H] +

[0155] Step 5. (S)-3-((5-Amino-1-(2-methoxy-5-((4-methylpiperazin-1-yl)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)hexan-1-ol (Compound 157). A mixture of (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (15 mg, 0.023 mmol) and 1-methylpiperazine (13.71 mg, 0.137 mmol) in DMF (0.5 mL) was stirred at RT for 2 h. Triethylamine trihydrofluoride (0.022 mL, 0.137 mmol) and DMSO (0.5 mL) were added. The reaction mixture was stirred at RT overnight. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 5% B for 0 min, 5 - 45% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing Compound 157 were combined and dried by centrifugal evaporation.

[0156] According to this example, the following compounds were prepared analogously: Compound 155, Compound 156, and Compound 158. Example I - Compound 164

Chemical formula

[0157] Step 1. A mixture of methyl 3-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.6 g, 1.287 mmol, prepared in the prior patent), Pd(dppf)2Cl2 (0.094 g, 0.129 mmol), K2CO3 (0.534 g, 3.86 mmol) and trimethylboroxine (0.899 mL, 6.43 mmol) was stirred at 120 °C overnight. After cooling, water was added to quench the reaction. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered and concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 10% MeOH to give methyl 3-((5-amino-7-hydroxy-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate as a white solid. LC-MS m / z 344.2 [M+H] + 1 H NMR(500MHz,DMSO-d6)δ 10.91(s,1H),7.87(dd,J = 8.5,2.0 Hz,1H),7.19-7.12(m,2H),6.08(s,2H),5.55(s,2H),3.88(s,3H),3.73(s,3H),2.21(s,3H)

[0158] Step 2. A solution of methyl 3-((5-amino-7-hydroxy-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (0.12 g, 0.350 mmol) in THF (20 mL) was cooled to 0 °C, and then treated with a dropwise addition of LiAlH4 (2 M in THF) (0.175 mL, 0.350 mmol). LCMS indicated the completion of the reaction after 2 hours. Methanol was slowly added to quench the reaction, and then the reaction mixture was stirred with Rochelle salt (1 M, 10 mL) for 1 hour. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 20% MeOH to give 5-amino-1-(5-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol as a white solid. LC-MS m / z 316.2 [M+H] + 1 H NMR(400MHz,DMSO-d6)δ 7.16(d,J = 8.3 Hz,1H),6.96(d,J = 8.3 Hz,1H),6.54(s,1H),6.09(s,2H),5.54(s,2H),4.96(t,J = 5.7 Hz,1H),4.28(d,J = 5.5 Hz,2H),3.80(s,3H),2.22(s,3H)

[0159] A solution of 3.5-amino-1-(5-(hydroxymethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-ol (74.8 mg, 0.237 mmol) and BOP (210 mg, 0.474 mmol) in DMSO (2 mL) was added to a solution of (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (506 mg, 1.423 mmol) and DBU (0.143 mL, 0.949 mmol) in DMSO (2 mL). The reaction mixture was heated at 60 °C for 6 h. Water was added to quench the reaction. The aqueous solution was extracted with EtOAc. The combined organic layers were dried, filtered, and concentrated. The crude material was purified by silica gel column in CH2Cl2 with 0 - 20% MeOH to give (S)-(3-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol as a pale yellow oil. LC-MS m / z 653.5 [M+H] +

[0160] Step 4. SOCl2 (0.333 mL, 4.59 mmol) was added to a solution of (S)-(3-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxyphenyl)methanol (0.15 g, 0.230 mmol) in THF (2 mL) at RT. The reaction mixture was stirred at RT for 20 min. The solvent was evaporated to give (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(5-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine as a white solid. LC-MS m / z 671.5 [M+H] +

[0161] Step 5.1 - 1-Methylpiperazine (17.90 mg, 0.179 mmol) was added to a solution of (S)-N7-(1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)-1-(5-(chloromethyl)-2-methoxybenzyl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (20 mg, 0.030 mmol) in DMF (0.5 mL). The reaction mixture was stirred at RT for 3 h. Triethylamine trihydrofluoride (0.029 mL, 0.179 mmol) was added in DMSO (0.5 mL). The reaction mixture was stirred at RT overnight. The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile: water containing NH4OAc; mobile phase B: 95:5 acetonitrile: water containing NH4OAc; gradient: hold at 10% B for 0 min, 10 - 50% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the desired product were combined and dried by centrifugal evaporation to give Compound 164.

[0162] According to this example, the following compounds were prepared analogously: Compound 159, Compound 161, and Compound 162. Example J - Compound 169

Chemical Structure

[0163] Step 1. A mixture of methyl 5-bromo-2-fluoro-4-methoxybenzoate (2.239 g, 8.51 mmol, prepared according to US 2015 / 0299104), tribasic potassium phosphate (5.42 g, 25.5 mmol) in 1,4-dioxane (38.3 ml) and H2O (4.26 ml) at RT was sparged with N2 for 30 minutes. Methylboronic acid (0.764 g, 12.77 mmol) and XPhos Pd G2 (0.167 g, 0.213 mmol) were added. The mixture was sparged with N2 for 2 minutes and stirred at 80 °C for 22 hours. The reaction was cooled to RT, diluted with EtOAc (200 mL), washed with H2O (200 mL) and saturated aqueous NaCl solution (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography (40 g silica gel; linear gradient 0 - 25% EtOAc - hexane). The combined fractions were concentrated and further purified by flash chromatography (40 g silica gel; linear gradient 0 - 25% EtOAc - hexane). The products from both columns were combined to give methyl 2-fluoro-4-methoxy-5-methylbenzoate (1.563 g, 93%). LC-MS m / z 199 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 7.67 (dd, J = 8.6, 0.7 Hz, 1H), 6.95 (d, J = 13.2 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 3H), 2.13 (s, 3H)

[0164] Step 2. To a solution of methyl 2-fluoro-4-methoxy-5-methylbenzoate (1.563 g, 7.89 mmol) in CCl4 (19.72 ml) at RT was added N-bromosuccinimide (1.474 g, 8.28 mmol) and 2,2'-azobis(2-methylpropionitrile) (0.130 g, 0.789 mmol). The suspension was stirred at 75 °C for 20 h. The reaction was cooled to RT and filtered. The solid was washed with CCl4 (2 x 2 mL). The combined filtrates were concentrated in vacuo. The crude material was purified by flash chromatography (40 g silica gel; linear gradient 0-25% EtOAc-hexane). The combined fractions were concentrated and further purified by flash chromatography (40 g silica gel; linear gradient 0-15% EtOAc-hexane). The product from both columns was combined to give methyl 5-(bromomethyl)-2-fluoro-4-methoxybenzoate (1.73 g, 79%). LC-MS m / z 277 / 279 [M+H] + 1 H NMR(400MHz,DMSO-d6)δ 8.00(d,J=8.5 Hz,1H),7.09(d,J=13.2 Hz,1H),4.67(s,2H),3.95(s,3H),3.82(s,3H)

[0165] Step 3. To a solution of methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (1.60 g, 5.55 mmol) (above, Scheme 2, Compound 11) in DMF (27.8 mL) at RT was added Cs2CO3 (5.43 g, 16.66 mmol). The reaction mixture was stirred at 0 °C for 10 min, then methyl 5-(bromomethyl)-2-fluoro-4-methoxybenzoate (1.539 g, 5.55 mmol) was added. The reaction mixture was stirred at 0 °C for 30 min, then the cooling bath was removed and the mixture was stirred at RT for 1 h. The reaction mixture was added to H2O (150 mL), and the solid was collected by vacuum filtration and washed with H2O (3 x 10 mL), MeOH (3 x 10 mL), and Et2O (3 x 10 mL) to give methyl 5-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-2-fluoro-4-methoxybenzoate (1.191 g, 44%) as an off-white solid. LC-MS m / z 484 / 486 [M+H] + 1 H NMR(400MHz,DMSO-d6)δ 11.82-11.58(m,1H),11.51-11.31(m,1H),7.59(d,J=8.3 Hz,1H),7.07(d,J=13.1 Hz,1H),5.68(s,2H),3.84(s,3H),3.79(s,3H),3.75(s,3H)

