DNA polymerase iiic inhibitors and uses thereof

Abstract

To provide compositions and methods for use in the treatment of Gram-positive bacterial infections.SOLUTION: The compound of formula I, or an optical isomer thereof, an isotopic isomer thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof. (A, B:N, CH. n:0 to 3. R0:H, CH2PO (OH) 2, etc. R1: (CH2) m {(V) o (CH2) p} q W (V: CH2, CH=CH, CO, etc. W:H, halo, substituted / unsubstituted C1-6 alkyl, and the like. m:1-5. O, p, q:0-4). R2:H, halo, CN, etc. R3:Cl, F, Br, I, OH, etc. ) SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 783,754, filed December 21, 2018, the contents of which are incorporated herein by reference. The present invention relates to compounds and methods useful for inhibiting the DNA polymerase IIIC (pol IIIC) enzyme. The present invention also provides pharmaceutically acceptable compositions comprising the compounds of the present invention, and methods of using the compositions in the treatment of Gram-positive bacterial infections.

[0002] Background of the Invention Bacterial pathogens pose a serious threat to public health. Aerobic and anaerobic Gram-positive bacteria with multiple resistance to a variety of antibiotics have emerged as a major treatment challenge. Two gram-positive pathogens, Staphylococcus aureus and Enterococcus faecalis / faecium, account for the majority of hospital-acquired illnesses (Muto, et al.). A third organism, Streptococcus pneumoniae, is a common community-acquired pathogen. These organisms are aerobic, meaning they grow in an oxygen-containing atmosphere.

[0003] Staphylococcus aureus is the most common cause of nosocomial bacteremia and skin / wound infections and the second most common cause of nosocomial lower respiratory tract infections. The emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has become a serious public health concern. MRSA strains have become increasingly multidrug-resistant over time. In many parts of the world, MRSA infections represent the majority of community-acquired sporadic staphylococcal infections. These strains are also associated with numerous outbreaks of localized (skin and skin structure) and invasive (bacteremic) infections.

[0004] Enterococcus faecalis and Enterococcus faecium cause nosocomial sepsis, endocarditis, and wound and urinary tract infections. A vancomycin-resistant phenotype was first reported in enterococci (vancomycin-resistant enterococci or VRE) in 1987, many years after the drug was introduced into widespread clinical use. Today, >30% of Enterococcus faecalis infections in ICUs are VRE. There are few or no treatment options for certain illnesses caused by VRE, including bloodstream infections, surgical site, and urinary tract infections. The incidence of VRE is approximately 20,000 patients per year in the United States alone.

[0005] Streptococcus pneumoniae is the most common bacterial cause of meningitis, community-acquired pneumonia, acute otitis media, and sinusitis. In the United States, S. pneumoniae is estimated to account for 3,000 to 6,000 cases of pneumococcal meningitis, 500,000 cases of pneumonia, over 12,000 cases of bacteremia, and 6 million cases of otitis media each year. Annual mortality from S. pneumoniae-induced diseases is estimated at 40,000 in the United States and 3 to 5 million worldwide. Penicillin-resistant S. pneumoniae (PRSP) is increasingly being identified. The emergence and spread of drug-resistant strains of S. pneumoniae complicates the treatment of these common infections. Anaerobic bacteria, i.e., bacteria that thrive in an oxygen-deficient atmosphere, are also a public health problem. Clostridium difficile has been increasingly associated with illness in human patients, often as a result of treatment with certain antibiotic drugs. The most common illness is called Clostridium difficile-associated diarrhea (CDAD).

[0006] One approach to solving the problem of multidrug-resistant bacteria involves the development of effective antibacterial agents that can selectively attack new bacterial targets. The DNA pol IIIC enzyme has been shown to be crucial for replicative DNA synthesis in Gram-positive bacteria (Kornberg, et al.). Because the DNA pol IIIC enzyme shows little homology to mammalian or Gram-negative bacterial DNA polymerases, it is an attractive target for inhibition in discovering new Gram-positive-selective antibacterial agents.

[0007] The DNA pol IIIC enzyme is specifically required for chromosome replication by low-G:C Gram-positive organisms (both aerobic and anaerobic bacteria). The DNA pol IIIC enzyme, encoded by the structural gene polC, is one of two specialized DNA polymerases essential for replication in Gram-positive bacteria. polC is absent from high-G:C eubacteria, Gram-negative eubacteria, and eukaryotic cells, but is highly conserved among a wide range of Gram-positive pathogens.

[0008] Thus, DNA pol III is essential for replication of host chromosomes in Gram-positive bacteria with low G:C content. If its function is blocked, chromosomal DNA fails to replicate and the bacterial host dies. This essential structure of pol IIIC is tightly conserved among a broad group of Gram-positive pathogens with low G:C content, including Staphylococcus, Streptococcus, Enterococcus, and Mycoplasma (Tarantino, et al., Antimicrobial Agents and Chemotherapy, August 1999, pp. 1982-87). Although DNA pol IIIC inhibitors have demonstrated Gram-positive antibacterial and in vivo protective activity, their development has been hindered by the lack of "druggable" characteristics of the compounds, such as compatibility with parenteral formulations or favorable pharmacokinetics. Thus, there remains a need to identify compounds that can be effectively used to inhibit DNA pol III C and, therefore, treat and inhibit bacterial infections.

[0009] Summary of the Invention The present invention relates to DNA pol IIIC inhibitors useful against Gram-positive microorganisms, including antibiotic-resistant strains such as vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, and the Gram-positive anaerobe Clostridium difficile.

[0010] In one aspect, the present invention provides a compound of the formula shown below: [ka] wherein A and B are independently N, CH, or R1; n is 0 to 3, R1 is (CH2) m -{(V) o -(CH2) p} q -W, V is CH2, CH=CH, C≡C, CO, O, S, SO, SO2, NR4, CHR5, OC(O), (O)CO, CONR6, NR7CO, SO2NH, NHSO2;C 3~8 is cycloalkyl, Each of R4, R6 and R7 is independently H or C 1~6 is alkyl, R5 is OH or C 1~6 alkyl, CH(R8R9), Each of R8 and R9 is independently H, halo, or C 1~6 is alkyl, W is H, halo, substituted or unsubstituted C 1~6 Alkyl, substituted or unsubstituted C 3~8Cycloalkyl, substituted or unsubstituted C 2~8 Heterocyclyl, substituted or unsubstituted C 6~14 Aryl, substituted or unsubstituted C 1~10 Heteroaryl, NH2, CN, OR 10 , S.R. 11 , C.O.R. 12 ,OCOR 13 , N.R. 14 COR 15 , N.R. 16 R 17 , N.R. 18 (CO)NHR 19 , CH(CO2R 20 )2, CO2R 21 , NHSO2R 22 ,CONR 23 R 24 , CH2CO2R 25 , S(O)R 26 or S(O2)R 27 and R 10 ~R 27 each independently represents H, substituted or unsubstituted C 1~6 Alkyl, substituted or unsubstituted C 3~8 Cycloalkyl, substituted or unsubstituted C 2~8 Heterocyclyl, substituted or unsubstituted C 6~14 Aryl, substituted or unsubstituted C 1~10 is heteroaryl, m is 1 to 5, o is 0 to 4, p is 0 to 4, and q is 0 to 4; R2 is H, halo, CN, substituted or unsubstituted C 1~6 Alkyl, CO2R 21 ,CONR 23 R 24 , substituted or unsubstituted C 2~8 Heterocyclyl or substituted or unsubstituted C 1~10 is heteroaryl, R3 is F, Cl, Br, I, C 1~6 Alkyl, OH, CN, C 1~6 -C substituted by one or more substituents selected from the group consisting of alkyl, CF3, CHF2, CF3CH2, OCH3 and OCF3 6~14Aryl or C 1~10 is heteroaryl, R0 is H, CH2OPO(OH)2, CH2OCONHCH2(CH2) t OPO(OH)2, CHOCOCH(CH2) t OPO(OH) 2、 COO(CH2) t OPO(OH)2, CH2OPO(OH)OPO(OH)2 or (CR 30 R 31 O) s -XY-(CR 30 R 31 ) t -OPO(OR 28 )(OR 29 ) and X is a direct bond or (C=O), Y is a direct bond or oxygen, s is 0 or 1, t is 1, 2, or 3; R 28 and R 29 are each independently hydrogen or a hydrolyzable ester group, and R 28 is hydrogen, R 29 HA-P(O)OR 32 OR 33 It can be, R 30 and R 31 are each independently hydrogen or C1-4 alkyl, R 32 and R 33 are each independently hydrogen or a hydrolyzable ester group), or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, an isotopic isomer thereof, a prodrug or a pharmaceutically acceptable salt thereof.

[0011] Specific compounds of the above formula are described herein. The present invention covers all enantiomeric, racemic, tautomeric and diastereomeric forms of the compounds described herein, and mixtures thereof. The invention further features a pharmaceutical composition including a compound of Formula I and a pharmaceutically acceptable carrier.

[0012] In another aspect, the invention features formulations of compounds of Formula I suitable for coating the surface of, for example, a medical device described herein. In such formulations, the compounds of the invention can be mixed with a suitable biocompatible coating agent or can be covalently or otherwise bound (e.g., electrostatically or as a ligand) to the coating agent.

[0013] In another aspect, the invention features a method for inhibiting bacterial growth, including contacting an area where bacteria tend to grow (e.g., a habitat or surface, such as a medical device) with a compound of Formula I. The invention also features a method for treating an animal for a gram-positive bacterial infection, comprising administering to the animal a therapeutically effective amount of a compound of formula I.

[0014] In various aspects of the present invention, the compounds of the present invention are useful for treating or preventing infection or inhibiting or preventing the growth of Gram-positive bacteria, including, but not limited to, Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; Enterococcus faecalis; Enterococcus faecium; vancomycin-resistant enterococci; Streptococcus pneumoniae; other microorganisms in the genera Bacillus, Staphylococcus, Streptococcus, and Enterococcus; and any other Gram-positive microorganism that produces DNA pol IIIC enzyme.

[0015] Detailed Description of the Invention The features and other details of the present invention are described in more detail below. It will be understood that the detailed embodiments described herein are shown for purposes of illustration and not as limitations of the present invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.

[0016] definition The term "alkyl" is defined as a branched or unbranched saturated acyclic hydrocarbon group, preferably having 1 to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, and hexyl. Alkyl groups can be unsubstituted or substituted as described herein.

[0017] The term "cycloalkyl" is defined as a monocyclic or bicyclic structure having only carbon atoms in the ring(s), each ring preferably having from 3 to 8 members. Exemplary cycloalkyl groups include cyclopropyl; cyclobutyl; cyclopentyl; and cyclohexyl. Cycloalkyl groups can be unsubstituted or substituted as described herein.

[0018] The term "heterocyclyl" is defined as a monocyclic, bicyclic, or polycyclic heterocyclic ring system that does not include an aromatic ring. Each ring preferably contains 2 to 8 carbon atoms and 1 to 4 oxygen, nitrogen, and / or sulfur atoms. Examples include aziridinyl, azetidinyl, morpholinyl, oxazolidinyl, oxazolinyl, oxecanyl, oxepanyl, oxiranyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, and tetrahydrothiopyranyl. Heterocyclyl groups can be unsubstituted or substituted as described herein.

[0019] The term "aryl" is defined as a monocyclic, bicyclic, or polycyclic carbocyclic ring system having one or more aromatic rings. Each ring preferably contains from 6 to 14 carbon atoms. Examples include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl. Aryl groups can be unsubstituted or substituted as described herein.

[0020] The term "heteroaryl" is defined as a monocyclic, bicyclic, or polycyclic heterocyclic ring system having one or more aromatic rings. Each ring preferably contains 1 to 10 carbon atoms and 1 to 4 oxygen, nitrogen, and / or sulfur atoms. Examples include benzimidazolyl, benzofuranyl, benzotriazolyl, furyl, imidazolyl, indolyl, isobezofuranyl, isoquinolinyl, isoxazolyl, oxazolyl, purinyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thienyl, triazinyl, and triazolyl. Heteroaryl groups can be unsubstituted or substituted as described herein.

[0021] The term "halo" is defined as fluoro, bromo, chloro, or iodo. The term "alkoxy" is defined as --OR, where R is an alkyl group. The term "aryloxy" is defined as --OR, where R is an aryl group. The term "alkylamino" is defined as --NHR, where R is an alkyl group. The term "arylamino" is defined as --NHR, where R is an aryl group. The term "alkylsulfonyl" is defined as -SOR2, ​​where R is an alkyl group. The term "arylsulfonyl" is defined as -SOR2, ​​where R is an aryl group. The term "alkylthio" is defined as --SR, where R is an alkyl group. The term "arylamino" is defined as --NHR, where R is an aryl group. The term "alkylthio" is defined as --SR, where R is an alkyl group. The term "arylthio" is defined as --SR, where R is an aryl group. The term "quaternary amino" refers to -NRRR'R'', where R, R', and R'' are independently alkyl, aryl, heteroaryl, and heterocyclyl. + It is defined as follows.

[0022] The term "substituted" means that one or more hydrogen atoms of a group or part of a group have been replaced with a C 1~6 Alkoxy, C 6~14 Aryloxy, sulfhydryl (-SH), C 1~6 Alkylthio, C 6~14 Arylthio, amino (-NH2), C 1~6 Alkylamino, C 6~14 Arylamino, disubstituted amino, quaternary amino, hydroxyl (-OH), carboxyl (-COOR), halo, cyano (-CN), azido (-N3), oxo, -C(O)-C 1~6 Alkyl, -C(O)-C 3~8 Cycloalkyl, -C(O)-C 6~14 Aryl, -C(O)-C 1~10 Heteroaryl, C(O)-C 2~8 Heterocyclyl, C 1~6 Alkyl sulfonyl, (SO2)OC 1~6 Alkyl, -(SO2)OC 3~8 Cycloalkyl, -(SO2)OC 3~8 Cycloalkyl, -(SO2)-C 6~14 Aryl, -(SO2)OC 6~14 Aryl, -(SO2)-C 1~10 Heteroaryl, -(SO2)OC 1~10 Heteroaryl, -(SO2)-C 2~8 Heterocyclyl and -(SO2)OC 2~8 Heterocyclyl is defined as being substituted by a substituent, including but not limited to:

[0023] Additionally, alkyl, aryl, cycloalkyl, heteroaryl and heterocyclyl groups can be C 6~14 Aryl, C 3~8 Cycloalkyl, C 1~10 Heteroaryl or C 2~8 The cycloalkyl, heteroaryl, and heterocyclyl groups may also be substituted with alkyl groups. The substituents may be as described for the parent group, for example, halogen, trifluoromethyl, hydroxyl, or carboxyl. As used herein, the terms "administration" or "administering" refer to the method of providing one or more unit doses of an antimicrobial pharmaceutical composition to an animal (e.g., topical, oral, intravenous, intraperitoneal, or intramuscular administration). The method of administration may vary depending on various factors, such as the components of the pharmaceutical composition, the potential or actual site of infection, the microorganism involved, and the severity of the actual microbial infection.

[0024] "Animal" refers to any animal susceptible to gram-positive bacterial infections. For example, animals may include humans, dogs, cats, pigs, cows, horses, goats, chickens, turkeys, sheep, rats, mice, and rabbits, as well as other animals kept commercially or as pets. The term "animal susceptible to microbial infections" is defined as an animal at increased risk of contracting a microbial infection compared to the general population. Examples of such animals include those that have recently undergone a surgical procedure or immunocompromised humans, such as those with AIDS (acquired immune deficiency syndrome) or those with transplants requiring immunosuppressive drugs. Such animals can be identified using methods known to those skilled in the art.

[0025] The term "coating agent" is defined as a biocompatible compound or mixture of compounds suitable for coating a surface. Suitable coating agents are known in the art. Exemplary coating agents include, but are not limited to, polymers such as polyethylene glycol, hypromellose, hydroxypropyl cellulose, polytetrafluoroethylene, methylcellulose, polyvinyl alcohol, or other biocompatible polymers. The term "effective amount" of a compound is defined as an amount that, when administered to a site of infection or potential infection, e.g., a habitat, e.g., a eukaryotic cell culture, or a patient, achieves a specific level of microbial inhibition or prevention of the establishment of a microbial infection, respectively.