[0166] Step 4. To a suspension of methyl 5-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-2-fluoro-4-methoxybenzoate (569 mg, 1.175 mmol) in DMSO (7834 μl) at RT was added (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine, HCl (691 mg, 1.763 mmol) (US 2020 / 0038403 A1, Figure 8, compound 71a) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (780 mg, 1.763 mmol), followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (879 μl, 5.88 mmol). The reaction was stirred at RT for 4 h. An additional amount of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (52 mg, 0.12 mmol) was added and the reaction was stirred at RT for 19 h. The reaction was added to a stirred flask containing H2O (80 mL), the insoluble material was collected by vacuum filtration, washed with H2O (2 x 5 mL), and then dissolved in EtOAc (100 mL). The resulting solution was washed with saturated aqueous NaCl (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography (80 g silica gel; linear gradient 0-40% EtOAc-CH2Cl2). This material was further purified by flash chromatography (40 g silica gel; linear gradient 0-50% EtOAc-hexane) to give methyl (S)-5-((3-bromo-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-2-fluoro-4-methoxybenzoate (455 mg, 47%) as a brown foam. LC-MS m / z 821 / 823 [M+H] + 11H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 7.55 (dd, J = 7.9, 1.5 Hz, 2H), 7.50 - 7.46 (m, 2H), 7.46 - 7.32 (m, 5H), 7.27 - 7.21 (m, 2H), 7.03 (d, J = 13.1 Hz, 1H), 6.68 (br d, J = 8.5 Hz, 1H), 5.77 - 5.69 (m, 1H), 5.67 - 5.59 (m, 1H), 4.70 - 4.60 (m, 1H), 3.74 (s, 6H), 3.65 (t, J = 6.5 Hz, 2H), 3.58 (s, 3H), 1.91 - 1.83 (m, 2H), 1.61 - 1.43 (m, 2H), 1.27 - 1.13 (m, 2H), 0.91 (s, 9H), 0.80 (t, J = 7.4 Hz, 3H)

[0167] Step 5. A solution of methyl (S)-5-((3-bromo-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-2-fluoro-4-methoxybenzoate (0.455 g, 0.554 mmol) in EtOH (22.15 ml) at RT was evacuated and then filled with N2 (3x), and then palladium carbon (10 wt% (anhydrous basis), wet carrier) (0.088 g) was added. The mixture was evacuated and then filled with H2, and stirred for 2 h under an atmosphere of H2 (balloon). The reaction mixture was purged with N2 for 30 min and then filtered through CELITE™ under a blanket of N2 and washed with EtOH (2 x 15 mL). The combined filtrates were concentrated under reduced pressure to give methyl (S)-5-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-2-fluoro-4-methoxybenzoate (423 mg, quant.) as a white foam. LC-MS m / z 743 [M+H] + 11H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.56 - 7.49 (m, 3H), 7.50 - 7.46 (m, 2H), 7.43 - 7.32 (m, 4H), 7.29 - 7.24 (m, 2H), 7.04 (d, J = 13.1 Hz, 1H), 5.85 - 5.78 (m, 1H), 5.73 - 5.66 (m, 1H), 4.68 - 4.58 (m, 1H), 3.76 (s, 3H), 3.77 (br s, 3H), 3.73 (s, 3H), 3.70 - 3.62 (m, 2H), 1.92 (br dd, J = 5.3, 3.2 Hz, 2H), 1.67 - 1.51 (m, 2H), 1.29 - 1.14 (m, 2H), 0.91 (s, 9H), 0.82 (t, J = 7.3 Hz, 3H)

[0168] In a mixture of THF (5112 μl) and MeOH (568 μl), a solution of methyl (S)-5-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-2-fluoro-4-methoxybenzoate (422 mg, 0.568 mmol) at 0 °C was added dropwise with lithium borohydride (2 M THF solution) (2840 μl, 5.68 mmol). The reaction was stirred at RT for 17 h. An additional amount of lithium borohydride (284 μL, 0.568 mmol) was added and the reaction was stirred at RT for 30 min. An additional amount of lithium borohydride (1.14 mL, 2.28 mmol) was added and the reaction was stirred at RT for 30 min and then at 40 °C for 5 h. The reaction was cooled to 0 °C and quenched by slowly adding MeOH (2 mL). The mixture was stirred at RT for 15 min, then diluted with H2O (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with saturated aqueous NaCl solution (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by flash chromatography (40 g silica gel; linear gradient 0-100% EtOAc-CH2Cl2) to give methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-fluoro-5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (197.6 mg, 49%) as a white foam. LC-MS m / z 715 [M+H] + 11H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 7.88 (s, 1H), 7.57 - 7.54 (m, 2H), 7.50 - 7.46 (m, 2H), 7.44 - 7.33 (m, 4H), 7.27 - 7.22 (m, 2H), 6.89 (d, J = 12.1 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.07 (d, J = 8.5 Hz, 1H), 5.70 - 5.64 (m, 1H), 5.61 - 5.55 (m, 1H), 5.03 (t, J = 5.6 Hz, 1H), 4.61 - 4.51 (m, 1H), 4.32 - 4.22 (m, 2H), 3.74 (s, 3H), 3.65 - 3.59 (m, 2H), 3.58 (s, 3H), 1.89 - 1.72 (m, 2H), 1.52 - 1.41 (m, 2H), 1.21 - 1.05 (m, 2H), 0.92 (s, 9H), 0.78 (t, J = 7.3 Hz, 3H)

[0169] Step 7. A solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-fluoro-5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (204 mg, 0.285 mmol) in THF (2853 μl) was added dropwise to a solution of thionyl chloride (104 μl, 1.427 mmol) in THF (2853 μl) at 0 °C. The reaction mixture was stirred at RT for 20 minutes and then concentrated under reduced pressure. The crude material was mixed with THF and concentrated under reduced pressure (2x) to give crude methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(5-(chloromethyl)-4-fluoro-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate. This material was used without further purification. LC-MS m / z 733 [M+H] +

[0170] Step 8. A solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(5-(chloromethyl)-4-fluoro-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.042 g, 0.057 mmol) in MeCN (1.140 ml) at RT was added to methylamine (2M THF solution) (0.086 ml, 0.171 mmol), and then N,N-diisopropylethylamine (0.060 ml, 0.342 mmol) was added. The reaction mixture was stirred at 60 °C for 2 h and then at 70 °C for 1 h. The reaction mixture was cooled to RT and concentrated. The residue was taken up in EtOAc (2 mL) and washed with saturated aqueous NaHCO3 (2 mL). The aqueous layer was extracted with EtOAc (2 x 2 mL). The combined organic layers were washed with saturated aqueous NaCl (2 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give crude methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(4-fluoro-2-methoxy-5-((methylamino)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate. This material was used without further purification. LC-MS m / z 728 [M+H] + Step 9. 4N HCl in 1,4-dioxane (570 μl) was added to a solution of the crude material obtained in Step 8 in 1,4-dioxane (570 μl) at RT. The reaction mixture was stirred at RT for 5 h and concentrated. The residue was mixed with 1,4-dioxane (0.3 mL) and concentrated to give crude methyl (S)-(1-(4-fluoro-2-methoxy-5-((methylamino)methyl)benzyl)-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate. This material was used without further purification. LC-MS m / z 490 [M+H] +

[0171] Step 10. In a mixture of 1,4-dioxane (570 μl) and MeOH (0.285 mL), to a solution of the crude material obtained in Step 9 at RT, 10 M aqueous NaOH (57.0 μl, 0.570 mmol) was added. The reaction mixture was stirred at 70 °C for 3 h. The reaction mixture was cooled to RT and neutralized by addition of acetic acid (32.6 μl, 0.570 mmol). The mixture was concentrated and then dissolved in a mixture of H2O (0.3 mL) and DMF (1.7 mL), filtered (0.45 μm nylon syringe filter), and purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: Hold at 4% B for 0 min, 4 - 44% B over 20 min, then hold at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing Compound 169 were combined and dried by centrifugal evaporation (10.4 mg, 41%).

[0172] These compounds were prepared similarly: Compound 165, Compound 166, Compound 167, Compound 168, Compound 171, Compound 172, Compound 173, Compound 175, and Compound 176 (in some examples, the following changes were made to Step 8: when the starting amine was a salt, an additional equivalent of i-Pr2NEt was added; the temperature of the reaction mixture ranged from 60 °C to 80 °C). Example K - Compound 170 [Chemical formula]

[0173] Step 1. A mixture of methyl 6-methoxy-5-methylnicotinate (491 mg, 2.71 mmol), NBS (627 mg, 3.52 mmol), and AIBN (111 mg, 0.677 mmol) in carbon tetrachloride (20 mL) was heated at 80 °C for 16 h. The reaction mixture was evaporated under reduced pressure and purified by silica gel column with a gradient of 0% to 50% ethyl acetate in hexane to give 5-(bromomethyl)-6-methoxynicotinate (493 mg). 1 H NMR (400 MHz, chloroform-d) δ 8.84 - 8.74 (m, 1H), 8.28 - 8.18 (m, 1H), 4.54 - 4.46 (m, 2H), 4.15 - 4.07 (m, 3H), 3.98 - 3.89 (m, 3H)

[0174] Step 2. To a mixture of methyl 5-(bromomethyl)-6-methoxynicotinate (233 mg, 0.896 mmol) and methyl (3-bromo-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (215 mg, 0.747 mmol) in DMF (5 mL) was added Cs2CO3 (730 mg, 2.240 mmol). After 16 h, the reaction was partitioned between ethyl acetate (50 mL) / LiCl (10% aqueous solution, 50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was isolated by trituration with methanol to give methyl 5-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-6-methoxynicotinate (133 mg). This material was used without further purification. LC-MS m / z 469.1 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 11.69 (br s, 1H), 11.45 (br s, 1H), 8.72 (d, J = 2.2 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 5.73 (s, 2H), 3.99 - 3.92 (m, 3H), 3.83 (s, 3H), 3.76 (s, 3H)

[0175] Step 3. A solution of methyl 5-((3-bromo-7-hydroxy-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-6-methoxynicotinate (215 mg, 0.460 mmol), (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (245 mg, 0.690 mmol), BOP (305 mg, 0.690 mmol), and DBU (0.312 mL, 2.071 mmol) in DMSO (5 mL) was stirred at RT for 16 h. BOP (305 mg, 0.690 mmol) and DBU (0.312 mL, 2.071 mmol) were added. The reaction mixture was stirred at RT for 3 h and partitioned between DCM (50 mL) and water (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel column with a gradient of 0% to 100% ethyl acetate in hexane to give methyl (S)-5-((3-bromo-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-6-methoxynicotinate (191 mg). LC-MS m / z 804.4 [M+H] +

[0176] Step 4. A suspension of methyl (S)-5-((3-bromo-7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-6-methoxynicotinate (191 mg, 0.237 mmol) and Pd-C (200 mg, 0.094 mmol) in MeOH (10 mL) was purged three times with N2 (evacuating in between) and then three times with H2 (evacuating in between). The mixture was stirred under hydrogen for 1 hour. The reaction mixture was filtered through CELITE™ and evaporated under reduced pressure to give methyl (S)-5-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-6-methoxynicotinate (172 mg), which was used without further purification. LC-MS m / z 726.3 [M+H] +

[0177] In a mixture of THF (3 mL) and methanol (0.600 mL), to a solution of methyl (S)-5-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-6-methoxynicotinate (172 mg, 0.237 mmol) was added LiBH4 (2 M in THF) (0.592 mL, 1.185 mmol). After 1 hour, an additional amount of LiBH4 (2 M in THF) (0.592 mL, 1.185 mmol) was added. After 16 hours, the reaction mixture was partitioned between ethyl acetate (50 mL) and 1% aqueous potassium / sodium tartrate solution (10 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography with a gradient of 0% to 100% ethyl acetate in hexane to afford methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-(hydroxymethyl)-2-methoxypyridin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (165 mg). LC-MS m / z 698.5 [M+H] +

[0178] Step 6. Dess-Martin periodinane (201 mg, 0.473 mmol) was added to a solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-(hydroxymethyl)-2-methoxypyridin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (165 mg, 0.236 mmol) in DCM (10 mL). After 30 minutes, the reaction mixture was evaporated under reduced pressure and dried under high vacuum. The crude material was purified by silica gel column chromatography with a gradient of 0% to 100% EtOAc in hexane to afford methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-formyl-2-methoxypyridin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (66 mg). LC-MS m / z 696.5 [M+H] +

[0179] Step 7. A solution was obtained by adding sodium triacetoxyborohydride (70.4 mg, 0.332 mmol) to a solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-formyl-2-methoxypyridin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (33 mg, 0.047 mmol) and N1,N1,N2-trimethylethane-1,2-diamine (24.23 mg, 0.237 mmol) in DCM (3 mL). After 2 hours, 2 mL of saturated sodium carbonate was added. The resulting mixture was diluted with 20 mL of methanol. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-2-methoxypyridin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (37 mg). This material was used without further purification. LC-MS m / z 782.5 [M+H] +

[0180] Step 8. To a solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((5-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-2-methoxypyridin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.037 g, 0.047 mmol) was added HCl (4N dioxane) (3 ml, 12.00 mmol). After 16 h, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum to give methyl (S)-(1-((5-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-2-methoxypyridin-3-yl)methyl)-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate hydrochloride (26 mg), which was used without further purification. LC-MS m / z 544.3 [M+H] +

[0181] Step 9. A solution of methyl (S)-(1-((5-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-2-methoxypyridin-3-yl)methyl)-7-((1-hydroxyhexan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (25.6 mg, 0.047 mmol) and NaOH (10N) (50 μl, 0.500 mmol) in dioxane (3 mL) was heated to 50 °C. After 24 h, the solvent was evaporated under reduced pressure, the residue was dried under high vacuum and diluted with 2 mL of DMF:HOAc (1:1). The crude material was purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water containing NH4OAc; mobile phase B: 95:5 acetonitrile:water containing NH4OAc; gradient: hold at 7% B for 0 min, 7 - 47% B over 20 min then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing compound 170 were combined and dried by centrifugal evaporation (11 mg). Example L - Compound 160

Chemical formula

[0182] Step 1. To methyl (1-(5-(chloromethyl)-2-methoxybenzyl)-7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (Compound 23, 130 mg, 0.344 mmol) in DMF (3 mL) were added 3-methoxyazetidine (90 mg, 1.032 mmol) and DIPEA (0.240 mL, 1.376 mmol). The reaction mixture was stirred at 25 °C overnight. The solvent was removed by V-10, and the residue was purified by silica gel (dry load) eluting with 0-20% DCM-MeOH to afford methyl (7-hydroxy-1-(2-methoxy-5-((3-methoxyazetidin-1-yl)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (130 mg, 0.303 mmol, 88% yield). LC-MS m / z 429.4 [M+H]+

[0183] Step 2. To methyl (7-hydroxy-1-(2-methoxy-5-((3-methoxyazetidin-1-yl)methyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (65 mg, 0.152 mmol) in DMSO (1.5 mL) were added (S)-3-amino-1-cyclopropylpropan-1-ol (34.9 mg, 0.303 mmol), DBU (0.091 mL, 0.607 mmol) and BOP (134 mg, 0.303 mmol). The mixture was stirred at 70 °C for 1 h. The mixture was treated with 5M NaOH (1 mL, 5.00 mmol) and heated at 70 °C for 1 h. The crude material was purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: water containing 5:95 acetonitrile:0.1% TFA; Mobile phase B: acetonitrile containing 95:5 0.1% TFA; Gradient: hold at 0%B for 0 min, 0-40%B over 20 min, then hold at 100%B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing Compound 160 were combined and dried by centrifugal evaporation. Example M - Compound 163

Chem.