[0026] The term "inhibiting" is defined as reducing the rate of microbial cell growth by at least 80%. In certain embodiments, growth can be inhibited by up to 90%, 95%, or even 99% or more. The degree of inhibition can be confirmed, for example, by an in vitro growth assay, e.g., by standard liquid culture techniques. Compounds that exhibit inhibition of colony formation at a minimum inhibitory concentration (MIC) of <100 μg / ml, more preferably <10 μg / ml, are particularly useful.

[0027] The term "habitat" is defined as any substance, liquid, or solid on or in which microorganisms may reside, or in which it is desirable to prevent the presence of microorganisms. Exemplary habitats include culture media (e.g., agar or broth), food, medical supplies (e.g., sterile fluids), medical devices (e.g., catheters), countertops, and other surfaces.

[0028] The term "microbial infection" is defined as the invasion of a host animal by a pathogenic microorganism. For example, an infection can include the overgrowth of a microorganism normally present in or on an animal, or the growth of a microorganism not normally present in or on an animal. More generally, a microbial infection can be any situation in which the presence of a population or populations of microorganisms causes damage to a host animal. Thus, an animal is "suffering from" a microbial infection when an excessive amount of a population of microorganisms is present in or on an animal, or when the presence of a population or populations of microorganisms causes damage to the animal's cells or other tissues. In one aspect, the number of microorganisms of a particular genus or species is at least 2, 4, 6, or 8 times the number normally found in the animal. Examples of microorganisms include, but are not limited to, gram-positive or any other class of bacteria.

[0029] "Pharmaceutically acceptable salts" refers to salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Other acids, such as oxalic acid, are not pharmaceutically acceptable per se but may be useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., magnesium), ammonium, and NR4 + (wherein R is C 1~4 Preferred salts include the hydrochloride, hydrobromide, sulfate, mesylate, maleate, tartrate, and fumarate salts. References below to compounds according to the invention include compounds of the general formula shown, as well as pharmaceutically acceptable salts thereof.

[0030] "Prevention" of microbial growth or infection is defined as application of a compound of the invention such that microbial growth or infection does not occur. The amount of a compound of the invention required to prevent microbial growth can be determined, for example, by an in vitro growth assay, e.g., by standard liquid culture techniques. The amount of a compound of the invention required to prevent microbial infection can be determined, for example, by an in vivo assay, e.g., by determining the amount of compound that must be administered to prevent infection in a research animal, e.g., a guinea pig, after inoculation with the microorganism. Generally, compounds that show prevention at suitable concentrations, e.g., <100 μg / ml, more preferably <10 μg / ml, are useful for further testing as therapeutic agents.

[0031] The term "treating" is defined as the medical management of a patient with the intent to result in the cure, amelioration, or prevention of a disease, condition, or disorder. This term encompasses active treatment, i.e., treatment specifically directed at ameliorating the disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the disease, condition, or disorder. In addition, this term encompasses palliative treatment, i.e., treatment designed to alleviate the symptoms of a disease, condition, or disorder but not cure it; preventative treatment, i.e., treatment directed at preventing a disease, condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed at ameliorating the disease, condition, or disorder. The term "treating" also encompasses symptomatic treatment, i.e., treatment directed at the systemic symptoms of a disease, condition, or disorder. The term "therapeutically effective amount" is defined as an amount that, when administered to an animal in need thereof, relieves at least some of the symptoms of a bacterial infection.

[0032] With respect to prevention, a "therapeutically effective amount" is an amount that, when administered to an animal susceptible to bacterial infection, helps to inhibit or otherwise reduce the likelihood of such infection.

[0033] The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

[0034] In one aspect, the present invention provides a compound of formula I [ka] The present invention relates to a compound according to the present invention. In some embodiments, R1 can be H, methyl, other substituted or unsubstituted alkyl, cyclic and heterocyclyl groups, as illustrated below. [ka]

[0035] In some embodiments, R1 can also be substituted or unsubstituted aryl and heteroaryl groups, as illustrated below. [ka] In some embodiments, R2 is H, halo, CN, substituted or unsubstituted C 1~6 Alkyl, CO2R 21 ,CONR 23 R 24 , substituted or unsubstituted C 2~8 Heterocyclyl or substituted or unsubstituted C 1~10 It is heteroaryl.

[0036] In some embodiments, R3 can be substituted or unsubstituted aryl and heteroaryl groups, as illustrated below. [ka]

[0037] antibacterial compounds Preferred compounds are: 5-((3,4-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 1-allyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butyl acetate, 1-(cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-[(3,4-cyclophenyl)methylamino]-1-phenyl-6H-pyrazolo[4,3-d]pyrimidin-7-one, 1-cyclopropyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, cyclopentyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, cetyl 5-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate, 5-[(3,4-cyclophenyl)methylamino]-1-(4-pyridyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one, 3-chloro-5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-cyclolobenzyl)amino)-3-fluoro-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 3-chloro-5-((3,4-dichlorobenzyl)amino)-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 1-(2-(4-acetylpiperazin-1-yl)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((4-methylmorpholin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(3-methylpicolinoyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-difluorobenzyl)amino)-3-fluoro-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((4-chloro-3-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1,3-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholinoethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(4-hydroxybutyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one), 5-((3,4-dichlorobenzyl)amino)-1-(thiazol-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((tetrahydrofuran-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(3-hydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyrazin-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(5-methoxypentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((2-methoxyethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-3-ylsulfonyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, ethyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate, isopropyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate, ethyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butanoate, methyl 3-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)propanoate, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-4-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride, 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(oxazole-4-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(thiazole-2-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methoxypicolinoyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methylpicolinoyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 3-chloro-5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 5-((3,4-difluorobenzyl)amino)-3-fluoro-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 3-chloro-5-((3,4-dichlorobenzyl)amino)-1-(1-nicotinoylpiperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, Ammonium (2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethoxy)methyl phosphate (5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-7-oxo-1H-pyrazolo[4,3-d]pyrimidin-6(7H)-yl)methyl dihydrogen phosphate, or an optical isomer thereof, an isotopic isomer thereof, a prodrug or a pharmaceutically acceptable salt thereof.

[0038] Synthesis method The following examples are given for the purpose of illustrating the present invention, but are not intended to limit the scope or spirit of the invention. Compounds of the present invention include those specifically disclosed herein above and below and can be prepared as described in the following schemes: For example, compounds of Formula I can be prepared as described in the following schemes, and it is known to those skilled in the art to prepare fragments and combinations thereof. In some schemes provided herein, compounds may be depicted in brackets. Those skilled in the art will recognize that bracketed compounds represent a mixture of isomers used or produced in the reaction.

[0039] Synthesis method Scheme A [ka]

[0040] General Procedure for Preparing Compounds in Scheme A Preparation of 1-methyl-4-nitro-1H-pyrazole-5-carboxylic acid (Step 1 in Scheme A) [ka] To a solution of fuming HNO3 (24.73 g, 392.50 mmol, 16.38 mL, 1.5 equiv.) in H2SO4 (119.84 g, 1.22 mol, 65.13 mL, 4.67 equiv.) was added 2-methylpyrazole-3-carboxylic acid (33 g, 261.67 mmol, 1 equiv.) portionwise at 20-25°C. The mixture was stirred at 30-40°C for 1 h and then at 75-80°C for 5 h. TLC showed that the starting material was consumed and one major new spot with greater polarity was detected. The reaction mixture was slowly poured into ice water (150 mL). Some solid precipitated. The solid was collected after filtration and washed with water (50 mL) and petroleum ether (50 mL). The solid was concentrated under reduced pressure and then used in the next step without further purification. 2-Methyl-4-nitro-pyrazole-3-carboxylic acid (37 g, 216.23 mmol, 82.64% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.30 (s, 1H), 3.96 (s, 3H).

[0041] Preparation of 1-methyl-4-nitro-1H-pyrazole-5-carbonyl chloride (Step 2 in Scheme A) [ka] A solution of 2-methyl-4-nitro-pyrazole-3-carboxylic acid (35 g, 204.55 mmol, 1 equiv.) and DMF (149.51 mg, 2.05 mmol, 157.38 μL, 0.01 equiv.) in SOCl (150 mL) was stirred at 85 °C for 1 h. TLC showed the reaction was complete. The reaction mixture was cooled and the solvent was removed under reduced pressure. The crude product was used in the next step without further purification. 2-Methyl-4-nitro-pyrazole-3-carbonyl chloride (38 g, 200.47 mmol, 98.01% yield) was obtained as a colorless oil.

[0042] Preparation of 1-methyl-4-nitro-1H-pyrazole-5-carboxamide (Step 3 in Scheme A) [ka] To NH3·H2O (150 mL) was added dropwise 2-methyl-4-nitro-pyrazole-3-carbonyl chloride (38 g, 200.47 mmol, 1 equiv.) at 0 °C. The mixture was stirred at 25 °C for 1 h. TLC and LC-MS showed the reaction was complete. Some solid was formed. After filtration, the solid was collected. The aqueous solution was extracted with EtOAc (80 μL × 5). The combined organic layers were washed with brine (50 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The combined crude was used in the next step without further purification. 2-Methyl-4-nitro-pyrazole-3-carboxamide (32 g, 188.10 mmol, 93.83% yield) was obtained as a pale yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.48 (s, 1H), 8.32 (s, 1H), 8.27 (s, 1H), 3.86 (s, 3H).

[0043] Preparation of 4-amino-1-methyl-1H-pyrazole-5-carboxamide (Step 4 in Scheme A) [ka] A mixture of 2-methyl-4-nitro-pyrazole-3-carboxamide (32 g, 188.10 mmol, 1 equiv.) and 10% Pd / C (3 g) in EtOH (600 mL) was stirred under H (45 psi) at 25 °C for 5 h. TLC showed that no starting material remained and one major new spot with greater polarity was detected. After filtration, the filtrate was concentrated under reduced pressure. The crude product was used in the next step without further purification. 4-amino-2-methyl-pyrazole-3-carboxamide (23 g, 164.12 mmol, 87.25% yield) was obtained as a purple solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.37 (s, 2H), 7.01 (s, 1H), 4.39 (s, 2H), 3.89 (s, 3H).

[0044] Preparation of 1-methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (Step 5 in Scheme A) [ka] To a mixture of 4-amino-2-methyl-pyrazole-3-carboxamide (23 g, 164.12 mmol, 1 equiv.) in CHCN (500 mL) was added CDI (34.60 g, 213.35 mmol, 1.3 equiv.) in portions over 1 h at 100 °C. The mixture was then heated at 100 °C for 12 h under N. A gray solid was formed. LC-MS showed no remaining starting material. Several new peaks were observed on LC-MS, and approximately 80% of the desired compound was detected. After filtration at 90 °C, the solid was collected. The crude product was used in the next step without further purification. 1-Methyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione (25 g, 150.48 mmol, 91.69% yield) was obtained as a gray solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.10 (s, 1H), 10.95 (s, 1H), 7.35 (s, 1H), 4.05 (s, 3H).

[0045] Preparation of 5,7-dichloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidine (Step 6 in Scheme A) [ka] To a solution of 1-methyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione (26 g, 156.50 mmol, 1 equiv.) in POCl3 (239.96 g, 1.56 mol, 145.43 mL, 10 equiv.) under N2 at 50 °C, DBU (142.95 g, 938.98 mmol, 141.53 mL, 6 equiv.) was added dropwise. The mixture was stirred at 85 °C for 12 h. LC-MS showed no starting material remained. The mixture was poured into ice water (1 L) and then extracted with EtOAc (200 mL × 6). The combined organic layers were washed with saturated NaHCO3 to pH = 7 and brine (100 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 80 g, eluting with a 0-20% ethyl acetate / petroleum ether gradient at 150 mL / min). The eluent was removed under reduced pressure to give 5,7-dichloro-1-methyl-pyrazolo[4,3-d]pyrimidine (14 g, 68.96 mmol, 44.06% yield) as a pale yellow oil. 1 H NMR (CDCl 3, 400 MHz) δ 8.17 (s, 1H), 4.40 (s, 3H).

[0046] Preparation of 5-chloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 7 in Scheme A) [ka] To a solution of 5,7-dichloro-1-methyl-pyrazolo[4,3-d]pyrimidine (14 g, 68.96 mmol, 1 equiv.) in dioxane (140 mL) and HO (100 mL) was added dropwise a solution of NaOH (2.76 g, 68.96 mmol, 1 equiv.) in HO (20 mL) at 0 °C. The mixture was then stirred at 100 °C for 6 h. TLC and LC-MS indicated that approximately 10% of 5,7-dichloro-1-methyl-pyrazolo[4,3-d]pyrimidine remained. The organic solvent was removed under reduced pressure. The aqueous solution was extracted with MTBE (120 mL × 2) to recover the starting material. The aqueous solution was then adjusted to pH = 5 with 2 N HCl. A white solid was formed. The solid was collected after filtration and concentrated under reduced pressure. The residue was used in the next step without further purification. 5-Chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (12.7 g, 68.80 mmol, 99.78% yield) was obtained as a white solid. 1 H NMR (CDCl 3, 400 MHz) δ 7.85 (s, 1H), 4.30 (s, 3H).

[0047] Preparation of Compounds in Scheme A (Step 8 in Scheme A) [ka] 5-Chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (541.76 μmol, 1 equiv.), R3(CH2) nA solution of NH (1.63 mmol, 3 equiv.) and base (no base or TEA or DIEA) or TFA in a solvent (t-BuOH or i-PrOH or NMP) (6 mL / mmol) was heated (100-160 °C) for a period of time (4-20 h). LC-MS and HPLC indicated the reaction was complete. The reaction mixture was quenched with HO and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC. Column: a) Luna C18 100mm x 30mm 5μm; b) Phenomenex Luna C18 150mm x 30mm 5μm; c) Waters Xbridge 150mm x 25mm 5μm; d) Nano-micro Kromasil C18 100mm x 30mm 5μm; e) Boston Prime C18 150mm x 30mm 5μm;f)Phenomenex Luna C18 150mm×30mm 5μm;g)Waters Xbridge 150mm×25mm 5μm;h)Xtimate C18 150mm×25mm 5μm;i)Xbridge 150mm×30mm 10μm. Mobile phase: a) [Water (0.1% TFA)-MeCN], B%: 1%–55%, 10 min; b) [Water (0.05% HCl)-MeCN], B%: 5%–35%, 8 min; c) [Water (10 mM NHHCO)-MeCN], B%: 5%–50%, 20 min; d) [Water (0.04% NH H0 + 10 mM NHHCO)-MeCN], B%: 15%–60%, 10.5 min; e) [Water (10 mM NHHCO)-MeCN], B%: 1%–25%, 10 min. The aqueous solution was lyophilized to give the desired product.

[0048] compound 1 Preparation of 5-((4-chloro-3-fluorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 8 in Scheme A) [ka] A solution of 5-chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 541.76 μmol, 1 equiv.) and (4-chloro-3-fluorophenyl)methanamine (259.38 mg, 1.63 mmol, 198.39 μL, 3 equiv.) in t-BuOH (3 mL) was heated at 100 °C for 16 h. LC-MS and HPLC showed that 5-chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was quenched with HO (5 mL) at 25 °C and then extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (Waters Xbridge 150 mm × 25 mm 5 μm column; mobile phase: [water (10 mM NH₄HCO₃)-MeCN]; B%: 25%–45%, 20 min). The aqueous solution was lyophilized to give 5-[(4-chloro-3-fluoro-phenyl)methylamino]-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (71.8 mg, 233.34 μmol, 43.07% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.60~7.50 (m, 2H), 7.38 (d, J = 10.4 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 4.53 (d, J = 4.4 Hz, 2H), 4.07 (s, 3H). HPLC: 99.03% (220 nm), 99.12% (215 nm), 96.82% (254 nm). MS (ESI): C 13 H 11 Calculated mass of ClFNO: 307.06, measured m / z: 308.0 [M+H] + .

[0049] compound 2 5-((3,4-Dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (0.8 g, 2.45 mmol, 64.54% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.09 (s, 1H), 7.57~7.55 (m, 2H), 7.50 (s, 1H), 7.31 (t, J = 2.0 Hz, 1H), 6.67 (s, 1H), 4.44 (d, J = 6.0 Hz, 2H), 4.04 (s, 3H). HPLC: 99.17% (220 nm), 98.91% (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 11 Calculated mass of Cl2N5O: 323.03, measured m / z: 324.0 [M+H] + .