[0184] Step 1. A mixture of methyl 3 - ((7 - hydroxy - 5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-4 - methoxybenzoate (0.53 g, 1.368 mmol) in DMSO (8 mL) was treated with (S)-1 - ((tert - butyldiphenylsilyl)oxy)pentan - 3 - amine (1.402 g, 4.10 mmol), 2,3,4,6,7,8,9,10 - octahydropyrimido[1,2 - a]azepine (0.619 mL, 4.10 mmol), followed by ((1H - benzo[d][1,2,3]triazol - 1 - yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V) (1.210 g, 2.74 mmol) and stirred overnight at RT. The reaction was diluted with EtOAc and washed with water. The solvent mixture was dried over Na2SO4. The solvent was removed and the substance was purified by a 40 g COMBIFLASH silica gel column. The 80% EtOAc / hexane fraction was concentrated to give methyl (S)-3 - ((7 - ((1 - ((tert - butyldiphenylsilyl)oxy)pentan - 3 - yl)amino)-5 - ((methoxycarbonyl)amino)-1H - pyrazolo[4,3 - d]pyrimidin - 1 - yl)methyl)-4 - methoxybenzoate (0.32 g, 0.450 mmol, yield 32.9%) as a white solid. LC / MS [M + H]=711.5

[0185] Step 2. To a solution of methyl (S)-3-((7-((1-((tert-butyldiphenylsilyl)oxy)pentan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (590 mg, 0.830 mmol) in THF (7469 μl) and MeOH (830 μl) was added lithium borohydride (2 M solution in THF) (4150 μl, 8.30 mmol) dropwise (gas evolved during addition). The reaction mixture was stirred at RT for 30 minutes. The reaction was cooled to 0 °C and quenched by the addition of H2O, resulting in the precipitation of a solid. The mixture was diluted with H2O (50 mL) and extracted with EtOAc (2 x 50 mL) (shaking the layers until all solids dissolved). The combined organic layers were washed with saturated aqueous NaCl (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by flash chromatography (packed as a CH2Cl2 solution; 40 g silica gel; linear gradient 0 - 100% EtOAc - CH2Cl2, then 0 - 10% MeOH - CH2Cl2). The impurities were eluted in the EtOAc gradient and the product was eluted in the MeOH gradient. The product fractions were concentrated to give the product as a white solid. This was taken up in THF (2 mL) and treated with thionyl chloride (0.062 mL, 0.843 mmol). The solvent was evaporated and the residue was redissolved in DMF (2 mL). Triethylamine trihydrofluoride (0.343 mL, 2.109 mmol) was added and the mixture was stirred at RT overnight until LCMS indicated completion of the reaction. Purification was performed on a COMBIFLASH 24g column. From the 5% MeOH / DCM fraction, methyl (S)-(1-(5-(chloromethyl)-2-methoxybenzyl)-7-((1-hydroxypentan-3-yl)amino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (140 mg, 0.302 mmol, 36% yield) was obtained as a thick oil.From the 15% MeOH / DCM fraction, (S)-3-((5-amino-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)pentan-1-ol (40 mg, 0.099 mmol, 12% yield) was obtained. LC / MS [M+H]=405.3

[0186] Step 3. To a solution of (S)-3-((5-amino-1-(5-(chloromethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)pentan-1-ol (0.099 mmol, 40 mg) in DMSO (1 mL) was added 1-methylpiperazine (0.494 mmol, 49.5 mg). The reaction mixture was heated at 80 °C for 1 h and purified by preparative LC / MS using the following conditions: column: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:0.05% TFA; mobile phase B: acetonitrile containing 95:5 acetonitrile:0.05% TFA; gradient: hold at 0% B for 0 min, 0 - 40% B over 20 min, then hold at 100% B for 0 min; flow rate: 20 mL / min; column temperature: 25 °C. Fraction collection was triggered by MS and UV signals. The fractions containing Compound 163 were combined and dried by centrifugal evaporation (white solid, 6.7 mg, 9% yield). Example N - Compound 178

Chemical Structure

[0187] Step 1. A stirred solution of 2-chloro-5-methylpyridin-4-ol (5.00 g, 34.8 mmol) in DMF (50 mL) was cooled to 0 °C. NaH (1.39 g, 34.8 mmol) was added. After 10 minutes, methyl iodide (2.61 mL, 41.8 mmol) was added. The reaction mixture was stirred at RT for 16 hours and partitioned between water and ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude material was obtained as a pale yellow oil and purified using Combi Flash (silica gel 60 - 120 mesh; 15% ethyl acetate in petroleum ether as eluent). The fraction was concentrated using high vacuum at 50 °C to give 2-chloro-4-methoxy-5-methylpyridine (5.2 g, 32.7 mmol, 94% yield) as a yellow liquid. LC-MS [M+H] + 158.2 1 1H NMR (400 MHz, DMSO-d6) δ = 8.00 (s, 1H), 7.33 (s, 1H), 3.89 (s, 3H), 2.17 (s, 3H)

[0188] Step 2. To a stirred solution of 2-chloro-4-methoxy-5-methylpyridine (5.750 g, 36.5 mmol) in DMF (100 mL) and methanol (100 mL), TEA (15.26 mL, 109 mmol) was added. After purging with nitrogen for 5 minutes, PdCl2(dppf)-CH2Cl2 adduct (5.96 g, 7.30 mmol) was added. The reaction mixture was stirred at 100 °C for 12 hours under CO gas (pressure 10 kg). The reaction mixture was filtered through a CELITE™ bed. The filtrate was washed with methanol and concentrated in vacuo to give the crude material as a pale yellow oil. This was purified using Combi Flash (silica gel 60 - 120 mesh; 25% ethyl acetate in petroleum ether as eluent). The product-containing fraction was concentrated using high vacuum at 50 °C to give methyl 4-methoxy-5-methylpicolinate (5.00 g, 27.6 mmol, 76% yield) as a brown solid. LC-MS [M+H] + 182.2 1 1H NMR (400 MHz, DMSO-d6) δ = 8.36 (s, 1H), 7.90 (s, 1H), 3.99 (s, 3H), 3.84 (s, 3H), 2.22 (s, 3H)

[0189] Step 3. To a solution of methyl 5-methoxy-4-methylpicolinate (5.00 g, 27.6 mmol) in carbon tetrachloride (100 mL) were added AIBN (0.906 g, 5.52 mmol) and NBS (5.89 g, 33.1 mmol). The reaction mixture was stirred at 65 °C for 16 h and concentrated in vacuo. The residue was dissolved in ethyl acetate and partitioned between water and ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude material was obtained as a pale yellow oil, which was purified using Combi Flash (silica gel 60 - 120 mesh; 25% ethyl acetate in petroleum ether as eluent). The product-containing fractions were concentrated using high vacuum at 50 °C to give methyl 4-(bromomethyl)-5-methoxypicolinate (5.1 g, 14.51 mmol, 52.6% yield) as a pale yellow solid. LC-MS [M+H] + : 260.1 1 1H NMR (400 MHz, DMSO-d6) δ = 8.59 (s, 1H), 8.13 (s, 1H), 4.66 (s, 2H), 4.03 (s, 3H), 3.86 (s, 3H)

[0190] Step 4. To a stirred solution of methyl (7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (1.600 g, 4.78 mmol) in DMF (20 mL) were added Cs2CO3 (3.11 g, 9.55 mmol) and methyl 4-(bromomethyl)-5-methoxypicolinate (1.242 g, 4.78 mmol). The reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was partitioned between water and ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude material was obtained as a pale yellow solid, which was purified using Combi Flash (silica gel 60-120 mesh; eluent: 10% ethyl acetate in chloroform). The product-containing fractions were concentrated using high vacuum at 50 °C to give methyl 4-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.100 g, 1.968 mmol, 41.2% yield) as an off-white solid. LC-MS m / z 515.2 [M+H] + 1 H NMR(400MHz,DMSO-d6)δ = 11.71(s,1H),11.40(s,1H) 8.51(s,1H),7.43(s,1H),5.73(s,2H),4.04(s,3H),3.80(s,3H),3.73(s,3H)

[0191] Step 5. To a stirred solution of methyl 4-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.100 g, 2.139 mmol) in DMSO (10 mL) were successively added DBU (0.967 mL, 6.42 mmol), BOP (1.419 g, 3.21 mmol) and (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (0.761 g, 2.139 mmol). The reaction mixture was stirred at 45 °C for 4 h and then partitioned between water and ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude material was obtained as a pale yellow oil, which was purified using Combi Flash (silica gel 60 - 120 mesh; 25% ethyl acetate in chloroform as eluent). The fractions were concentrated using high vacuum at 50 °C to give methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.10 g, 1.188 mmol, 55.5% yield) as a yellow solid. LC-MS m / z 852.8 [M+H] +

[0192] Step 6. To a stirred solution of methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.30 g, 1.526 mmol) in methanol (15 mL) was added 10% palladium on carbon (0.812 g, 0.763 mmol). The reaction mixture was stirred at RT under H2 for 14 h. The mixture was filtered through a CELITE™ bed. The filtrate was washed with methanol and DCM (400 mL) and concentrated in vacuo at 50 °C to afford methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.050 g, 1.418 mmol, 93% yield) as a brown solid. LC-MS m / z 726.3 [M+H] +

[0193] Step 7. To a stirred solution of methyl (S)-4-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.00 g, 1.378 mmol) in THF (10 mL):methanol (3 mL) at 0 °C was added LiBH4 (10.33 mL, 20.66 mmol). The reaction mixture was stirred at 45 °C for 16 h, quenched with ammonium chloride solution, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude material was obtained as an off-white solid. The crude material was purified using Combi Flash (silica gel 60 - 120 mesh; eluent: 5% methanol in chloroform). The fractions were concentrated using high vacuum at 50 °C to give methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((2-(hydroxymethyl)-5-methoxypyridin-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.170 g, 0.173 mmol, 12.55% yield) as an off-white solid. LC-MS m / z 698.3 [M+H] +

[0194] Step 8. Thionyl chloride (0.105 mL, 1.433 mmol) was added to a stirred solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((2-(hydroxymethyl)-5-methoxypyridin-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.200 g, 0.287 mmol) in THF (3 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated to give methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((2-(chloromethyl)-5-methoxypyridin-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.226 g, 0.271 mmol, 95% yield) as a yellow oil. LC-MS m / z 718.2 [M+H] +

[0195] Step 9. To a stirred solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((2-(chloromethyl)-5-methoxypyridin-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.112 g, 0.156 mmol) in DMF (2 mL) were added methylamine HCl (0.021 g, 0.313 mmol) and K2CO3 (0.065 g, 0.469 mmol). The reaction mixture was stirred at 50 °C for 14 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in methanol (2 mL). HCl (5.21 μl, 0.172 mmol) was added in water (1 mL). The reaction mixture was stirred at RT and concentrated under reduced pressure. The crude was taken up in 1,4-dioxane (1 mL) and to this was added NaOH (0.044 g, 1.100 mmol) in water (1 mL). The reaction mixture was stirred at 70 °C for 3 h. The reaction mixture was partitioned between water and ethyl acetate. The organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude material was obtained as a pale brown oil. The crude material was purified by preparative LC / MS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:10 mM NH4OAc; mobile phase B: 95:5 acetonitrile:10 mM NH4OAc in water; gradient: hold at 10% B for 0 min, 10 - 45% B over 25 min, then hold at 100% B for 5 min; flow rate: 15 mL / min; column temperature: 25 °C) to give compound 178 (0.04 g, yield 2.4%).

[0196] Compound 177 was prepared similarly. Example O - Compound 174

Chemical Structure

[0197] Step 1. To a stirred solution of 2-methylpyridin-3-ol (10.0 g, 92 mmol) in acetonitrile (150.0 mL) was slowly added a solution of NBS (33.4 g, 188 mmol) in acetonitrile (350.0 mL) over 1 hour. The reaction mixture was stirred at 85 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude material, which was adsorbed on silica gel and purified by ISCO COMBIFLASH™ chromatography, eluting with 0 - 100% ethyl acetate in chloroform to afford 4,6-dibromo-2-methylpyridin-3-ol (11.0 g, 39.6 mmol, 43.2% yield) as a pale yellow solid. LC-MS m / z 268.0 [M+H] + 1 H NMR(300MHz,DMSO-d6)δ = 9.98(s,1H),7.70(s,1H),2.41(s,3H)

[0198] Step 2. To a stirred solution of 4,6-dibromo-2-methylpyridin-3-ol (10.0 g, 37.5 mmol) in THF (150.0 mL) was added n-BuLi (31.5 mL, 79 mmol) at -78 °C. The reaction mixture was stirred at the same temperature for 3 hours. To this mixture was added H2O (30.0 mL, 1665 mmol), followed by 1.5 N HCl solution (30.0 mL) at the same temperature. The reaction mixture was stirred at the same temperature for 10 minutes, diluted with saturated ammonium chloride solution, and extracted with DCM. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford 6-bromo-2-methylpyridin-3-ol (5.1 g, 25.5 mmol, 68.1% yield) as a light brown solid. LC-MS m / z 188.1 [M] + 1 H NMR(300MHz,DMSO-d6)d = 10.10(br s,1H),7.24(d,J=8.7 Hz,1H),7.08(d,J=8.3 Hz,1H),2.34 - 2.23(m,3H)

[0199] Step 3. To a stirred solution of 6-bromo-2-methylpyridin-3-ol (4.0 g, 21.27 mmol) in acetonitrile (40.0 mL) was added Cs2CO3 (20.79 g, 63.8 mmol). To this mixture was added MeI (1.995 mL, 31.9 mmol). The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was partitioned between EtOAc and water. The organic layer was washed with brine solution, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude compound. The crude compound was rinsed with petroleum ether and the filtrate was concentrated in vacuo to give 6-bromo-3-methoxy-2-methylpyridine (4.0 g, 18.81 mmol, 88% yield) as a brown solid. LC-MS m / z 202.0 [M+H] + 1 H NMR(300MHz, chloroform-d)δ = 7.23-7.14(m,1H),6.90(d,J=8.7 Hz,1H),3.75(s,3H),2.37(s,3H)

[0200] Step 4. To a stirred solution of 6-bromo-3-methoxy-2-methylpyridine (4.0 g, 19.80 mmol) in DMF (40.0 mL):MeOH (40.0 mL) were added TEA (8.28 mL, 59.4 mmol), PdCl2(dppf)-CH2Cl2 (3.23 g, 3.96 mmol) under nitrogen purge. The reaction mixture was stirred at 100 °C under CO gas (pressure 10 bar) in an autoclave for 16 h. The reaction mixture was concentrated in vacuo to give a residue. The residue was diluted with DCM and then filtered through a CELITE(™) bed and washed with an excess amount of DCM. The filtrate was concentrated in vacuo to give the crude compound. The crude compound was purified by ISCO Combiflash chromatography eluting with 0-100% ethyl acetate in petroleum ether to give methyl 5-methoxy-6-methylpicolinate (2.62 g, 14.32 mmol, 72.3% yield) as a pale brown solid. LC-MS m / z 182.0 [M+H] + 11H NMR (300 MHz, DMSO-d6) δ = 7.98 - 7.91 (m, 1H), 7.49 - 7.40 (m, 1H), 3.92 - 3.87 (m, 3H), 3.86 - 3.80 (m, 3H), 2.42 - 2.36 (m, 3H)