[0050] compound 3 5-(Benzylamino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (70.8 mg, 271.41 μmol, 41.75% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.57 (s, 1H), 7.35~7.25 (m, 5H), 6.93 (s, 1H), 4.50 (d, J = 5.2 Hz, 2H), 4.07 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 13 Calculated mass of NO: 255.11, measured m / z: 256.1 [M+H] + .

[0051] compound 4 1-Methyl-5-((pyridin-2-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (90.4 mg, 352.76 μmol, 54.26%) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.67 (d, J = 5.2 Hz, 1H), 8.12 (t, J = 7.6 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.58 (t, J = 6.4 Hz, 1H), 7.52 (s, 1H), 7.07 (s, 1H), 4.71 (d, J = 3.6 Hz, 2H), 4.07 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 12 H 12 Calculated mass of NO: 256.11, measured m / z: 257.2 [M+H] + .

[0052] compound 5 1-Methyl-5-(3-pyridylmethylamino)-6H-pyrazolo[4,3-d]pyrimidin-7-one) was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (149.1 mg, 581.82 μmol, 89.50% yield) as a yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.81 (s, 1H), 8.72 (d, J = 4.4 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 7.87 (dd, J = 7.6 Hz, 5.2 Hz, 1H), 7.52 (s, 1 HPLC: 97.28% (220 nm), 96.72% (215 nm), 100.00% (254 nm). MS(ESI): C 12 H 12 Calculated mass of NO: 256.11, measured m / z: 257.1 [M+H] + .

[0053] compound 6 1-Methyl-5-((pyridin-4-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (89.9 mg, 350.81 μmol, 53.96%) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.80 (d, J = 4.8 Hz, 2H), 7.91 (d, J = 5.6 Hz, 2H), 7.46 (s, 1H), 7.11 (s, 1H), 4.75 (d, J = 3.6 Hz, 2H), 4.06 (s, 3H). HPLC: 96.79% (220 nm), 96.31% (215 nm), 98.37% (254 nm). MS (ESI): C 12 H 12 Calculated mass of NO: 256.11, measured m / z: 257.1 [M+H] + .

[0054] compound 7 5-((3,4-Difluorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (0.1288 g, 442.22 μmol, 81.63%) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.54 (s, 1H), 7.42~7.35 (m, 2H), 7.18 (d, J = 3.6 Hz, 1H), 6.67 (s, 1H), 4.60 (d, J = 5.6 Hz, 2H), 4.06 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 11 Calculated mass of F2N5O: 291.09, measured m / z: 292.1 [M+H] + .

[0055] compound 8 5-((3,4-Dichlorophenethyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (0.119 g, 351.87 μmol, 98.20%) as a yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.58~7.53 (m, 3H), 7.26 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.40 (s, 1H), 4.06 (s, 3H), 3.50 (s, 2H), 2.85 (t, J = 6.8 Hz, 2H). HPLC: 98.20% (220 nm), 97.79% (215 nm), 98.10% (254 nm). MS (ESI): C 14 H 13 Calculated mass of Cl2N5O: 337.05, measured m / z: 338.1 [M+H] + .

[0056] compound 9 5-((3,4-Dichlorophenyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (46.3 mg, 149.29 μmol, 27.56% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.06 (s, 1H), 8.86 (s, 1H), 8.10 (d, J = 2.4 Hz, 1H), 7.76 (s, 1H), 7.58~7.53 (m, 1H), 7.51~7.47 (m, 1H), 4.12 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 12 Calculated mass of H9Cl2N5O: 309.02, measured m / z: 310.0 [M+H] + .

[0057] compound 10 1-Methyl-5-(phenylamino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (100 mg, 391.73 μmol, 60.26% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.52 (s, 1H), 7.70 (s, 1H), 7.62 (d, J = 7.6 Hz, 2H), 7.33 (t, J = 8.0 Hz, 2H), 7.01 (t, J = 7.6 Hz, 1H), 4.11 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 99.20 % (254 nm). MS (ESI): C 12 H 11 Calculated mass of NO: 241.10, measured m / z: 242.1 [M+H] + .

[0058] compound 11 1-Methyl-5-((pyrimidin-2-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (31 mg, 120.50 μmol, 22.24% yield) as a light brown solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.81 (d, J = 4.8 Hz, 2H), 7.55 (s, 1H), 7.43 (t, J = 4.8 Hz, 1H), 6.88 (s, 1H), 4.69 (d, J= 4.4 Hz, 2H), 4.07 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 11 H 11 Calculated mass of NO: 257.10, measured m / z: 258.1 [M+H] + .

[0059] compound 12 1-Methyl-5-((pyrazin-2-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (30.6 mg, 118.95 μmol, 21.96% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.66 (s, 1H), 8.60~8.59 (m, 1H), 8.53 (s, 1H), 7.52 (d, J = 1.2 Hz, 1H), 6.81 (t, J = 5.2 Hz, 1H), 4.64 (d, J = 5.2 Hz, 1H), 4.06 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 11 H 11 Calculated mass of NO: 257.10, measured m / z: 258.1 [M+H] + .

[0060] compound 13 5-(((1H-indazol-5-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (56.6 mg, 191.13 μmol, 35.28% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 13.01 (s, 1H), 10.78 (s, 1H), 8.03 (s, 1H), 7.69 (s, 1H), 7.55 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 1.2 Hz, HPLC: 99.72% (220 nm), 99.71% (215 nm), 100.00% (254 nm). MS (ESI): C 14 H 13 Calculated mass of NO: 295.12, measured m / z: 296.1 [M+H] + .

[0061] compound 14 5-(((1H-indol-5-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (38.4 mg, 130.47 μmol, 24.08% yield) as an orange solid. 1H NMR (DMSO-d6, 400 MHz) δ 11.08 (s, 1H) 7.61 (s, 1H), 7.52 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 2.8 Hz, 1H), 7.09 (dd, J = 1.6 HPLC: 96.92% (220 nm), 96.82% (215 nm), 99.08% (254 nm). MS (ESI): C 15 H 14 Calculated mass of NO: 294.12, measured m / z: 295.1 [M+H] + .

[0062] compound 15 1-Methyl-5-((thiazol-4-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (66.9 mg, 255.06 μmol, 58.85% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 9.09 (d, J = 2.0 Hz, 1H), 7.55 (s, 1H), 7.51 (d, J = 0.8 Hz, 1H), 6.49 (s, 1H), 4.59 (d, J = 5.2 Hz, 1H), 4.06 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 10 H 10 Calculated mass of N6OS: 262.06, measured m / z: 263.0 [M+H] + .

[0063] compound 16 5-(((1H-pyrazol-3-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (118.8 mg, 484.42 μmol, 89.42% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.67 (d, J = 1.6 Hz, 1H), 7.62 (s, 1H), 7.13 (s, 1H), 6.24 (d, J = 1.6 Hz, 1H), 4.49 (s, 2H), 4.08 (s, 3H). HPLC: 98.38% (220 nm), 97.78% (215 nm), 100.00% (254 nm). MS (ESI): C 14 H 18 N 10 Calculated mass of O: 245.10, measured m / z: 246.1 [M+H] + .

[0064] compound 17 5-(((2H-1,2,3-triazol-4-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (55.9 mg, 227.03 μmol, 41.91% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.78 (s, 1H), 7.60 (s, 1H), 6.75 (s, 1H), 4.55 (d, J = 4.4 Hz, 2H), 4.07 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C9H 10 Calculated mass of NO: 246.10, measured m / z: 247.1 [M+H] + .

[0065] compound 18 5-((Benzo[d]thiazol-2-ylmethyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (73.3 mg, 234.67 μmol, 43.32% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.02 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.49 (t, J = 8.4 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.03 (s, 1H), 4.90 (d, J = 5.6 Hz, 2H), 4.08 (s, 3H). HPLC: 96.43% (220 nm), 96.13% (215 nm), 97.34 % (254 nm). MS (ESI): C 14 H 12 Calculated mass of N6OS: 312.08, measured m / z: 313.1 [M+H] + .

[0066] compound 19 5-(((1H-Benzo[d]imidazol-5-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (44.5 mg, 149.58 μmol, 22.01% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 9.41 (s, 1H), 7.80 (d, J = 8.8 Hz,1H), 7.75 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 3.2 Hz, 1H), 6.70 (m, 1H), 4.65 (t, J = 6.0 Hz, 2H), 4.06 (s, 3H). HPLC: 99.26% (220 nm), 98.34% (215 nm), 98.34% (254 nm). MS (ESI): C 14 H 13 Calculated mass of NO: 295.12, measured m / z: 296.2 [M+H] + .

[0067] compound 20 1-Methyl-5-((thiazol-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (19.7 mg, 75.11 μmol, 13.86% yield) as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 10.17 (s, 1H), 8.93 (s, 1H), 7.82 (s, 1H), 7.59 (s, 1H), 6.65 (t, J = 4.8 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 4.07 (s, 3H). HPLC: 96.83% (220 nm), 96.38% (215 nm), 96.83% (254 nm). MS (ESI): C 10 H 10 Calculated mass of N6OS: 262.06, measured m / z: 263.0 [M+H] + .

[0068] compound 21 1-Methyl-5-((oxazol-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (39.5 mg, 160.42 μmol, 29.61% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.30 (s, 1H), 7.59 (s, 1H), 7.06 (s, 1H), 6.70 (s, 1H),4.55 (d, J = 5.2 Hz, 2H), 4.07 (s, 3H). HPLC: 96.01% (220 nm), 95.85% (215 nm), 100.00% (254 nm). MS (ESI): C 10 H 10 Calculated mass of N6O2: 246.09, measured m / z: 247.1 [M+H] + .

[0069] compound 22 5-(((1H-imidazol-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (26.4 mg, 107.27 μmol, 19.80% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.57 (s, 1H), 6.94 (s, 2H), 6.51 (s, 1H), 4.44 (d, J = 5.2 Hz, 2H), 4.06 (s, 3H). HPLC: 99.65% (220 nm), 98.40% (215 nm), 100.00% (254 nm). MS (ESI): C 10 H 11 Calculated mass of NO: 245.10, measured m / z: 246.1 [M+H] + .

[0070] compound 23 1-Methyl-5-((pyridazin-3-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (4.9 mg, 19.05 μmol, 3.52% yield) as a light brown solid. 1 H NMR (DMSO-d 6,400 MHz) δ 9.15 (dd, J = 2.8 Hz, 4.0 Hz, 1H), 7.69~7.64 (m, 2H), 7.54 (s, 1H), 7.05~6.96 (m, 1H), 4.78 (d, J = 5.2 Hz, 2H), 4.07 (s, 3H). HPLC: 99.18% (220 nm), 98.00% (215 nm), 98.57% (254 nm). MS (ESI): C 11 H 11 Calculated mass of NO: 257.10, measured m / z: 258.1 [M+H] + .

[0071] compound 24 5-((3-chloro-4-fluorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (139 mg, 451.72 μmol, 83.38% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.55~7.54 (m, 2H), 7.37~7.35 (m, 2H), 6.87 (s, 1H), 4.47 (d, J = 5.2 Hz, 2H), 4.07 (s, 3H). HPLC: 98.59 % (220 nm), 97.69 % (215 nm), 99.20 % (254 nm). MS (ESI): C 13 H 11 Calculated mass of ClFNO: 307.06, measured m / z: 308.0 [M+H] + .

[0072] compound 25 5-((4-chlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (23.1 mg, 79.73 μmol, 14.72% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.84 (s, 1H), 7.62 (s, 1H), 7.46~7.35 (m, 4H), 4.57 (d, J = 3.6 Hz, 2H), 4.09 (s, 3H). HPLC: 99.48% (220 nm), 99.72% (215 nm), 98.92% (254 nm). MS (ESI): C 13 H 12 Calculated mass of ClNO: 289.07, measured m / z: 290.1 ​​[M+H] + .

[0073] compound 26 5-((3-chlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (157.9 mg, 391.09 μmol, 72.19% yield, TFA) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.57 (s, 1H), 7.40 (s, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.33~7.28 (m, 2H), 7.04 (s, 1H), 4.51 (d, J = 5.4 Hz, 2H), 4.07 (s, 3H). HPLC: 97.49% (220 nm), 97.20% (215 nm), 98.84% (254 nm). MS (ESI): C 13 H 12 Calculated mass of ClNO: 289.07, measured m / z: 290.1 ​​[M+H] + .

[0074] compound 27 5-((2-chlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (93.2 mg, 313.84 μmol, 57.93% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.55 (s, 1H), 7.47~7.45 (m, 1H), 7.41~7.38 (m, 1H), 7.34~7.29 (m, 2H), 6.68~6.67 (m, 1H), 5.55 (d, J = 5.6 Hz,2H), 4.07 (s, 3H). HPLC: 97.56% (220 nm), 97.45% (215 nm), 98.61% (254 nm). MS (ESI): C 13 H 12 Calculated mass of ClNO: 289.07, measured m / z: 290.1 ​​[M+H] + .

[0075] compound 28 5-((2,4-Dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (92.6 mg, 276.14 μmol, 50.97% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.62 (d, J = 1.8 Hz, 1H), 7.53 (s, 1H), 7.42~7.35 (m, 2H), 6.63 (s, 1H), 4.51 (d, J = 6.0 Hz, 2H), 4.06 (s, 3H). HPLC: 96.67% (220 nm), 96.29% (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 11 Calculated mass of Cl2N5O: 323.03, measured m / z: 324.0 [M+H] + .

[0076] compound 29 5-((2,3-Dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (55 mg, 169.67 μmol, 31.32% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 11.14 (s, 1H), 7.56~7.53 (m, 2H), 7.33 (d, J = 5.2 Hz, 2H), 6.64 (s, 1H), 5.56 (d, J = 6.0 Hz, 2H), 4.06 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 11 Calculated mass of Cl2N5O: 323.03, measured m / z: 324.0 [M+H] + .

[0077] compound 30 5-((2,6-Dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (26.5 mg, 81.75 μmol, 15.09% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.55 (s, 1H), 7.61 (s, 1H), 7.54~7.52 (m, 2H), 7.40~7.38 (m, 1H), 6.24 (s, 1H), 4.68 (d, J = 4.0 Hz, 2H), 4.06 (s, 3H). HPLC: 100.00% (220 nm), 100.00 % (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 11 Calculated mass of Cl2N5O: 323.03, measured m / z: 324.0 [M+H] + .

[0078] compound 31 5-((3-chloro-4-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (106.2 mg, 349.63 μmol, 64.54% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.56 (s, 1H), 7.38 (s, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 6.82 (s, 1H), 4.46 (d, J = 5.2 Hz, 2H), 4.08 (s, 3H), 2.30 (s, 3H). HPLC: 97.09% (220 nm), 96.49% (215 nm), 98.94% (254 nm). MS (ESI): C 14 H 14 Calculated mass of ClNO: 303.09, measured m / z: 304.0 [M+H] + .

[0079] compound 32 5-((3,4-Dimethoxybenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (91.3 mg, 289.54 μmol, 59.38% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.57 (s, 1H), 6.98 (s, 1H), 6.93~6.83 (m, 2H), 6.74 (s, 1H), 4.39 (d, J = 5.2 Hz, 2H), 4.07 (s, 3H), 3.73 (d, J = 5.2 Hz, 6H). HPLC: 99.37% (220 nm), 99.36% (215 nm), 100.00% (254 nm). MS (ESI): C 15 H 17 Calculated mass of N5O3: 315.13, measured m / z: 316.1 [M+H] + .

[0080] compound 33 5-((3,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (117 mg, 412.95 μmol, 84.69% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.56 (s, 1H), 7.09 (d, J = 5.6 Hz, 2H), 7.05 (d, J = 5.6 Hz, 1H), 6.59 (s, 1H), 4.39 (d, J = 4.8 Hz, 2H), 4.07 (s, 3H), 2.19 (d, J = 4.8 Hz, 6H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 15 H 17 Calculated mass of NO: 283.14, measured m / z: 284.1 [M+H] + .

[0081] compound 34 5-((4-chloro-3-methoxybenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (92.1 mg, 288.04 μmol, 59.08% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.55 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 1.6 Hz, 1H), 6.91 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 6.56 (s, HPLC: 100.00% (220 nm), 98.52% (215 nm), 100.00% (254 nm). MS (ESI): C 14 H 14 Calculated mass of ClN5O2: 319.08, measured m / z: 320.1 [M+H] + .

[0082] compound 35 5-((4-chloro-3-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (102.3 mg, 336.79 μmol, 69.07% yield) as an off-white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.56 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 6.97 (s, 1H), 4.46 (d, J = 4.0 Hz, 2H), 4.07 (s, 3H), 2.31 (s, 3H). HPLC: 99.90% (220 nm), 99.86% (215 nm), 98.42% (254 nm). MS (ESI): C 14 H 14 Calculated mass of ClNO: 303.09, measured m / z: 304.1 [M+H] + .

[0083] compound 36 5-(((4,5-Dichloropyridin-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one-2,2,2-trifluoroacetate was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (35.8 mg, 110.10 μmol, 33.87% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.20 (s, 1H), 8.73 (s, 1H), 7.69 (s, 1H), 7.53 (s, 1H), 6.73 (s, 1H), 4.58 (d, J = 5.6 Hz, 2H), 4.07 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 96.16% (254 nm). MS (ESI): C 12 H 10 Calculated mass of Cl2N6O: 324.03, measured m / z: 325.0 [M+H] + .

[0084] compound 37 5-(((4,5-Dichloro-1H-imidazol-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (23.2 mg, 73.85 μmol, 16.41% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.57 (s, 1H), 6.55 (s, 1H), 4.42 (d, J = 5.6 Hz, 2H), 4.07 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 10 Calculated mass of H9Cl2N7O: 313.02, measured m / z: 314.0 [M+H] + .

[0085] compound 38 5-(((4-chloro-5-ethyl-1H-imidazol-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 8 in Scheme A. [ka] The procedure afforded the desired compound (8.4 mg, 27.01 μmol, 9.97% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.59 (s, 1H), 7.19 (s, 1H), 4.61 (d,J = 2.4 Hz, 2H), 4.09 (s, 3H), 2.56 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). HPLC:98.96% (220 nm), 98.49% (215 nm), 99.11% (254 nm).MS (ESI): C 12 H 15 Calculated mass of Cl2N7O: 307.09, measured m / z: 308.1 [M+H] + .

[0086] compound 39 5-((1,3-Dihydroisobenzofuran-5-yl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as step 8 in Scheme A. [ka] The procedure afforded the desired compound (26.8 mg, 92.37 μmol, 42.63% yield) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.54 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 5.00 (s, 2H), 4.96 (s, 2H), 4.11 (s, 3H). HPLC: 97.64 % (220 nm), 96.24 % (215 nm), 98.52% (254 nm). MS (ESI): C 14 H 13 Calculated mass of N5O2: 283.11, measured m / z: 284.1 [M+H] + .

[0087] Scheme B-1 [ka]

[0088] compound 40 Methyl 5-((3,4-dichlorobenzyl)amino)-1-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate was prepared according to the procedure described herein as steps 1-2 in Scheme B-1.

[0089] Preparation of 5-((3,4-dichlorobenzyl)amino)-3-iodo-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-1) [ka] HBF (677.22 mg, 7.71 mmol, 480.30 μL, 2.5 equiv) was added to a mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (1 g, 3.08 mmol, 1 equiv) and NIS (694.03 mg, 3.08 mmol, 1 equiv) in MeCN (8 mL). The mixture was stirred at 100 °C for 4 h. LC-MS showed the reaction was complete. Some yellow solid was formed. After filtration and washing with HO (8 mL) and EtOAc (8 mL), the solid was collected. The compound 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (730 mg, 1.62 mmol, 52.58% yield) was obtained as a yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.21 (s, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.39 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.70 (t, J = 6.0 Hz, 1H), 4.46 (d, J = 9.6 Hz, 2H), 4.07 (s, 3H).

[0090] Preparation of methyl 5-((3,4-dichlorobenzyl)amino)-1-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylate (Step 2 in Scheme B-1) [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (300 mg, 666.58 μmol, 1 equiv.) and TEA (269.80 mg, 2.67 mmol, 371.12 μL, 4 equiv.) in MeOH (10 mL) and DMF (3 mL) was added Pd(dppf)Cl (97.55 mg, 133.32 μmol, 0.2 equiv.) under a N atmosphere. The suspension was degassed and purged with CO three times. The mixture was stirred at 80 °C under CO (50 psi) for 5 h. LC-MS showed the reaction was complete. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 4 g, elution with a 0-100% ethyl acetate / petroleum ether gradient at 65 mL / min). The eluent was removed under reduced pressure. 50 mg of the resulting crude product was further purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 45%-70%, 10 min). The solvent was removed by lyophilization. The compound methyl 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carboxylate (9.8 mg, 25.54 μmol, yield 3.83%, purity 99.62%) was obtained as a white solid and used for further analysis. 1 H NMR (DMSO-d 6,400 MHz) δ 11.32 (s, 1H), 7.72 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 6.88 (s, 1H), 4.47 (d, J = 5.6 Hz, 2H), 4.13 (s, 3H), 3.83 (s, 3H). HPLC: 99.62% (220 nm), 99.52 % (215 nm), 99.35 % (254 nm). MS (ESI): C 15 H 13 Calculated mass of Cl2N5O3: 381.04, measured m / z: 382.1 [M+H] + .

[0091] Preparation of Compounds in Scheme B-1 (Step 3 in Scheme B-1) [ka] A mixture of methyl 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carboxylate (784.93 μmol, 1 equiv.) and LiOH·HO (2.35 mmol, 3 equiv.) in MeOH (2 mL) and HO (2 mL) (or MeNH (2 M in EtOH, 15.7 mmol, 7.85 mL, 20 equiv.)) was stirred at 25 °C for a period of time (1 to 12 h). LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC. Column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm. Mobile phase: [water (0.1% TFA)-MeCN]; B%: 35% to 65%, 10 min). The solvent was removed under lyophilization to give the desired product.

[0092] compound 41 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxylic acid (Step 3 in Scheme B-1) [ka] The procedure afforded the desired compound (14.1 mg, 37.32 μmol, 4.75% yield, 97.44% purity) as a light brown solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.25 (s, 1H), 10.64 (s, 1H), 7.73 (s, 1H), 7.59~7.55 (m, 1H), 7.47~7.32 (m, 1H), 6.77 (s, 1H), 4.55~4.47 (m, 2H), 4.12 (s, 3H). HPLC: 97.44% (220 nm), 97.47 % (215 nm), 98.21 % (254 nm). MS (ESI): C 14 H 11 Calculated mass of Cl2N5O3: 367.02, measured m / z: 368.0 [M+H] + .

[0093] compound 42 Preparation of 5-((3,4-dichlorobenzyl)amino)-N,1-dimethyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxamide (Step 3 in Scheme B-1) [ka] A mixture of methyl 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carboxylate (50 mg, 130.82 μmol, 1 equiv.) in MeNH2 (2 M in EtOH, 1.31 mL, 20 equiv.) was stirred at 25 °C for 12 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 35% to 55%, 10 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-N,1-dimethyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carboxamide (5.8 mg, 15.02 μmol, yield 11.48%, purity 98.71%) was obtained as a brown solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.42 (s, 1H), 7.94 (d, J = 4.8 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.03 (t, J = 5.6 HPLC: 98.71% (220 nm), 98.32 % (215 nm), 96.98 % (254 nm). MS (ESI): C 15 H 14 Calculated mass of Cl2N6O2: 380.06, measured m / z: 381.1 [M+H] + .

[0094] compound 43 5-((3,4-Dichlorobenzyl)amino)-N,N,1-trimethyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxamide was prepared according to the procedure described herein as Step 3 in Scheme B-1. [ka] The procedure afforded the desired compound (3.3 mg, 7.80 μmol, 5.96% yield) as a brown solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.24 (s, 1H), 7.59~7.57 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 6.73 (s, 1H), 4.44~4.45 (m, 2H), 4.09 (s, 3H), 2.96 (s, 3H), 2.82 (s, 3H). HPLC: 93.37% (220 nm), 90.08 % (215 nm), 92.84 % (254 nm). MS (ESI): C 16 H 16 Calculated mass of Cl2N6O2: 394.07, measured m / z: 395.1 [M+H] + .

[0095] Scheme B-2 [ka]

[0096] compound 44 Preparation of 5-((3,4-dichlorobenzyl)amino)-1,3-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-2) [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.2 g, 444.38 μmol, 1 equiv.), 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (334.71 mg, 1.33 mmol, 372.73 μL, 50% in THF, 3 equiv.), Pd(dppf)Cl (48.77 mg, 66.66 μmol, 0.15 equiv.), and KCO (184.26 mg, 1.33 mmol, 3 equiv.) in dioxane (10 mL) was stirred at 120 °C under N for 10 h. LC-MS indicated the reaction was complete. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 35% to 50%, 10 min) to give 5-[(3,4-dichlorophenyl)methylamino]-1,3-dimethyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (22.4 mg, 66.23 μmol, 14.90% yield) as an off-white solid. 1 H NMR (DMSO-d 6, 6.56 (s, 1H), 4.46 (d, J = 4.8 Hz, 2H), 3.98 (s, 3H), 2.20 (s, 3H). HPLC: 97.11% (220 nm), 95.73% (215 nm), 100.00% (254 nm). MS (ESI): C 14 H 13 Calculated mass of Cl2N5O: 337.05, measured m / z: 338.2 [M+H] + . Scheme B-3 [ka]

[0097] compound 45 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile (Step 1 in Scheme B-3) [ka] A mixture of 3-bromo-5-[(3,4-dichlorophenyl)methylamino]-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (50 mg, 124.05 μmol, 1 equiv.) and CuCN (12.22 mg, 136.46 μmol, 29.81 μL, 1.1 equiv.) in DMSO (1 mL) was stirred at 160° C. for 4 h. LC-MS showed the reaction was complete. The reaction mixture was filtered to remove insoluble material. The filtrate was first purified by preparative HPLC (column: Welch Xtimate C18 100 mm × 25 mm 3 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30%–60%, 12 min), and then secondly purified by preparative HPLC (column: Welch Xtimate C18 150 mm × 25 mm 5 μm; mobile phase: [water (10 mM NH4HCO3)-MeCN]; B%: 40%–65%, 10.5 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile (5.3 mg, 14.75 μmol, 11.89% yield, 97.20% purity) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.63~7.59 (m, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.14 (s, 3H). HPLC: 97.20% (220 nm), 96.95 % (215 nm), 95.24 % (254 nm). MS (ESI): C 14 H 10 Calculated mass of Cl2N6O: 348.03, measured m / z: 349.0 [M+H] + .

[0098] Scheme B-4 [ka]

[0099] compound 46 Preparation of 5-((3,4-dichlorobenzyl)amino)-3-(2,5-dihydrofuran-2-yl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-4) [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.3 g, 666.58 μmol, 1 equiv.), 2,3-dihydrofuran (233.60 mg, 3.33 mmol, 252.00 μL, 5 equiv.), AcOK (163.54 mg, 1.67 mmol, 2.5 equiv.), Pd(OAc) (29.93 mg, 133.32 μmol, 0.2 equiv.), and PPh (17.48 mg, 66.66 μmol, 0.1 equiv.) in DMF (1 mL) was degassed and purged with N three times, and then the mixture was stirred under a N atmosphere at 100 °C for 12 h. LCMS and HPLC indicated the reaction was complete. The reaction mixture was cooled to room temperature and quenched with HO (5 mL) at 0 °C. The mixture was extracted with EtOAc (8 mL × 3). The combined organic layers were washed with brine (5 mL × 1), dried over NaSO, filtered, and concentrated under reduced pressure. 0.45 g (crude) of 5-[(3,4-dichlorophenyl)methylamino]-3-(2,5-dihydrofuran-2-yl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.6 g, crude) was obtained as a pale yellow solid. 0.15 g of the crude product was further purified by preparative HPLC (column: Xtimate C18 100 mm × 30 mm 3 μm; mobile phase: [water (0.04% HCl)-MeCN]; B%: 40% to 55%, 10 min). MeCN was removed under reduced pressure at 30 °C. The aqueous solution was dehydrated by lyophilization to give 5-[(3,4-dichlorophenyl)methylamino]-3-(2,5-dihydrofuran-2-yl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (24.9 mg, 62.01 μmol, 9.30% yield, 97.685% purity) as a pale yellow solid, which was used further. 1 H NMR (DMSO-d 6,400 MHz) δ 7.63~7.58 (m, 2H), 7.35 (d, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.08 (d, J = 4.4 Hz, 1H), 5.88 (dd, J = 18.0 Hz, 2.0 Hz, 2H), 4.70~4.67 (m, 1H), 4.57 (s, 1H), 4.46 (s, 2H), 4.02(s, 3H). HPLC: 97.69% (220 nm), 97.64% (215 nm), 98.97% (254 nm). MS (ESI): C 17 H 15 Calculated mass of Cl2N5O2: 391.06, measured m / z: 392.1 [M+H] + .

[0100] compound 47 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-methyl-3-(tetrahydrofuran-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme B-4) [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-3-(2,5-dihydrofuran-2-yl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.3 g, 229.45 μmol, 1 equiv.) in EtOH (50 mL) and THF (50 mL) was added Rh(PPh3)3Cl (21.23 mg, 22.95 μmol, 0.1 equiv.). The suspension was degassed and purged with H2 three times. The mixture was stirred under H2 (20 psi) at 30 °C for 16 h. LCMS and HPLC showed the reaction was complete. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 35% to 55%, 12 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-3-tetrahydrofuran-2-yl-6H-pyrazolo[4,3-d]pyrimidin-7-one (20.7 mg, 49.82 μmol, yield 21.71%, purity 94.90%) was obtained as a yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.61~7.57 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 6.92~6.81 (m, 1H), 4.93 (t, J = 6.8 Hz, 1H), 4.46 (s, 2H), 4.01 (s, 3H), 3.82~3.79 (m, 1H), 3.72~3.69 (m, 1H), 2.23~2.20 (m, 1H), 2.07~2.05 (m, 2H), 2.03~1.87 (m, 1H). HPLC: 94.90% (220 nm), 94.45% (215 nm), 98.07% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O2: 393.08, measured m / z: 394.0 [M+H] + . Scheme B-5 [ka]

[0101] compound 48 Preparation of 5-((3,4-dichlorobenzyl)amino)-3-(2-hydroxyethyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0102] Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-3-(2-ethoxyvinyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-5) [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.5 g, 1.11 mmol, 1 equiv.), 2-[(E)-2-ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (880.18 mg, 4.44 mmol, 4 equiv.), KCO (460.62 mg, 3.33 mmol, 3 equiv.), and Pd(dppf)Cl (121.94 mg, 166.64 μmol, 0.15 equiv.) in dioxane (12 mL) and HO (3 mL) was stirred at 100 °C under N for 10 h. LCMS and HPLC indicated the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 12 g, elution with a 0-100% ethyl acetate / petroleum ether gradient at 50 mL / min). The eluent was removed under reduced pressure. The compound 5-[(3,4-dichlorophenyl)methylamino]-3-[(E)-2-ethoxyvinyl]-1-methyl-6H-pyrazolo-[4,3-d]pyrimidin-7-one (0.34 g, 862.39 μmol, 77.63% yield) was obtained as an off-white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.09 (s, 1H), 7.62~7.56 (m, 3H), 7.34 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 6.62 (t, J = 5.6 Hz, 1H), 5.77 (d, J = 12.8 Hz, 1H), 5.25 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 3.91 (s, 3H), 3.82 (t, J = 6.8 Hz, 2H), 1.21 (t, J = 6.8 Hz, 3H).

[0103] compound 49 Preparation of 5-((3,4-dichlorobenzyl)amino)-3-(2-ethoxyethyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme B-5) [ka]

[0104] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-3-[(E)-2-ethoxyvinyl]-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.15 g, 380.46 μmol, 1 equiv.) and Rh(PPh3)3Cl (35.20 mg, 38.05 μmol, 0.1 equiv.) in EtOH (50 mL) and THF (20 mL) was stirred under H2 (20 psi) at 30 °C for 5 h. HPLC and LCMS indicated the reaction was complete. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 35%–55%, 10 min). The eluate was removed by lyophilization, and the compound 5-[(3,4-dichlorophenyl)methylamino]-3-(2-ethoxyethyl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (71.7 mg, 180.94 μmol, 47.56% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.08 (s, 1H), 7.62 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.34 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 6.62 (s, 1H), 4.44 (d, HPLC: 99.64% (220 nm), 99.62% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl2N5O2: 395.09, measured m / z: 396.1 [M+H] + .