[0201] Step 5. To a stirred solution of methyl 5-methoxy-6-methylpicolinate (2.5 g, 13.80 mmol) in chloroform (25.0 mL) were added NBS (2.95 g, 16.56 mmol) and AIBN (0.453 g, 2.76 mmol). The reaction mixture was stirred at 65 °C for 16 h. The reaction mixture was filtered through a CELITE™ bed, washed with an excess of DCM, and the filtrate was concentrated under reduced pressure to give the crude compound. The crude compound was purified by ISCO Combiflash chromatography, eluting with 0 - 100% ethyl acetate in petroleum ether to give a pale brown solid, which was stirred in water for 15 min, followed by filtration of the solid and drying under vacuum to give methyl 6-(bromomethyl)-5-methoxypicolinate (1.6 g, 5.84 mmol, 42.4% yield) as a pale brown solid. LC-MS m / z 262.0 [M+H] + 1 1H NMR (300 MHz, DMSO-d6) δ = 11.17 - 10.94 (m, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.66 - 7.57 (m, 1H), 4.73 - 4.58 (m, 2H), 3.99 - 3.97 (m, 3H), 3.87 - 3.84 (m, 3H), 2.57 - 2.56 (m, 1H), 2.57 (s, 5H)

[0202] Step 6. To a stirred solution of methyl (7-hydroxy-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (2.0 g, 5.97 mmol) in DMF (20.0 mL) was added Cs2CO3 (3.89 g, 11.94 mmol). To this mixture was added methyl 6-(bromomethyl)-5-methoxypicolinate (1.552 g, 5.97 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour. The reaction mixture was partitioned between EtOAc and water. The organic layer was washed with brine solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by ISCO combiflash chromatography eluting with 0 - 100% ethyl acetate in chloroform to give methyl 6-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (1.08 g, 1.764 mmol, 29.6% yield) as a pale brown solid. LC-MS m / z 515.0 [M+H] +

[0203] Step 7. To a stirred solution of methyl 6-((7-hydroxy-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (0.32 g, 0.622 mmol) in DMSO (3.0 mL) were added DBU (0.281 mL, 1.867 mmol), BOP (0.413 g, 0.933 mmol) and (S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-amine (0.266 g, 0.747 mmol). The reaction mixture was stirred at 45 °C for 3 h. The reaction mixture was treated with water. The precipitate was collected and dried under vacuum to give the crude compound. The crude compound was purified by ISCO combiflash chromatography eluting with 0-100% ethyl acetate in petroleum ether to give methyl (S)-6-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (0.189 g, 0.220 mmol, 35.3% yield) as a pale brown solid. LC-MS m / z 852.2 [M+H] +

[0204] Step 8. To a stirred solution of methyl (S)-6-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-3-iodo-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (0.16 g, 0.188 mmol) in MeOH (5.0 mL) was added Pd-C (0.100 g, 0.094 mmol). The reaction mixture was stirred at RT under hydrogen gas (bladder) for 4 h. The reaction mixture was filtered through a CELITE™ bed, washed with an excess of methanol:DCM (1:1), and the filtrate was concentrated in vacuo to afford the crude compound. The crude compound was triturated with diethyl ether and petroleum ether, and the solid was dried under vacuum to give methyl (S)-6-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (0.118 g, 0.135 mmol, 71.8% yield) as a pale brown solid. LC-MS m / z 726.3 [M+H] +

[0205] Step 9. To a stirred solution of methyl (S)-6-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-5-methoxypicolinate (0.1 g, 0.138 mmol) in THF (3.5 mL):MeOH (1.5 mL), LiBH4 (2 M in THF) (0.344 mL, 0.689 mmol) was added. The reaction mixture was stirred at 45 °C for 16 h. To this mixture, LiBH4 (2 M in THF) (0.689 mL, 1.378 mmol) was added. The reaction mixture was stirred at 45 °C for 18 h and quenched with saturated aqueous NH4Cl. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to give methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((6-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.11 g, 0.128 mmol, 93% yield) as an off-white semi-solid. LC-MS m / z 698.3 [M+H] +

[0206] Step 10. To a stirred solution of methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-((6-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.1 g, 0.143 mmol) in MeOH (1.5 mL) was added aqueous HCl solution (0.1 mL, 1.152 mmol) at 0 °C. The reaction mixture was stirred at RT for 2 h. The reaction mixture was concentrated to dryness under reduced pressure and distilled with DCM to give the crude compound. The crude compound was triturated with diethyl ether and petroleum ether and the solid was dried under vacuum to give methyl (S)-(7-((1-hydroxyhexan-3-yl)amino)-1-((6-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate, HCl (85 mg, 0.141 mmol, 98% yield) as a pale green semi-solid. LC-MS m / z 460.2 [M+H] +

[0207] Step 11. To a stirred solution of methyl (S)-(7-((1-hydroxyhexan-3-yl)amino)-1-((6-(hydroxymethyl)-3-methoxypyridin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate, HCl (80 mg, 0.161 mmol) in dioxane (1.0 mL):water (1.0 mL) was added NaOH (32.3 mg, 0.807 mmol). The reaction mixture was stirred at 70 °C for 90 minutes. The organic layer was separated and concentrated under reduced pressure to give the crude compound. The crude material was purified by reverse phase preparative HPLC (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: water containing 5:95 acetonitrile:10 mM NH4OAc; mobile phase B: water containing 95:5 acetonitrile:10 mM NH4OAc; gradient: hold at 7% B for 0 minutes, 7 - 25% B over 20 minutes, then hold at 100% B for 5 minutes; flow rate: 15 mL / min; column temperature: 25 °C) to give Compound 174 (26.4 mg, 0.064 mmol, 40.0% yield). Example P - Compound 179 [Chemical formula]

[0208] Step 1. A 0.25 M solution of lithium diisobutyl-tert-butoxyaluminum hydride in THF / hexane (50 mL, 12.50 mmol) was added dropwise to a solution of methyl (S)-3-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzoate (1.87 g, 2.58 mmol) in THF (25.8 mL) at 0 °C over 5 minutes. The reaction mixture was stirred at 25 °C overnight (3 h, 98% conversion). The solution was diluted with cold water and extracted three times with AcOEt. Finally, the organic layer was dried over Na2SO4 and evaporated in vacuo. The material was purified by silica gel (hexane-EtOAc 0-100%) to afford methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (1.56 g, 2.238 mmol, 87% yield). LC-MS m / z 697.5 [M+H]+

[0209] Step 2. Methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(5-(hydroxymethyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.51 g, 0.732 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) in a 25 mL round-bottom flask to obtain a clear solution at 25 °C. After cooling the solution to 0 °C, Et3N (0.306 ml, 2.195 mmol) and Ms-Cl (0.114 ml, 1.464 mmol) were added. After 15 minutes, the reaction was complete and quenched with ice water and DCM. The organic layer was washed with brine and dried over Na2SO4. The solution was concentrated to give methyl (S)-(7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(2-methoxy-5-(methoxymethyl)benzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (0.35 g, 67.3% yield). The material was used without purification.

[0210] Step 3. (3S,4S)-4-Aminotetrahydro-2H-pyran-3-ol hydrochloride (70 mg, 0.456 mmol) and DIPEA (0.073 mL, 0.418 mmol) were added to (S)-3-((7-((1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-5-((methoxycarbonyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-methoxybenzyl methanesulfonate (108 mg, 0.139 mmol) in DMF (1 mL). The reaction mixture was stirred at 25 °C for 12 h. Formation of the first intermediate methyl (7-(((S)-1-((tert-butyldiphenylsilyl)oxy)hexan-3-yl)amino)-1-(5-((((3S,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)methyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate was confirmed by LC / MS. HCl was added to the reaction mixture in 1,4-dioxane (3 mL, 12.00 mmol), followed by stirring at 25 °C for 2 h. The solvent was removed, and formation of the second intermediate methyl (7-(((S)-1-hydroxyhexan-3-yl)amino)-1-(5-((((3S,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)methyl)-2-methoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate was confirmed by LC / MS. The solvent was removed, the residue was diluted with 5 M NaOH in MeOH, and then stirred at 80 °C for 1 h. The target product was confirmed by LC / MS, and the solvent was removed.