[0105] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-1-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-3-yl)acetaldehyde (Step 3 in Scheme B-5) [ka] To a mixture of 5-[(3,4-dichlorophenyl)methylamino]-3-[(E)-2-ethoxyvinyl]-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.15 g, 380.46 μmol, 1 equiv.) in DCM (10 mL) was added TFA (1 mL) dropwise at 0° C. The mixture was then stirred at 20° C. for 5 h. LCMS showed the reaction was complete. The mixture was poured into water (10 mL), and the organic layer was separated. The aqueous solution was extracted with DCM (20 mL × 5). The combined organic layers were washed with saturated NaHCO to pH = 7, dried over NaSO, filtered, and concentrated under reduced pressure. The residue was used in the next step without further purification. The compound 2-[5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-3-yl]acetaldehyde (0.1 g, 273.07 μmol, 71.77% yield) was obtained as a yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.08 (s, 1H), 9.67 (s, 1H), 7.62~7.55 (m, 2H), 7.34 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 6.59 (s, 1H), 4.42 (d, J = 5.6 Hz, 2H), 4.05 (s, 3H), 3.75 (d, J = 2.0 Hz, 2H).

[0106] Preparation of 5-((3,4-dichlorobenzyl)amino)-3-(2-hydroxyethyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 4 in Scheme B-5) [ka]

[0107] To a solution of 2-[5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-3-yl]acetaldehyde (0.1 g, 273.07 μmol, 1 equiv.) in MeOH (5 mL) was added NaBH (30.99 mg, 819.22 μmol, 3 equiv.) portionwise at 0 °C. The mixture was then stirred at 20 °C for 1 h. LCMS showed the reaction was complete. The mixture was slowly quenched with ice-water (5 mL) and extracted with EtOAc (10 mL × 4). The combined organic layers were washed with brine (5 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 20% to 40%, 10 min). The eluate was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-3-(2-hydroxyethyl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (83.8 mg, 227.58 μmol, 83.34% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.64 (d, J = 2.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.63 (s, 1H), 4.46 (d, J = 5.6 Hz, 2H), 4.00 (s, 3H), 3.67 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H). HPLC: 100.00% (220 nm), 99.85% (215 nm), 100.00% (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O2: 367.06, measured m / z: 368.1 [M+H] + .

[0108] Scheme B-6 [ka]

[0109] compound 50 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-(oxazol-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0110] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-6) [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 251.10 μmol, 1 equiv.) and NIS (73.44 mg, 326.43 μmol, 1.3 equiv.) in MeCN (2 mL) was added HBF (55.12 mg, 627.76 μmol, 39.10 μL, 2.5 equiv.) dropwise. The mixture was stirred at 100 °C for 10 h. TLC showed the reaction was complete. HO (5 mL) was added to the reaction mixture. The reaction mixture was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (5 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (0.12 g, 228.95 μmol, 91.18% yield) was obtained as a brown solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.70 (s, 1H), 7.60~7.57 (m, 2H), 7.40 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H), 6.72 (t, J = 5.6 Hz, 1H), 4.56 (d, J = 5.2 Hz, 2H), 4.46 (d, J = 5.2 Hz, 2H), 3.78 (t, J = 6.0 Hz, 2H), 3.40~3.38 (m, 4H).

[0111] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-(oxazol-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme B-6) [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-3-iodo-6H-pyrazolo[4,3-d]pyrimidin-7-one (55 mg, 104.93 μmol, 1 equiv.), tributyl(oxazol-2-yl)stannane (375.77 mg, 1.05 mmol, 10 equiv.), and Pd(dppf)Cl (15.36 mg, 20.99 μmol, 0.2 equiv.) in dioxane (2 mL) was degassed and purged with N three times, and then the mixture was stirred at 100 °C under a N atmosphere for 12 h. LCMS and HPLC indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.04% HCl)-MeCN]; B%: 20% to 50%, 10 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-(oxazol-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (14.0 mg, 28.83 μmol, yield 27.48%, purity 95.832%) was obtained as a pale yellow solid. 1H NMR (DMSO-d 6, 400 MHz) δ 8.22 (s, 1H), 7.81 (s, 1H), 7.58~7.57 (m, 1H), 7.47~7.41 (m, 2H), 7.07 (s, 1H), 4.66 (s, 2H), 4.50 (s, 2H), 3.85 (s, 2H), 3.59 (s, 2H), 3.45 (s, 2H). HPLC: 95.83% (220 nm), 95.77% (215 nm), 98.53% (254 nm). MS (ESI): C 19 H 18 Calculated mass of Cl2N6O4: 464.08, measured m / z: 465.0 [M+H] + .

[0112] Scheme B-7 [ka]

[0113] compound 51 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile

[0114] compound 52 Preparation of 3-bromo-5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-7) [ka] To a mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (230 mg, 577.54 μmol, 1 equiv.) and NBS (113.07 mg, 635.29 μmol, 1.1 equiv.) in MeCN (3 mL) was added HBF (126.79 mg, 1.44 mmol, 89.92 μL, 2.5 equiv.). The mixture was stirred at 100 °C for 30 min. TLC showed the reaction was complete. The reaction mixture was quenched with HO (5 mL) and extracted with EtOAc (8 mL × 3). The combined organic layers were washed with saturated NaHCO (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 4 g, elution with 0 to 100% ethyl acetate / methanol at 30 mL / min). The eluent was removed under reduced pressure. The compound 3-bromo-5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (190 mg, crude) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.27 (s, 1H), 7.66 (d, J = 1.6 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.76 (t, J = 5.6 Hz, 1H), 4.57~4.52 (m, 3H), 4.47 (d, J = 5.6 Hz, 2H), 3.78 (t, J = 5.6 Hz, 2H), 3.41~3.37 (m, 4H). HPLC: 99.39 % (220 nm), 99.22 % (215 nm), 99.59% (254 nm). MS (ESI): C 16 H 16 Calculated mass of BrCl2N5O3: 474.98, measured m / z: 476.0 [M+H] + .

[0115] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile (Step 2 in Scheme B-7) [ka] A mixture of 3-bromo-5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (40 mg, 83.83 μmol, 1 equiv.), Zn(CN) (29.53 mg, 251.50 μmol, 3 equiv.), dppf (7.44 mg, 13.41 μmol, 0.16 equiv.), and Pd(dba) (6.14 mg, 6.71 μmol, 0.08 equiv.) in DMA (0.5 mL) was degassed and purged with N three times, and then the mixture was stirred at 170 °C under N for 40 min. LC-MS indicated the reaction was complete. After filtration, the filtrate was purified by preparative HPLC (column: Phenomenex Gemini-NX 150 30 mm × 5 μm; mobile phase: [water (10 mM NH4HCO3)-MeCN]; B%: 20% to 50%, 8 min). The solvent was removed by freeze-drying. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile (27.7 mg, 64.67 μmol, yield 19.28%, purity 98.81%) was obtained as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.63 (d, J = 1.6 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.05 (t, J = 5.6 Hz, 1H), 4.66 (t, J = HPLC: 98.81 % (220 nm), 98.60 % (215 nm), 98.43 % (254 nm). MS (ESI): C 17 H 16 Calculated mass of Cl2N6O3: 422.07, measured m / z: 423.0 [M+H] + .

[0116] Scheme B-8 [ka]

[0117] compound 53 Preparation of 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0118] Preparation of 5-chloro-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-8) [ka] A mixture of 5-chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.5 g, 2.71 mmol, 1 equiv.) and Select F (2.88 g, 8.13 mmol, 3 equiv.) in MeCN (15 mL) was stirred at 100° C. for 40 h. LCMS and HPLC showed approximately 20% of 5-chloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one remained, with approximately 40% being the desired peak. The solvent was removed under reduced pressure. The residue was dissolved in water (20 mL) and stirred at 20° C. for 15 min. After filtration, the solid was collected. The residue was used in the next step without further purification. The compound 5-chloro-3-fluoro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.4 g, 1.97 mmol, 72.90% yield) was obtained as a yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 4.13 (s, 3H).

[0119] Preparation of 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme B-8) [ka]

[0120] A mixture of 5-chloro-3-fluoro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 493.65 μmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (173.81 mg, 987.30 μmol, 131.67 μL, 2 equiv.) in t-BuOH (3 mL) was stirred at 100 °C under N for 10 h. LCMS and HPLC indicated the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (column: Luna C8 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 35% to 60%, 10 min). The eluate was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1-methyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (15.6 mg, 45.59 μmol, 9.24% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.28 (s, 1H), 7.60~7.58 (m, 2H), 7.32 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.68 (t, J = 5.6 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 3.95 (s, 3H). HPLC: 95.42% (220 nm), 94.90% (215 nm), 100.00% (254 nm). MS (ESI): C 13 H 10 Calculated mass of Cl2FN5O: 341.02, measured m / z: 342.0 [M+H] + . Scheme B-9 [ka]

[0121] Compound 54 and Compound 55 Preparation of 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and 3-chloro-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0122] Preparation of 5-chloro-3-fluoro-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and 3,5-dichloro-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 1 in Scheme B-9) [ka] To a mixture of 5-chloro-3-fluoro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (100 mg, 493.65 μmol, 1 equiv.) and 3,5-dichloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (493.65 μmol, 1 equiv.) in MeCN (1 mL) was added NaI (295.97 mg, 1.97 mmol, 4 equiv.) and TMSCl (214.52 mg, 1.97 mmol, 250.61 μL, 4 equiv.) at 0° C. The mixture was then stirred at 25° C. for 5 h. TLC indicated the reaction was complete. The reaction mixture was quenched with HO (1 mL) and concentrated under reduced pressure. The aqueous solution was extracted with EtOAc (1 mL × 3). The combined organic layers were washed with brine (1 mL), dried over NaSO, filtered, and concentrated under reduced pressure. A mixture of the compounds 5-chloro-3-fluoro-1,6-dihydropyrazolo[4,3-d]pyrimidin-7-one (100 mg, crude) and 3,5-dichloro-1,6-dihydropyrazolo[4,3-d]pyrimidin-7-one was obtained as a brown solid. MS (ESI): Calculated mass for C5H2ClFN4O 187.99 m / z Found 189.0 [M+H] + C5H2Cl2N4O 203.96 m / z Measured value 205.0 [M+H] + .

[0123] Preparation of 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and 3-chloro-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme B-9) [ka] To a mixture of 5-chloro-3-fluoro-1,3a,6,7a-tetrahydropyrazolo[4,3-d]pyrimidin-7-one (130 mg, 682.19 μmol, 1 equiv.) and 3,5-dichloro-1,3a,6,7a-tetrahydropyrazolo[4,3-d]pyrimidin-7-one (682.19 μmol, 1 equiv.) in t-BuOH (4 mL) was added (3,4-dichlorophenyl)methanamine (240.19 mg, 1.36 mmol, 181.96 μL, 2 equiv.). The mixture was stirred at 100° C. for 12 hours. HPLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Xtimate C18 100 mm × 25 mm 3 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30% to 50%, 12 min). The solvent was removed by lyophilization. 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1,6-dihydropyrazolo[4,3-d]pyrimidin-7-one (compound 54) (6.6 mg, 18.94 μmol, yield 2.78%, purity 94.16%) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 13.19 (s, 1H), 11.19 (s, 1H), 7.60~7.58 (m, 2H), 7.34 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.68 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 6.0 Hz, 2H). HPLC: 94.16 % (220 nm), 90.65 % (215 nm), 90.27 % (254 nm). MS (ESI): C 12 Calculated mass of H8Cl2FN5O: 327.01, measured m / z: 328.0 [M+H] + 3-Chloro-5-[(3,4-dichlorophenyl)methylamino]-1,6-dihydropyrazolo[4,3-d]pyrimidin-7-one (compound 55) (8.2 mg, 23.34 μmol, 4.45% yield, 98.1% purity) was obtained as an off-white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 13.85 (s, 1H), 11.17 (s, 1H), 7.64 (d, J = 1.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.73 (t, J = 5.6 Hz, 1H), 4.49 (d, J = 6.4 Hz, 2H). HPLC: 98.10 % (220 nm), 98.04 % (215 nm), 99.41 % (254 nm). MS (ESI): C 12 Calculated mass of H8Cl3N5O: 342.98, measured m / z: 344.0 [M+H] + .

[0124] Scheme C-1 [ka]

[0125] General Procedure for Preparing Compounds in Scheme C-1 Preparation of 1-allyl-4-nitro-pyrazole (Step 1 in Scheme C-1) [ka] To a solution of 4-nitro-1H-pyrazole (200 g, 1.77 mol, 1 equiv.) in DMF (1 L) at 25 °C under N was added 3-bromoprop-1-ene (235.29 g, 1.94 mol, 1.1 equiv.) and KCO (488.74 g, 3.54 mol, 2 equiv.). The mixture was then stirred at 80 °C for 1 h. TLC showed the reaction was complete. The reaction mixture was quenched with ice water (500 mL) at 0 °C and then extracted with MTBE (500 mL × 3). The combined organic layers were washed with brine (300 mL × 6), dried over NaSO, filtered, and concentrated under reduced pressure to give 1-allyl-4-nitro-pyrazole (270 g, 1.76 mol, 99.71% yield) as a brown oil. 1 H NMR (CDCl 3,400 MHz) δ 8.16 (s, 1H), 8.09 (s, 1H), 6.07~6.00 (m, 1H), 5.44~5.34 (m, 2H), 4.78 (d, J = 6.0 Hz, 2H).

[0126] Preparation of 2-allyl-4-nitro-pyrazole-3-carboxylic acid (Step 2 in Scheme C-1) [ka] To a solution of 1-allyl-4-nitro-pyrazole (50 g, 326.50 mmol, 1 equiv.) in THF (500 mL) was added dropwise LiHMDS (1 M, 489.75 mL, 1.5 equiv.) under N2 at -78 °C. The mixture was then stirred at -78 °C for 1 h. Dry CO2 was then bubbled through the mixture for 30 min. TLC showed that approximately 30% of the starting material remained and a new spot with greater polarity was formed. The reaction mixture (5 batches) was quenched with water (2.5 L) at 0 °C and then adjusted to pH = 8-9 with saturated Na2CO3. The mixture was extracted with MTBE (1 L × 3) to recover 1-allyl-4-nitro-pyrazole. The aqueous layer was then adjusted to pH = 1 with 2 N HCl and then extracted with EtOAc (2 L × 3). The combined organic layers were washed with brine (1 L), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-allyl-4-nitro-pyrazole-3-carboxylic acid (200 g, 1.01 mol, 62.14% yield) as a brown oil. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.34 (s, 1H), 6.02~5.94 (m, 1H), 5.25 (dd, J = 10.4 Hz, 1.2 Hz, 1H), 5.10 (dd, J = 17.2 Hz, 1.2 Hz, 1H), 4.90 (d, J = 5.6 Hz, 2H).

[0127] Preparation of 2-allyl-4-nitro-pyrazole-3-carboxamide (Step 3 in Scheme C-1) [ka] A solution of 2-allyl-4-nitro-pyrazole-3-carboxylic acid (65 g, 329.70 mmol, 1 equiv.) in SOCl (200 mL) and DMF (5 mL) was stirred at 80 °C for 2 h. TLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give 1-allyl-5-(chlorosylmethyl)-4-nitro-pyrazole (80 g, crude) as a brown oil. A solution of 2-allyl-4-nitro-pyrazole-3-carbonyl chloride (80 g, 371.07 mmol, 1 equiv.) in DCM (50 mL) was added dropwise to NH HO (200 mL) at 0 °C, and the mixture was stirred at 25 °C for 1 h. TLC showed the reaction was complete. Some solid was formed. After filtration, the solid was collected, and the filtrate was extracted with EtOAc (200 mL × 3). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The combined residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 330 g, eluting with a 0-50% ethyl acetate / petroleum ether gradient at 200 mL / min) to afford 2-allyl-4-nitro-pyrazole-3-carboxamide (22 g, 112.15 mmol, 30.22% yield) as a pale yellow solid. 1 H NMR (DMSO-d6,400MHz) δ 8.48 (s, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 6.0~5.92 (m, 1H), 5.24 (dd, J = 10.4 Hz, 0.8 Hz, 1H), 5.13 (dd, J = 17.2 Hz, 1.0 Hz, 1H), 4.81 (br d, J = 5.6 Hz, 2H).

[0128] Preparation of 2-allyl-4-amino-pyrazole-3-carboxamide (Step 4 in Scheme C-1) [ka] To a solution of 2-allyl-4-nitro-pyrazole-3-carboxamide (22 g, 112.15 mmol, 1 equiv.) in MeOH (150 mL) and HO (50 mL) was added NH4Cl (12.00 g, 224.30 mmol, 2 equiv.) and Fe (18.79 g, 336.45 mmol, 3 equiv.) at 25 °C. The mixture was stirred at 85 °C for 2 h. TLC showed that 2-allyl-4-nitro-pyrazole-3-carboxamide was completely consumed, and one major new spot with high polarity was detected. The reaction mixture was filtered, and the filter cake was washed with MeOH (100 mL). The combined filtrate was concentrated under reduced pressure. The residue was diluted with water (50 mL) and extracted with DCM and i-PrOH (v:v = 3:1, 100 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give 2-allyl-4-amino-pyrazole-3-carboxamide (17.5 g, 105.31 mmol, 93.90% yield) as a brown oil. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.37 (s, 2H), 7.08 (s, 1H), 5.93~5.84 (m, 1H), 5.04 (dd, J = 10.4 Hz, 1.6 Hz, 1H), 4.96 (d, J = 5.2 Hz, 2H), 4.88 (dd, J = 17.2, 1.6 Hz, 1H), 4.35 (s, 2H).

[0129] Preparation of 1-allyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione (Step 5 in Scheme C-1) [ka] To a solution of 2-allyl-4-amino-pyrazole-3-carboxamide (17.5 g, 105.31 mmol, 1 equiv.) in MeCN (250 mL) was added CDI (22.20 g, 136.90 mmol, 1.3 equiv.) in portions over 1 h at 100 °C under N. The mixture was then heated at 100 °C for 12 h. LC-MS showed the reaction was complete. The reaction mixture was filtered at 100 °C, and the cake was washed with MeCN (100 mL × 3) to give 1-allyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione (15.2 g, 79.09 mmol, 75.11% yield) as an off-white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.14 (s, 1H), 11.00 (s, 1H), 7.40 (s, 1H), 6.03~5.94 (m, 1H), 5.14 (d, J = 7.2 Hz, 1H), 5.03~4.97 (m, 3H).

[0130] Preparation of 1-allyl-5,7-dichloro-pyrazolo[4,3-d]pyrimidine (Step 6 in Scheme C-1) [ka]

[0131] To a solution of 1-allyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione (15 g, 78.05 mmol, 1 equiv.) in POCl3 (119.68 g, 780.54 mmol, 72.53 mL, 10 equiv.) under N2 at 50 °C, DBU (71.30 g, 468.32 mmol, 70.59 mL, 6 equiv.) was added dropwise. The mixture was then stirred at 85 °C for 12 h. TLC showed that 1-allyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione was completely consumed, and one new spot with low polarity was detected. The reaction mixture was cooled to room temperature and slowly poured into ice water (200 mL) at 0 °C, followed by extraction with EtOAc (200 mL × 3). The combined organic layers were washed with saturated Na2CO3 to pH = 7-8, then washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 1-allyl-5,7-dichloro-pyrazolo[4,3-d]pyrimidine (17 g, 74.21 mmol, 95.08% yield) as a brown oil. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.56 (s, 1H), 6.12~6.05 (m, 1H), 5.35-5.30 (dd, J = 4.8, 1.6 Hz, 2H), 5.21 (dd, J = 10.8 Hz, 0.8 Hz, 1H), 4.94 (dd, J = 17.2 Hz, 1.2 Hz, 1H).

[0132] Preparation of 1-allyl-5-chloro-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 7 in Scheme C-1) [ka] To a solution of 1-allyl-5,7-dichloro-pyrazolo[4,3-d]pyrimidine (6.8 g, 29.69 mmol, 1 equiv.) in dioxane (60 mL) was added NaOH (1 M, 29.69 mL, 1 equiv.) at 25 °C. The mixture was stirred at 100 °C for 10 hours. TLC and LC-MS showed the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was adjusted to pH = 5 with 2 N HCl. Some solid was produced. The solid was collected after filtration and then concentrated under reduced pressure. The residue was used in the next step without further purification. Compound 1-allyl-5-chloro-6H-pyrazolo[4,3-d]pyrimidin-7-one (5.5 g, 26.11 mmol, 87.97% yield) was obtained as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 13.32 (s, 1H), 8.00 (s, 1H), 6.07~5.99 (m, 1H), 5.18~5.13 (m, 3H), 4.99 (d, J = 17.2 Hz, 1H).

[0133] compound 56 Preparation of 1-allyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 8 in Scheme C-1) [ka] A solution of 1-allyl-5-chloro-6H-pyrazolo[4,3-d]pyrimidin-7-one (4 g, 18.99 mmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (10.03 g, 56.97 mmol, 7.60 mL, 3 equiv.) in t-BuOH (60 mL) was heated at 100° C. for 10 h. LC-MS showed the reaction was complete. The solvent was removed under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 0.005 N HCl (50 mL×10). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was triturated with EtOAc (30 mL). After filtration, the solid was collected. The compound 1-allyl-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (3 g, 8.49 mmol, 44.70% yield, 99.09% purity) was obtained as a white solid, 59.6 mg of which was used further. 1 H NMR (DMSO-d6, 400 MHz) δ 11.08 (s, 1H), 9.58~7.56 (m, 3H), 7.31 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.56 (t, J = 5.6 Hz, 1H), 6.01~5.97 (m, 1H), 5.11 (d, J = 10.4 Hz, 1H), 5.03 (d, J = 5.6 Hz, 2H), 4.96 (d, J = 15.6 Hz, 1H), 4.46 (d, J = 6.0 Hz, 2H), 99.08% (215 nm), 100.00% (254 nm).MS (ESI): C 15 H 13 Calculated mass of Cl2N5O: 349.05, measured m / z: 350.0 [M+H] + .

[0134] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)acetaldehyde (Step 9 in Scheme C-1) [ka] A mixture of 1-allyl-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (1 g, 2.86 mmol, 1 equiv.) and KOsO·2HO (105.21 mg, 285.55 μmol, 0.1 equiv.) in THF (20 mL) and HO (20 mL) was stirred at 25 °C for 30 min. NaIO (1.83 g, 8.57 mmol, 3 equiv.) was then added, and the mixture was stirred at 25 °C for 1.5 h. TLC showed the reaction was complete. After filtration, the filtrate was extracted with EtOAc (30 mL × 3). The combined organic layers were washed with saturated NaSO (10 mL), brine (10 mL × 1), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was used in the next step without further purification. The compound 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (0.8 g, 2.27 mmol, 79.55% yield) was obtained as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.15 (s, 1H), 9.65 (s, 1H), 7.65 (s, 1H), 7.59~7.57 (m, 2H), 7.32 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 6.0 Hz, 1H), 5.36 (s, 2H), 4.48 (d, J = 6.0 Hz, 2H).

[0135] Preparation of Compounds in Scheme C-1 (Step 10 in Scheme C-1) [ka]

[0136] To a solution of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (4.26 mmol, 1 equiv.) and R'NHR'' (5.11–85.2 mmol, 1.2–20 equiv.) in MeOH or DCM (4 mL / mmol) was added NaBHCN (5.11–12.78 mmol, 1.2–3 equiv.) portionwise at 0°C. The mixture was then stirred at 0°C–80°C for a period of time (2–10 h). LCMS and HPLC indicated the reaction was complete. The reaction mixture was quenched with HO at 0°C and then extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC. Column:a)Luna C18 100mm×30mm 5μm;b)Phenomenex Luna C18 150mm×30mm 5μm;c)Nano-micro Kromasil C18 100mm×30mm 5μm;d)Boston Prime C18 150mm×30mm 5μm;e)Phenomenex Luna C18 150mm×30mm 5μm;f)Xbridge 150mm×30mm 10μm. Mobile phase: a) [water (0.1% TFA)-MeCN]; B%: 15%–55%, 10 min; b) [water (0.05% HCl)-MeCN]; B%: 1%–55%, 8, 10, or 12 min; c) [water (0.04% NH₃·H₂O + 10 mM NH₄HCO₃)-MeCN]; B%: 40%–60%, 10.5 min. MeCN was removed under reduced pressure. The aqueous solution was dehydrated by lyophilization.

[0137] compound 57 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholinoethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride (Step 10 in Scheme C-1) [ka] To a mixture of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (1.5 g, 4.26 mmol, 1 equiv.) and morpholine (1.11 g, 12.78 mmol, 1.12 mL, 3 equiv.) in MeOH (15 mL) was added NaBHCN (401.49 mg, 6.39 mmol, 1.5 equiv.) portionwise at 0 °C, and the mixture was stirred at 25 °C for 2 h. LC-MS showed the reaction was complete. The mixture was quenched with ice-water (10 mL) at 0 °C, and the organic solvent was removed under reduced pressure. The aqueous solution was extracted with EtOAc (20 mL × 5). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The filtrate was purified by preparative HPLC (HCl condition: Phenomenex Luna C18 250 mm × 50 mm 10 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 5% to 35%, 20 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-(2-morpholinoethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (460 mg, 1.08 mmol, yield 25.36%, purity 99.38%) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.75 (s, 1H), 7.70 (s, 1H), 7.62~7.59 (m, 2H), 7.52 (s, 1H), 7.35 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 4.86 (t, J = 6.0 Hz, 2H), 4.54 (d, J = 5.2 Hz, 2H), 3.97~3.93 (m, 2H), 3.74~3.68 (m, 2H), 3.65~3.60 (m, 2H), 3.48~3.45 (m, 2H), 3.13~3.10 (m, 2H). HPLC: 99.38% (220 nm), 99.42% (215 nm), 98.29% (254 nm). MS (ESI): C 18 H 21 Calculated mass of Cl3N6O2: 422.10, measured m / z: 423.1 [M+H] + .

[0138] compound 58 5-((3,4-Dichlorobenzyl)amino)-1-(2-(4-methylpiperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (58.7 mg, 134.53 μmol, 31.59% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.36~10.84 (m, 1H), 9.38~9.32 (m, 1H), 7.60~7.58 (m, 3H), 7.3 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.71~6.68 (m, 1H), 4.56 (t, J = 6.0 Hz, 2H), 4.47 (d, J = 5.6 Hz, 2H), 3.36~3.30 (m, 2H), 3.07~3.04 (m, 2H), 2.95~2.84 (m, 4H), 2.74 (s, 3H), 2.38~2.27 (m, 2H). HPLC: 99.58% (220 nm), 99.44% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 23 Calculated mass of Cl2N7O: 435.13, measured m / z: 436.2 [M+H] + .

[0139] compound 59 5-((3,4-Dichlorobenzyl)amino)-1-(2-(1,1-dioxidothiomorpholino)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (22.3 mg, 47.31 μmol, 11.11% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.62 (s, 1H), 7.5~7.54 (m, 2H), 7.31 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 4.64 (t, J = 6.0 Hz, 2H), 4.46 (s, 2H), 3.29~3.27 (m, 6H), 3.21~3.18 (m, 4H). HPLC: 99.85% (220 nm), 94.04% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 20 Calculated mass of Cl2N6O3S: 470.07, measured m / z: 471.1 [M+H] + .

[0140] compound 60 5-((3,4-Dichlorobenzyl)amino)-1-(2-(4-methyl-3-oxopiperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (69.1 mg, 153.45 μmol, 36.03% yield) as an off-white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.25 (s, 1H), 7.65 (s, 1H), 7.61~7.55 (m, 2H), 7.33 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.81 (s, 1H), 4.72 (s, 2H), 4.48 (d, HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N7O2: 449.11, measured m / z: 450.2 [M+H] + .

[0141] compound 61 5-((3,4-Dichlorobenzyl)amino)-1-(2-(methylamino)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (30.5 mg, 83.05 μmol, 19.50% yield) as an off-white solid. 1 HNMR (DMSO-d 6, 400 MHz) δ 11.25 (s, 1H), 8.45 (s, 2H), 7.64 (s, 1H), 7.57~7.54 (m, 2H), 7.28 (dd, J = 6.0 Hz, 2.0 Hz, 1H), 6.79~6.76 (m, 1H), 4.68 (t, J = 5.6 Hz, 2H), 4.45 (d, J = 6.0 Hz, 2H), 3.39~3.36 (m, 2H), 2.56 (s, 3H). HPLC: 98.94% (220 nm), 98.62% (215 nm), 97.58% (254 nm). MS (ESI): C 15 H 16Calculated mass of Cl2N6O: 366.08, measured m / z: 367.1 [M+H] + .

[0142] compound 62 1-[2-(tert-butylamino)ethyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (20.3 mg, 48.83 μmol, 24.46% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.59~7.57 (m, 2H), 7.54 (s, 1H), 7.32 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 6.58 (s, 1H), 4.47 (d, J = 6.0 Hz, 2H), 4.42 (t, J = HPLC: 98.11% (220 nm), 98.18% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 22 Calculated mass of Cl2N6O: 408.12, measured m / z: 409.1 [M+H] + .

[0143] compound 63 1-(2-aminoethyl)-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (34.9 mg, 98.81 μmol, 34.80% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.30 (s, 1H), 7.89 (s, 3H), 7.66 (s, 1H), 7.58 (m, 2H), 7.32 (q, J = 2.0 Hz,1H), 6.84 (t, J = 5.2 Hz,1H), 4.65 (t, J = 6.0 Hz,2H), 4.46 (s, 2H), 3.27 (t, J = 6.0 Hz,2H). HPLC: 99.19% (220 nm), 98.71% (215 nm), 100.00% (254 nm). MS (ESI): C 14 H 14 Calculated mass of Cl2N6O: 352.06, measured m / z: 353.1 [M+H] + .

[0144] compound 64 1-(2-(4-Acetylpiperazin-1-yl)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (0.293 g, 585.05 μmol, 20.60% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.01 (s, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 7.64~7.61 (m, 2H), 7.37 (m, 1H), 4.90~4.87 (m, 2H), 4.59 (d, J = 4.4 Hz, HPLC: 99.87% (220 nm), 99.77% (215 nm), 100.00% (254 nm). MS (ESI): C 20 H 24 Calculated mass of Cl3N7O2: 463.13, measured m / z: 464.1 [M+H] + .

[0145] compound 65 5-((3,4-Dichlorobenzyl)amino)-1-(2-(4-hydroxypiperidin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (25.4 mg, 51.62 μmol, 18.18% yield, HCl) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 10.03 (s, 1H), 7.68~7.60 (m, 3H), 7.34~7.33 (m, 2H), 4.84 (s, 2H), 4.50 (d, J = 10.4 Hz, 2H), 4.01 (s, 1H), 3.94~3.90 (m, 1H), 3.33 (s, 1H), 3.17 (s, 1H), 2.98 (s, 1H), 1.98~1.87 (m, 3H), 1.73~1.60 (m, 2H), 1.17 (s, 1H). HPLC: 95.87% (220 nm), 95.31% (215 nm), 99.13% (254 nm). MS (ESI): C 19 H 23 Calculated mass of Cl3N6O2: 436.12, measured m / z: 437.1 [M+H] + .

[0146] compound 66 1-(2-(5-((3,4-Dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)piperidine-4-carboxamide hydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (24.9 mg, 51.41 μmol, 18.10% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 9.48 (s, 1H), 7.71~7.67 (m, 1H), 7.63~7.57 (m, 2H), 7.41 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.01 (s, 1H), 6.93 (s, 1H), 4.84 (t, J = 6.0 Hz, 2H), 4.50 (d, J = 5.6 Hz, 2H), 3.58 (s, 4H), 3.03~2.88 (m, 2H), 2.39 (s, 1H), 1.92 (d, J = 13.4 Hz, 2H), 1.82~1.66 (m, 2H). HPLC: 95.87% (220 nm), 95.70% (215 nm), 94.38% (254 nm). MS (ESI): C 20 H 24 Calculated mass of Cl3N7O2: 463.13, measured m / z: 464.2 [M+H] + .