[0211] The crude material was purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: held at 3% B for 0 min, increased from 3 - 43% B over 30 min, then held at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the target compound were combined and dried by centrifugal evaporation.

[0212] The substance was further purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing 0.05% TFA; Mobile phase B: 95:5 acetonitrile: water containing 0.05% TFA; Gradient: held at 0% B for 0 min, increased from 0 - 40% B over 25 min, then held at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the target compound were combined and dried by centrifugal evaporation.

[0213] The substance was further purified by preparative LC / MS using the following conditions: Column: XBridge C18, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile: water containing NH4OAc; Mobile phase B: 95:5 acetonitrile: water containing NH4OAc; Gradient: held at 1% B for 0 min, increased from 1 - 41% B over 25 min, then held at 100% B for 0 min; Flow rate: 20 mL / min; Column temperature: 25 °C. Fraction collection was triggered by the MS signal. The fractions containing the target compound were combined and dried by centrifugal evaporation to obtain Compound 179 (15.9 mg, 0.031 mmol, yield 22.38%).

[0214] The following compounds were prepared analogously: Compound 180, Compound 181, Compound 182, Compound 183, and Compound 184. Example Q - Starting Materials and Intermediates

[0215] The following charts show schemes for creating compounds that may be useful as starting materials or intermediates for the preparation of TLR7 agonists disclosed herein. The schemes may be applied to the creation of other similar compounds that may be used as starting materials or intermediates. The reagents used are well-known in the art and, in many cases, their use is shown in the foregoing examples. Chart 1

Chem.

Chem.

Chem.

[0216] Biological Activity The biological activity of the compounds disclosed herein as TLR7 agonists may be quantified by the following procedure.

[0217] Human TLR7 Agonist Activity Assay This procedure describes a method for quantifying the human TLR7 (hTLR7) agonist activity of the compounds disclosed herein.

[0218] Modified human embryonic kidney HEK-Blue™ TLR7 cells (Invivogen) with 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 h. Compounds (100 nl) were added to the wells containing HEK-Blue™ TLR cells, and the treated cells were incubated at 37 °C, 5% CO2. Eighteen hours after treatment, 10 microliters of freshly prepared Quanti-Blue™ reagent (Invivogen) was added to each well and incubated for 30 min (37 °C, 5% CO2), and SEAP levels were measured using an Envision plate reader (OD = 620 nm). The half maximal effective concentration value (EC 50 ; the compound concentration that elicits a response midway between the assay reference value and the maximum) was calculated.

[0219] Induction of Type I Interferon Gene (MX-1) and CD69 in Human Blood Induction of the type I interferon (IFN) MX-1 gene and the 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.

[0220] 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 and predotted at 10 nL per well using an Echo to a final concentration of 1 μM (10 nL in 10 μL of blood). After mixing on a shaker for 30 s, the plates were covered and left overnight = 17 h in a 37 °C chamber. Fixation / Lysis buffer was prepared (5x in H20 → 1x, warmed at 37 °C; Cat# BD 558049), and Palm buffer was maintained (on ice) for later use.

[0221] Surface antibodies were prepared for surface marker staining (CD69): 0.045 μl hCD14-FITC (ThermoFisher Cat # MHCD1401) + 0.6 μl hCD19-ef450 (ThermoFisher Cat # 48-0198-42) + 1.5 μl hCD69-PE (cat# BD555531) + 0.855 μl FACS buffer. Added at 3 μl / well, centrifuged at 1000 rpm for 1 minute, mixed on a shaker for 30 seconds, and placed on ice for 30 minutes. After 30 minutes, the stimulation was stopped with 70 μL of pre-warmed 1x fixation / permeabilization buffer, resuspended using Felix mate (changed the tip for each plate 15 times), and incubated at 37 °C for 10 minutes.

[0222] Centrifuged at 2000 rpm for 5 minutes, aspirated with an HCS plate washer, mixed on a shaker for 30 seconds, then washed with 70 μL of dPBS and pelleted twice (2000 rpm, 5 minutes), washed with 50 μL of FACS buffer and pelleted once (2000 rpm, 5 minutes). Mixed on a shaker for 30 seconds. For intracellular marker staining (MX-1): 50 μl of BD Perm buffer III was added and mixed on a shaker for 30 seconds. Incubated on ice for 30 minutes (in the dark). Washed twice with 50 μL of FACS buffer (centrifuged at 2300 rpm for 5 minutes after permeabilization), then mixed on a shaker for 30 seconds. Resuspended in 20 μL of FACS buffer containing MX1 antibody ((4812)-Alexa 647: Novus Biologicals #NBP2-43704AF647) (20 μl FACS buffer + 0.8 μl hIgG + 0.04 μl MX-1). Centrifuged at 1000 rpm for 1 minute, mixed on a shaker for 30 seconds, incubated the sample at RT in the dark for 45 minutes, and then washed with 2x FACS buffer (centrifuged at 2300 rpm for 5 minutes after permeabilization). Resuspended in 20 μl of FACS buffer (total 35 μL per well), covered with foil, placed at 4 °C, and read the next day. The plate was read with an iQuePlus. The results were loaded into the toolset and an IC50 curve was created with CurveMaster. 100% on the y-axis was set to 1 μM of resiquimod.

[0223] Induction of TNF-alpha and Type I IFN Response Genes in Mouse Blood The induction of TNF-alpha and type I IFN response genes is a downstream event that occurs upon activation of the TLR7 pathway. The following is an assay for measuring their induction in mouse whole blood in response to a TLR7 agonist.

[0224] Heparinized mouse whole blood was diluted in RPMI 1640 medium containing Pen-Strep at a ratio of 5:4 (50 μL of whole blood and 40 μL of medium). Ninety microliters of the diluted blood was transferred to the wells of a Falcon flat-bottom 96-well tissue culture plate, and the plate was incubated at 4 °C for 1 hour. The test compound in 100% DMSO stock was diluted 20-fold in the same medium for the concentration-response assay and then 10 μL of the diluted test compound was added to the wells to give a final DMSO concentration of 0.5%. To the control wells, 10 μL of medium containing 5% DMSO was added. The plate was then incubated at 37 °C in a 5% CO2 incubator for 17 hours. After incubation, 100 μL of medium was added to each well. The plate was centrifuged, 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 RT for 5 - 10 minutes, followed by the addition of 2 μL of proteinase K (20 mg / mL) to each well. The plate was then shaken at RT for 15 - 20 minutes. The plate was then stored at -80 °C until further processing.

[0225] The frozen samples were thawed, and mRNA was extracted using the Invitrogen mRNA Catcher Plus Kit (Cat# K1570-02) according to the manufacturer's instructions. Using half of the mRNA obtained from RNA extraction, cDNA was synthesized in a 20 μL reverse transcriptase reaction using 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.

[0226] Definition "Aliphatic" means a straight-chain 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.

[0227] "Alkyl" means a saturated aliphatic moiety following the same convention for specifying the applicable number of 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,

Chem.

[0228] "Alkenyl" means an aliphatic moiety having at least one carbon-carbon double bond following the same convention for specifying the applicable number of 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.

[0229] "Alkynyl" means an aliphatic moiety having at least one carbon-carbon triple bond following the same convention for specifying the applicable number of carbon atoms. Examples of C2-C4 alkynyl groups include, but are not limited to, ethynyl (ethynyl), propargyl (prop-2-ynyl), 1-propynyl, but-2-ynyl, and the like.

[0230] "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

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0231] "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 having 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.

[0232] "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.

[0233] "Halogen" or "halo" means fluorine, chlorine, bromine or iodine, unless a more narrow meaning is indicated.