[0147] compound 67 5-((3,4-Dichlorobenzyl)amino)-1-(2-(4-(dimethylamino)piperidin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (42.0 mg, 76.61 μmol, 26.98% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.05 (s, 1H), 10.42~10.30 (m, 1H), 7.69 (s, 1H), 7.63~7.50 (m, 2H), 7.34 (d, J = 8.0 Hz, 1H), 4.90~4.82 (m, 2H), 4.51 (d, J = 5.2 Hz, 2H), 3.80~3.70 (m, 4H), 3.32 (s, 1H), 2.99 (s, 2H), 2.71 (d, J = 4.8 Hz, 6H), 2.27 (d, J = 14.0 Hz, 2H), 1.99 (d, J = 12.4 Hz, 2H). HPLC: 98.01% (220 nm), 97.80% (215 nm), 97.50% (254 nm). MS (ESI): C 21 H 29 Calculated mass for Cl4N7O: 463.17, measured m / z: 464.2 [M+H] + .

[0148] compound 68 1-(2-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (26.3 mg, 55.26 μmol, 19.46% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 11.21~10.52 (m, 1H), 7.70 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 4.86~4.74 (m, 2H), 4.66~4.52 (m, 4H), 4.13 (d, J = 10.4 Hz, 1H), 3.78~3.66 (m, 2H), 3.54~3.47 (m, 2H), 3.13~2.96 (m, 1H), 2.28~1.95 (m, 2H). HPLC: 99.12% (220 nm), 98.61% (215 nm), 99.33% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl3N6O2: 434.10, measured m / z: 435.0 [M+H] + .

[0149] compound 69 5-((3,4-Dichlorobenzyl)amino)-1-(2-(5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (26.9 mg, 57.25 μmol, 28.80% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.66 (s, 1H), 7.58 (s, 2H), 7.32 (d, J = 8.4 Hz, 1H), 6.99 (s, 1H), 4.77 (s, 2H), 4.49 (d, J = 5.6 Hz, 2H), 4.33 (s, HPLC: 95.42% (220 nm), 94.46% (215 nm), 93.04% (254 nm). MS (ESI): C 20 H 25 Calculated mass for Cl4N7O: 447.13, measured m / z: 448.1 [M+H] + .

[0150] compound 70 5-((3,4-Dichlorobenzyl)amino)-1-(2-(3-hydroxyazetidin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (33.8 mg, 71.91 μmol, 25.32% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 10.33~10.04 (m, 1H), 7.70 (d, J = 2.8 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.36 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 4.68~4.65 (m, HPLC: 94.83% (220 nm), 94.29% (215 nm), 95.83 % (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl3N6O4: 408.09, measured m / z: 409.1 [M+H] + .

[0151] compound 71 5-((3,4-Dichlorobenzyl)amino)-1-(2-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (28.3 mg, 52.38 μmol, 18.45% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.67 (s, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.34 (dd, J = 2.4 Hz, 8.6 Hz, 1H), 4.79 (t, J = 6.0 Hz, 2H), 4.54 (d, J = 5.2 Hz, 2H), 3.75~3.73 (m, 10H), 3.21 (s, 2H), 2.50 (d, J = 1.6 Hz, 2H). HPLC: 99.82% (220 nm), 99.75% (215 nm), 99.72% (254 nm). MS (ESI): C 20 H 27 Calculated mass of Cl4N7O2: 465.14, measured m / z: 466.1 [M+H] + .

[0152] compound 72 4-(2-(5-((3,4-Dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-N,N-dimethylmorpholine-2-carboxamide hydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (26 mg, 48.14 μmol, 16.95% yield, 98.662% purity, HCl) as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 11.03 (s, 1H), 7.68 (s, 1H), 7.59 (s, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.24 (s, 1H), 4.88 (s, 2H), 4.69~4.56 (m, 1H), 4.51 (s, 2H), 4.13~4.02 (m, 1H), 3.96~3.85 (m, 1H), 3.68 (s, 4H), 3.23~3.09 (m, 2H), 3.00 (s, 3H), 2.90~2.82 (m, 3H). HPLC: 98.66% (220 nm), 98.61% (215 nm), 99.05% (254 nm). MS (ESI): C 21 H 26 Calculated mass of Cl3N7O3: 493.14, measured m / z: 494.1 [M+H] + .

[0153] compound 73 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-((dimethylamino)methyl)morpholino)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Step 10 in Scheme C-1. [ka] The procedure afforded the desired compound (33.7 mg, 66.33 μmol, 23.36% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 10.37 (s, 2H), 7.68 (s, 1H), 7.60 (s, 2H), 7.34 (d, J = 8.8 Hz, 2H), 4.87 (s, 2H), 4.52 (s, 2H), 4.30 (s, 2H), 4.07~4.05 (m, 2H), 3.90~3.88 (m, 1H), 3.40~3.24 (m, 2H), 3.20~3.16 (m, 4H), 3.79 (s, 6H). HPLC: 94.95% (220 nm), 93.86% (215 nm), 99.71% (254 nm). MS (ESI): C 21 H 29 Calculated mass of Cl4N7O2: 479.16, measured m / z: 480.2 [M+H] + .

[0154] compound 74 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(piperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0155] Preparation of tert-butyl 4-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)piperazine-1-carboxylate (Step 10 in Scheme C-1) [ka] A mixture of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (0.18 g, 511.11 μmol, 1 equiv.), tert-butyl piperazine-1-carboxylate (114.23 mg, 613.33 μmol, 1.2 equiv.), and AcOH (3.07 mg, 51.11 μmol, 2.92 μL, 0.1 equiv.) in MeOH (4 mL) was stirred at 25° C. for 2 h. NaBHCN (38.54 mg, 613.33 μmol, 1.2 equiv.) was then added portionwise at 0° C., and the mixture was stirred at 25° C. for 2 h. LC-MS indicated the reaction was complete. The mixture was quenched with ice water (5 mL), and the organic solvent was removed under reduced pressure. The aqueous solution was extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was used in the next step without further purification. The compound tert-butyl 4-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethyl]piperazine-1-carboxylate (0.2 g, 382.83 μmol, 74.90% yield) was obtained as a light brown solid. MS (ESI): C 23 H 29 Calculated mass of Cl2N7O3: 521.17, measured m / z: 522.2 [M+H] + .

[0156] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(piperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka] To a mixture of tert-butyl 4-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethyl]piperazine-1-carboxylate (0.2 g, 382.83 μmol, 1 equiv.) in EtOAc (5 mL), HCl / EtOAc (4 M, 2.87 mL, 30 equiv.) was added, and the mixture was stirred at 25 °C for 2 h. LC-MS and HPLC indicated the reaction was complete. Some solid was formed. The solid was collected after filtration. The solid was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 u; mobile phase: [water (0.1% TFA)-MeCN]; B%: 20% to 35%, 10 min). The compound 5-[(3,4-dichlorophenyl)methylamino]-1-(2-piperazin-1-ylethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (60.2 mg, 142.55 μmol, 37.24% yield) was obtained as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.22~11.05 (m, 1H), 8.57~8.54 (m, 2H), 7.60~7.58 (m, 3H), 7.32 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.72 (t, J = HPLC: 100.00% (220 nm), 99.04% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 21 Calculated mass of Cl2N7O: 421.12, measured m / z: 422.1 [M+H] + .

[0157] compound 75 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2,3-dihydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka]

[0158] To a solution of 1-allyl-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 285.55 μmol, 1 equiv.) (Step 8 in Scheme C-1) in THF (0.5 mL) and HO (0.5 mL) was added KOsO·2HO (10.52 mg, 28.55 μmol, 0.1 equiv.). After 30 min, NMO (100.35 mg, 856.65 μmol, 90.41 μL, 3 equiv.) was added. The mixture was stirred at 25 °C for 3 h. LC-MS and HPLC indicated the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was extracted with EtOAc (8 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 25% to 40%, 10 min) to give 5-((3,4-dichlorobenzyl)amino)-1-(2,3-dihydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (48.5 mg, 126.23 μmol, yield 44.21%, purity 100%) as a yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.05~11.03 (m, 1H), 7.62~7.54 (m, 3H), 7.32 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 6.67~6.62 (m, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.42 (t, J = 6.0 Hz, 2H), 3.93~3.90 (m, 1H), 3.34~3.31 (m, 2H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 96.43% (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O3: 383.06, measured m / z: 384.1 [M+H] + .

[0159] compound 76 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-hydroxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka]

[0160] To a mixture of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (0.15 g, 425.92 μmol, 1 equiv.) (Step 9 in Scheme C-1) in MeOH (3 mL) was added NaBH (24.17 mg, 638.89 μmol, 1.5 equiv.) portionwise at 0 °C. The mixture was then stirred at 25 °C for 1 h. LC-MS showed the reaction was complete. The mixture was quenched with saturated NH Cl (1 mL) at 0 °C, and then the organic solvent was removed under reduced pressure. The residue was dissolved in EtOAc (30 mL) and washed with brine (5 mL × 1). The organic layer was dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 25% to 40%, 10 min). The eluate was removed by lyophilization. The compound 5-((3,4-dichlorobenzyl)amino)-1-(2-hydroxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (54.4 mg, 152.85 μmol, yield 35.89%, purity 99.52%) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.60~7.57 (m, 3H), 7.33 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.83 (s, 1H), 4.50~4.45 (m, 4H), 3.74 (t, J = 6.0 Hz, 2H). HPLC: 99.52% (220 nm), 98.91% (215 nm), 100.00% (254 nm). MS (ESI): C 14 H13 Calculated mass of Cl2N5O2: 353.04, measured m / z: 354.1 [M+H] + .

[0161] compound 77 Preparation of 5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 10 in Scheme C-1) [ka]

[0162] A mixture of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (0.15 g, 425.92 μmol, 1 equiv.), tert-butyl piperazine-1-carboxylate (95.19 mg, 511.11 μmol, 1.2 equiv.), and AcOH (2.56 mg, 42.59 μmol, 2.44 μL, 0.1 equiv.) in DCM (5 mL) was stirred for 1 h at 25° C. NaBH(OAc) (108.32 mg, 511.11 μmol, 1.2 equiv.) was then added, and the mixture was stirred at 25° C. for 3 h. LC-MS and HPLC showed no trace of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde. LC-MS showed mainly one new peak, which was 5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one. The mixture was quenched with ice water (5 mL) and then extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 20% to 45%, 10 min). After lyophilization, the resulting product was washed with MeOH (2 mL) and EtOAc (1 mL) to give the compound 5-[(3,4-dichlorophenyl)methylamino]-1,6-dihydropyrazolo[4,3-d]pyrimidin-7-one (24.3 mg, 78.35 μmol, 18.40% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.04~10.88 (m, 1H), 7.69 (s, 1H), 7.60~7.58 (m, 2H), 7.34 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.78~6.71 (m, 1H), 4.49 (d, J = 6.0 Hz, 2H). HPLC: 98.28% (220 nm), 97.37% (215 nm), 100.00% (254 nm). MS (ESI): C 12Calculated mass of H9Cl2N5O: 309.02, measured m / z: 310.0 [M+H] + .

[0163] compound 78 Preparation of 1,1'-(azanediylbis(ethane-2,1-diyl))bis(5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 10 in Scheme C-1) [ka] A mixture of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetaldehyde (0.1 g, 283.95 μmol, 1 equiv) and AcONH (218.73 mg, 2.84 mmol, 10 equiv) in MeOH (3 mL) was stirred at 25° C. for 2 h. NaBHCN (26.77 mg, 425.92 μmol, 1.5 equiv) was then added portionwise at 0° C., and the mixture was stirred at 25° C. for 10 h. LC-MS and HPLC showed the reaction was complete. The product was primarily 5-[(3,4-dichlorophenyl)methylamino]-1-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethylamino]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one. The mixture was quenched with ice water (5 mL), and the organic solvent was removed under reduced pressure. The aqueous solution was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 u; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30%–55%, 10 min). The compound 5-[(3,4-dichlorophenyl)-methylamino]-1-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethylamino]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (38.1 mg, 55.27 μmol, 19.46% yield) was obtained as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 11.31 (s, 2H), 8.74 (s, 2H), 7.64 (s, 2H), 7.60~7.57 (m, 4H), 7.31 (dd, J = 8.4 Hz, 2.0 Hz, 2H), 6.80~6.78 (m, 2H), 4.70 (t, J = 6.0 Hz, 4H), 4.48 (d, J = 6.0 Hz, 4H), 3.47 (t, J = 5.6 Hz, 4H). HPLC: 100.00% (220 nm), 99.46% (215 nm), 100.00% (254 nm). MS (ESI): C 28 H 25 Cl4N 11 Calculated mass of O2: 687.09, measured m / z: 688.2 [M+H] + .

[0164] Scheme C-2 [ka]

[0165] General Procedure for Preparing Compounds in Scheme C-2 Preparation of Compound (Step 1 in Scheme C-2) [ka] A mixture of 1-(R'-aminoethyl)-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (283.12 μmol, 1 equiv.), R''COOH (283.12 μmol to 368.06 μmol, 1 equiv. to 1.3 equiv.), EDCI (339.74 μmol, 1.2 equiv.), HOBt (56.62 μmol, 0.2 equiv.), and DIEA (849.36 μmol, 147.94 μL, 3 equiv.) in DMF (3 mL / mmol) was stirred at 20°C to 25°C for 10 to 12 h. The reaction mixture was stirred at 0°C to 25°C for a period of time (1 to 3 h). LC-MS indicated the reaction was complete. The reaction mixture was quenched with HO and then extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC. Column: Luna C18 100 mm × 30 mm 5 μm or Nano-micro Kromasil C18 100 mm × 30 mm 5 μm; Mobile phase: [water (0.1% TFA)-MeCN]; B%: 20% to 45%, 10 or 20 min). The aqueous solution was lyophilized to give the desired compound.

[0166] compound 79 Preparation of N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-3,5-dimethylisoxazole-4-carboxamide (Step 1 in Scheme C-2) [ka]

[0167] A mixture of 1-(2-aminoethyl)-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 283.12 μmol, 1 equiv.), 3,5-dimethylisoxazole-4-carboxylic acid (39.96 mg, 283.12 μmol, 48.14 μL, 1 equiv.), EDCI (65.13 mg, 339.74 μmol, 1.2 equiv.), HOBt (7.65 mg, 56.62 μmol, 0.2 equiv.), and DIEA (109.77 mg, 849.36 μmol, 147.94 μL, 3 equiv.) in DMF (1 mL) was stirred at 25° C. for 12 hours. LC-MS showed the reaction was complete. After filtration, the filtrate was purified by preparative HPLC (column: Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30%–45%, 10 min) to obtain the compound N-[2-[5-[(3,4-dichlorophenyl)-methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethyl]-3,5-dimethyl-isoxazole-4-carboxamide (29.2 mg, 58.90 μmol, yield 20.80%, purity 96.077%) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.96 (t, J = 5.2 Hz, 1H), 7.60 (s, 1H), 7.57 (d, J = 6.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 6.73-6.72 (m, 1H), 4.55 (t, J = HPLC: 96.08% (220 nm), 96.58% (215 nm), 100.00% (254 nm). MS (ESI): C 20 H 19 Calculated mass of Cl2N7O3: 475.09, measured m / z: 476.1 [M+H] + .

[0168] compound 80 N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-N,3,5-trimethylisoxazole-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (29.5 mg, 60.16 μmol, 44.19% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.86~10.33 (m, 1H), 7.63~7.49 (m, 3H), 7.34 (d, J = 7.6 Hz, 1H), 6.56~6.39 (m, 1H), 4.61 (s, 2H), 4.50 (d, J = 5.6 Hz, 2H), 3.87 (t, J = 5.2 Hz, 2H), 2.85 (s, 3H), 2.23 (s, 3H), 1.98 (s, 3H). HPLC: 98.05% (220 nm), 88.25% (215 nm), 100.00% (254 nm). MS (ESI): C 21 H 21 Calculated mass of Cl2N7O3: 489.11, measured m / z: 490.2 [M+H] + .