[0234] "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.

[0235] "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.

[0236] For example, when using "unsubstituted or substituted" or "optionally substituted", that is, expressing as "unsubstituted or substituted C1-C5 alkyl" or "optionally substituted heteroaryl", etc., it is indicated that the moiety may be substituted. Such a moiety may have one or more independently selected substituents, preferably 1 to 5 in number, more preferably 1 to 2 in number. The substituents and substitution patterns may be selected by those skilled in the art considering the moiety to which the substituent is attached, and provide compounds that are chemically stable and can be synthesized by techniques known in the art and the methods described herein. When a moiety is specified as "unsubstituted or substituted" or "optionally substituted", in a preferred embodiment, such a moiety is unsubstituted.

[0237] "Arylalkyl", "(heterocycloaliphatic)alkyl", "arylalkenyl", "arylalkynyl", "biarylalkyl", and the like, in some cases, mean an alkyl, alkenyl, or alkynyl moiety substituted with aryl, heterocycloaliphatic, biaryl, etc., and in some cases, a moiety having an open (unsatisfied) valence with an alkyl, alkenyl, or alkynyl moiety, such as benzyl, phenethyl, N-imidazolylethyl, N-morpholinoethyl, and the like. Conversely, "alkylaryl", "alkenylcycloalkyl", and the like, in some cases, mean an aryl, cycloalkyl, or other moiety substituted with alkyl, alkenyl, etc., and in some cases, a moiety such as methylphenyl (tolyl) or allylcyclohexyl. "Hydroxyalkyl", "haloalkyl", "alkylaryl", "cyanoaryl", and the like, in some cases, mean an alkyl, aryl, or other moiety substituted with one or more specific substituents (in some cases, hydroxyl, halo, etc.).

[0238] 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.

[0239] When the moiety to be replaced is an aliphatic moiety, preferred substituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo, hydroxyl, cyano, nitro, alkoxy, -O(hydroxyalkyl), -O(haloalkyl), -O(cycloalkyl), -O(heterocycloalkyl), -O(aryl), alkylthio, arylthio, =O, =NH, =N(alkyl), =NOH, =NO(alkyl), -CO2H, -C(=O)NHOH, -C(=O)O(alkyl), -C(=O)O(hydroxyalkyl), -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -OC(=O)(alkyl), -OC(=O)(hydroxyalkyl), -OC(=O)O(alkyl), -OC(=O)O(hydroxyalkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NH(hydroxyalkyl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, -NHC(=NH)NH2, -OSO2(alkyl), -SH, -S(alkyl), -S(aryl), -S(=O)alkyl, -S(cycloalkyl), -SO2(alkyl), -SO2NH2, -SO2NH(alkyl), and -SO2N(alkyl)2. More preferred substituents are halo, hydroxyl, cyano, nitro, alkoxy, -O(aryl), =O, =NOH, =NO(alkyl), -OC(=O)(alkyl), -OC(=O)O(alkyl), -OC(=O)NH2, -OC(=O)NH(alkyl), -OC(=O)N(alkyl)2, azide, -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl), -NHC(=O)(alkyl), -NHC(=O)H, -NHC(=O)NH2, -NHC(=O)NH(alkyl), -NHC(=O)N(alkyl)2, and -NHC(=NH)NH2. Particularly preferred substituents are phenyl, cyano, halo, hydroxyl, nitro, C1-C4 alkoxy, O(C2-C4alkanediyl)OH, and O(C2-C4alkanediyl)halo.

[0240] 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.

[0241] 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.

[0242] Unless a particular stereoisomer is clearly indicated (e.g., by bolding or dashing the valence bond at the relevant stereocenter in the structural formula, or by depicting the double bond as having an E or Z configuration in the structural formula, or by using nomenclature or symbols that specify stereochemistry), all stereoisomers are included within the scope of the invention as pure compounds as well as mixtures thereof. Unless otherwise specified, racemates, individual enantiomers (whether optically pure or partially resolved), diastereomers, geometric isomers, and combinations thereof, and mixtures thereof are all encompassed by the present invention.

[0243] One of ordinary skill in the art will recognize that the compounds 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.

[0244] "Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (e.g., in the human body) to produce the parent compound or a salt thereof, or that itself has activity similar to that of the parent compound. Suitable esters include C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl esters, particularly methyl, ethyl, or n-propyl esters.

[0245] "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 may be an acid addition salt, e.g., a sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate, methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like. When the compound has one or more acidic groups, the salt may be a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like. Polymorphic forms and solvates are also included within the scope of the present invention.

[0246] "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.

[0247] The terms "treat", "treating", and "treatment", as used in the context of treating a disease or disorder, are intended to include reducing or suppressing one or more of the disorders, diseases, or conditions, or symptoms associated with the disorder, disease, or condition; or delaying the progression, spread, or worsening of the disease, disorder, or condition, or one or more of their symptoms. "Treatment of cancer" refers to one or more of the following effects: (1) inhibition of tumor growth to some extent, including (i) delay and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintenance of tumor size; (4) reduction in tumor size; (5) inhibition of tumor cell infiltration into peripheral organs, including (i) reduction, (ii) delay, or (iii) complete prevention; (6) inhibition of metastasis, including (i) reduction, (ii) delay, or (iii) complete prevention; (7) enhancement of the anti-tumor immune response, which may result in (i) maintenance of tumor size, (ii) reduction in tumor size, (iii) delay in tumor growth, (iv) reduction, delay, or prevention of infiltration; and / or (8) reduction in the severity or number of one or more symptoms associated with the disorder to some extent.

[0248] In the formulas herein, a wavy line ([[]]

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0249] In the formulas of this specification, a valence bond that crosses an aromatic ring between two carbons thereof means that the group bonded to that valence bond may be at any of the positions of the aromatic ring that can be vacated by the removal of hydrogen that is implicitly there (or explicitly there if fully written). As an example, the following formula:

Chemical formula

Chemical formula

[0250] In another example,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0251] 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 limiting, of isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include 13 C and 14C may be mentioned. The isotopically labeled compounds of the present invention can generally be prepared by using appropriate isotopically labeled reagents instead of the unlabeled reagents used in other cases, by conventional techniques known to those skilled in the art, or by procedures similar to those described herein. As an example, a C1-C3 alkyl group may be non-deuterated, partially deuterated, or fully deuterated, and "CH3" may include CH3, 13 CH3, 14 CH3, CH2T, CH2D, CHD2, CD3, etc. In one embodiment, the various elements in the compound are present in their natural isotopic abundances.

[0252] 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.

[0253] Acronym and Abbreviation This is a table assigning meanings to the acronyms and abbreviations used herein.

Table 37

Table 38

[0254] Reference 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 incorporated herein by reference for all purposes.

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[0327] The foregoing detailed description of the invention includes sections that relate primarily or exclusively to specific parts or aspects of the invention. This is for clarity and convenience, and it is to be understood that a particular feature may be relevant not only in the section in which it is disclosed but also in other sections, and that the disclosure herein includes all appropriate combinations of the information described in different sections. Similarly, although the various figures and descriptions herein relate to specific embodiments of the invention, if a specific feature is disclosed in the context of a particular figure or embodiment, it is to be understood that such feature may be used in combination with other features in the context of another figure or embodiment, or generally in the invention, to the extent appropriate.

[0328] Furthermore, although the invention has been particularly described with respect to certain preferred embodiments, the invention is not limited to such preferred embodiments. On the contrary, the scope of the invention is defined by the appended claims.

Claims

1. A compound having a structure represented by the following formula (Ie): 【Chemical 1】 [wherein: R1 is 【Chemical 2】 ; R4 is [Chemical Formula 3] ; R5 is H or Me]

2. A compound having a structure represented by the following formula (If): [wherein, 【Chemical Formula 4】 ; R １ is 【Chemical Formula 5】 W is

3. 【Chemical Formula 6】 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 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, the pharmaceutical composition according to any one of claims 3 to 5. ​ ​ ​

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