[0169] compound 81 6-chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-3-hydroxy-N-methylpyridazine-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (34.2 mg, 65.30 μmol, 23.98% yield) as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 13.40 (s, 1H), 7.61~7.51 (m, 3H), 7.35 (d, J = 8.0 Hz, 1H), 7.25 (s, 0.5H), 6.67 (s, 0.5H), 6.57~6.42 (m, 1H), 4.73~4.60 (m, 1H), 4.58~4.52 (m, 1H), 4.51 (d, J = 4.0 Hz, 2H), 3.88~3.78 (m, 1H), 3.69~3.60 (m, 2H), 2.95 (s, 2H), 2.74 (s, 1H). HPLC: 97.50% (220 nm), 96.618% (215 nm), 100.00% (254 nm). MS (ESI): C 20 H 17 Calculated mass of Cl3N8O3S: 522.05, measured m / z: 523.1 [M+H] + .

[0170] compound 82 N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-N-methylisoxazole-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (25.1 mg, 54.30 μmol, 24.92% yield) as an off-white solid. 1H NMR (DMSO-d6, 400 MHz) δ 9.10 (s, 1H), 8.56 (s, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.57~7.51 (m, 2H), 7.37~7.31 (m, 1H), 6.57~6.47 (m, HPLC: 99.73% (220 nm), 99.65% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 17 Calculated mass of Cl2N7O3: 461.08, measured m / z: 462.1 [M+H] + .

[0171] compound 83 6-chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-3-hydroxypyridazine-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (19 mg, 37.27 μmol, 18.81% yield) as a pale yellow solid. 1H NMR (DMSO- d6, 400 MHz) δ 13.92 (s, 1H), 11.10 (s, 1H), 9.38 (t, J = 6.4 Hz, 1H), 7.93 (s, 1H), 7.60~7.56 (m, 3H), 7.33~7.30 (m, 1H), 6.55 (s, 1H), 4.58 (t, J = 5.6 Hz, 2H), 4.46 (d, J = 5.6 Hz, 2H), 3.76 (t, J = 5.6 Hz, 2H), 96.43% (254 nm). MS (ESI): C 19 H 15 Calculated mass of Cl3N8O3: 508.03, measured m / z: 509.1 [M+H] + .

[0172] compound 84 3-chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-6-hydroxypyridazine-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (3.7 mg, 6.08 μmol, 2.15% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 13.36 (s, 1H), 8.83 (s, 1H), 7.60~7.57 (m, 3H), 7.32 (d, J =8.4 Hz, 1H), 6.64 (s, 1H), 6.83~6.62 (m, 1H), 4.55 (t, J = 5.6 Hz, 2H), 4.47 (t, J = 5.6 Hz, 2H), 3.62 (s, 2H). HPLC: 97.29% (220 nm), 97.48% (215 nm), 98.52% (254 nm). MS (ESI): C 19 H15 Calculated mass of Cl3N8O3: 508.03, measured m / z: 509.1 [M+H] + .

[0173] compound 85 3,6-Dichloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-N-methylpyridazine-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (35 mg, 64.55 μmol, 11.85% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.92 and 7.43 (s, 1H), 7.67~7.55 (m, 3H), 7.38~7.30 (m, 1H), 6.83~6.59 (m, 1H), 4.73~4.66 (m, 1H), 4.51~4.47 (m, 3H), 3.97~3.88 (m, 1H), 3.77~3.66 (m, 1H), 3.65~3.52 (m, 1H), 3.0 and 2.71 (s, 3H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 20 H 16 Calculated mass of Cl4N8O2: 540.02, measured m / z: 541.1 [M+H] + .

[0174] compound 86 N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)isoxazole-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (20.4 mg, 45.24 μmol, 10.65% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.26 (s, 1H), 8.09 (s, 1H), 7.79 (t, J = 5.6 Hz, 1H), 7.60~7.54 (m, 3H), 7.33 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.65 (s, 1H), 4.48 (d, J = 3.6 Hz, 4H), 3.51 (d, J = 5.6 Hz, 2H). HPLC: 99.41% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 15 Calculated mass of Cl2N7O3: 447.06, measured m / z: 448.1 [M+H] + .

[0175] compound 87 3-chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethyl)-6-hydroxy-N-methylpyridazine-4-carboxamide was prepared according to the procedure described herein as Step 1 in Scheme C-2. [ka] The procedure afforded the desired compound (21.4 mg, 40.86 μmol, 17.04% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 13.14 (s, 1H), 7.60~7.54 (m, 2H), 7.35 (t, J = 6.4 Hz, 1H), 6.76 and 6.44 (s, 1H), 6.52 (s, 1H), 4.68 (t, J = 5.6 Hz, 1H), 4.59 (t, J = 6.0 Hz, 1H), 4.50 (s, 2H), 3.89 (d, J = 4.0 Hz, 1H), 3.71 (t, J = 5.6 Hz, 1H), 2.92 (s, 1H), 2.76 (s, 2H). HPLC: 99.82% (220 nm), 99.84% (215 nm), 100.00% (254 nm). MS (ESI): C 20 H 17 Calculated mass of Cl3N8O3: 522.05, measured m / z: 523.1 [M+H] + .

[0176] Scheme C-3 [ka]

[0177] General Procedure for Preparing Compounds in Scheme C-3 Preparation of Compound (Step 1 in Scheme C-3) [ka] To a solution of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (5.42 mmol, 1 equiv.), ROH (5.42 mmol–16.26 mmol, 1 equiv.–3 equiv.), and PPh (5.96 mmol–10.84 mmol, 1.1 equiv.–2 equiv.) in THF (3 mL / mmol–10 mL / mmol) at 0 °C, DIAD (5.96 mmol–10.84 mmol, 1.1 equiv.–2 equiv.) was added dropwise. The mixture was then stirred at 0 °C–25 °C for 2–16 h. TLC indicated the reaction was complete. The mixture was quenched with ice water, and the organic layer was separated. The aqueous solution was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 20 g or 40 g, eluting with a gradient of 0-100% ethyl acetate / petroleum ether or 0-20% MeOH / ethyl acetate ether at 35 mL / min or 80 mL / min). The eluent was removed under reduced pressure to give the desired product.

[0178] Preparation of Compound (Step 2 in Scheme C-3) [ka] To a solution of 5-chloro-7-methoxy-1-R1-1H-pyrazolo[4,3-d]pyrimidine (4.36 mmol, 1 equiv.) in MeOH (3 mL / mmol–5 mL / mmol) or THF (3 mL / mmol–5 mL / mmol) and HO (3 mL / mmol–5 mL / mmol) was added LiOH·HO (13.08 mmol–21.80 mmol, 3 equiv.–5 equiv.). The mixture was stirred at 20–25 °C for 2–16 h. TLC indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH 6 with 3 N HCl and then extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the desired product.

[0179] Preparation of Compound (Step 3 in Scheme C-3) [ka]

[0180] A mixture of 5-chloro-1-R1-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (3.87 mmol, 1 equiv.), R3NH2 (162.66 mmol–216.88 mmol, 1.5 equiv.–2 equiv.), and DIEA (11.61 mmol, 3 equiv.; DIEA was added only when R3NH2 was the HCl salt) in t-BuOH, t-AmOH, or NMP (3 mL / mmol–10 mL / mmol) was stirred at 100–160°C for 3–40 h. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC. Column:a) Phenomenex Luna C18 250mm×50mm 10μm;b)Phenomenex Luna C18 100mm×30mm 5μm;c)Phenomenex Luna C18 150mm×30mm 5μm;d)Luna C18 100mm×30mm 5μm;e)Boston Prime C18 150mm×30mm 5μm;f)Nano-micro Kromasil C18 100mm×30mm 5μm;g)Welch Xtimate C18 100mm×25mm 3μm;h)Xtimate C18 100mm×30mm 3μm;i)Kromasil C18(250 50mm 10μm);j)Phenomenex Gemini-NX 150 30 mm × 5 μm; k) Welch Xtimate C18 150 mm × 25 mm 5 μm; l) Xtimate C18 150 mm × 25 mm 5 μm; m) Xtimate C18 100 mm × 30 mm 3 μm. Mobile phase: a) [water (0.1% TFA)-MeCN]; B%: 10% to 70%, 10 or 20 min; b) [water (0.05% HCl)-MeCN]; B%: 10% to 70%, 10 or 20 min; c) [water (10 mM NH4HCO3)-MeCN]; B%: 10% to 85%, 8 or 20 min. The solvent was removed by lyophilization to give the desired product.

[0181] compound 88 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0182] Preparation of 2-(2-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethanol (Step 1 in Scheme C-3) [ka] To a solution of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (1 g, 5.42 mmol, 1 equiv.), 2-(2-hydroxyethoxy)ethanol (1.15 g, 10.84 mmol, 1.03 mL, 2 equiv.), and PPh3 (2.13 g, 8.13 mmol, 1.5 equiv.) in THF (10 mL) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 2 h. TLC showed the reaction was complete. The reaction mixture was quenched with HO (15 mL) and then concentrated under reduced pressure to remove the organic solvent. The aqueous solution was extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 20 g, eluting with a 0-60% ethyl acetate / petroleum ether gradient at 60 mL / min). The eluent was removed under reduced pressure. The compound 2-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]ethanol (1.19 g, 4.36 mmol, 80.55% yield) was obtained as a yellow oil. 1H NMR (DMSO-d6, 400 MHz) δ 8.26 (s, 1H), 4.66 (t, J = 5.6 Hz, 2H), 4.52–4.45 (m, 1H), 4.16 (s, 3H), 3.84 (t, J = 5.6 Hz, 2H), 3.37–3.35 (m, 4H). The compound 2-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)ethoxy]ethanol (440 mg, 1.61 mmol, 29.78% yield) was obtained as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.64 (s, 1H), 4.64~4.61 (m, 2H), 4.12 (s, 3H), 3.91 (t, J = 5.2 Hz, 2H), 3.44~3.41 (m, 4H).

[0183] Preparation of 5-chloro-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme C-3) [ka] A mixture of 2-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]ethanol (1.19 g, 4.36 mmol, 1 equiv.) and LiOH·HO (732.45 mg, 17.46 mmol, 4 equiv.) in THF (10 mL) and HO (10 mL) was stirred at 20 °C for 5 h. TLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove THF, and the aqueous solution was then adjusted to pH = 4 with 3 N HCl. The aqueous solution was extracted with EtOAc (25 mL × 4). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give 5-chloro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (1.3 g, crude) as a yellow oil. 1H NMR (DMSO-d6, 400 MHz) δ 8.06 (s, 1H), 7.78 (s, 1H), 4.62 (t, J = 5.2 Hz, 2H), 4.38 (t, J = 4.8 Hz, 2H), 3.83~3.75 (m, 5H).

[0184] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 3 in Scheme C-3) [ka] A mixture of (3,4-dichlorophenyl)methanamine (1.36 g, 7.73 mmol, 1.03 mL, 2 equiv.) and 5-chloro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (1 g, 3.87 mmol, 1 equiv.) in t-BuOH (6 mL) was stirred at 100 °C for 16 h. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 250 mm × 50 mm 10 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 10% to 40%, 20 min). The aqueous solution was lyophilized to give 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.99 g, 2.45 mmol, 63.34% yield, 98.50% purity) as a white solid, 30.1 mg of which was used further. 1H NMR (DMSO-d6, 400 MHz) δ 7.61~7.57 (m, 3H), 7.33 (dd, J = 2.0, 8.4 Hz, 1H), 6.82 (s, 1H), 4.57 (t, J = 5.6 Hz, 2H), 4.49 (d, J = 5.2 Hz, HPLC: 97.84% (220 nm), 98.14% (215 nm), 98.77% (254 nm). MS (ESI): C 16 H 17 Calculated mass of Cl2N5O3: 397.07, measured m / z: 398.0 [M+H] + .

[0185] compound 89 5-((3,4-Dichlorobenzyl)amino)-1-(4-hydroxybutyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (378.3 mg, 986.58 μmol, 46.04% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.07 (s, 1H), 7.62~7.58 (m, 2H), 7.56 (s, 1H), 7.34 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.62 (s, 1H), 4.48 (d, J = 5.6 Hz, HPLC: 99.69% (220 nm), 99.76% (215 nm), 100.00% (254 nm). MS (ESI): C 16 H 17Calculated mass of Cl2N5O2: 381.08, measured m / z: 382.2 [M+H] + .

[0186] compound 90 4-(5-((3,4-Dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butyl acetate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (0.03 g, 70.71 μmol, 77.48% yield) as a white solid. 1 H NMR (CDCl 3, 400 MHz) δ 11.28 (s, 1H), 7.57 (s, 1H), 7.43 (d, J = 1.6 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H), 5.45 (s, 1H), 4.54 (d, J = 5.2 Hz, 2H), 4.46 (t, J = 6.8 Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 1.94 (s, 3H), 1.83~1.91 (m, 2H), 1.49~1.53 (m, 2H). HPLC: 98.46% (220 nm), 98.15% (215 nm), 98.76% (254 nm). MS (ESI): C 18 H 19 Calculated mass of Cl2N5O3: 423.1, measured m / z: 424.0 [M+H] + .

[0187] compound 91 5-((3,4-Dichlorobenzyl)amino)-1-(thiazol-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (71.4 mg, 175.31 μmol, 42.66% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.75 (d, J = 3.2 Hz, 1H), 7.70 (s, 1H), 7.68 (d, J = 3.2 Hz, 1H), 7.62 ~ 7.57 (m, 2H), 7.34 (dd, J = 2.0 Hz, 8.4 HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 16 H 12 Calculated mass of Cl2N6OS: 406.02, measured m / z: 406.9 [M+H] + .

[0188] compound 92 5-((3,4-Dichlorobenzyl)amino)-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (290.5 mg, 741.49 μmol, 15.42% yield) as an off-white solid. 1H NMR (DMSO- d6, 400 MHz) δ 11.13 (s, 1H), 8.29 (s, 1H), 7.96 (s, 1H), 7.63~7.54 (m, 3H), 7.32 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.59 (s, 1H), 5.55 (s, 2H), 4.47 (d, J = 5.6 Hz, 2H). HPLC: 99.86% (220 nm), 99.88% (215 nm), 100.00% (254 nm). MS (ESI): C 16 H 12 Calculated mass of Cl2N6O2: 390.04, measured m / z: 391.0 [M+H] + .

[0189] compound 93 5-((3,4-Dichlorobenzyl)amino)-1-((5-oxopyrrolidin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (63.7 mg, 156.41 μmol, 46.52% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.64 (s, 1H), 7.61~7.58 (m, 3H), 7.33 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.68 (s, 1H), 4.50~4.46 (m, 3H), 4.42~4.36 (m, HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 16Calculated mass of Cl2N6O2: 406.07, measured m / z: 407.1 [M+H] + .

[0190] compound 94 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-oxopyrrolidin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (90.4 mg, 210.81 μmol, 59.38% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.59~7.57 (m, 2H), 7.54 (s, 1H), 7.33 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 6.78 (m, 1H), 4.51 (t, J = 5.2 Hz, 2H), 4.47 (d, J = 5.6 Hz, 2H), 3.56 (t, J = 5.6 Hz, 2H), 3.17 (t, J = 6.8 Hz, 2H), 2.05 (t, J = 8.4 Hz, 2H), 1.79~1.77 (m, 2H). HPLC: 98.24% (220 nm), 98.21% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 18 Calculated mass of Cl2N6O2: 420.09, measured m / z: 421.1 [M+H] + .

[0191] compound 95 1-(2-(2-(2-butoxyethoxy)ethoxy)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (77.5 mg, 155.50 μmol, 29.37% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.61~7.57 (m, 3H), 7.33 (dd, J = 1.6 Hz, 2.0 Hz, 1H), 6.72 (s, 1H), 4.56 (t, J = 5.6 Hz, 2H), 4.48 (d, J = 5.6 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 3.48~3.44 (m, 2H), 3.43~3.37 (m, 6H), 3.32 (t, J = 6.8 Hz, 2H), 1.47~1.38 (m, 2H), 1.31~1.22 (m, 2H), 0.85 (t, J = 7.2 Hz, 3H). HPLC: 99.05% (220 nm), 97.64% (215 nm), 100.00% (254 nm). MS (ESI): C 22 H 29 Calculated mass for Cl2N5O4: 497.16, measured m / z: 498.2 [M+H] + .

[0192] compound 96 5-((3,4-Dichlorobenzyl)amino)-1-(((2R,3R,4R,5S)-3,4,5-trihydroxypiperidin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (20 mg, 41.73 μmol, 6.69% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.97~8.95 (m, 1H), 8.67 (br s, 1H), 7.70 (s, 1H), 7.60~7.58 (m, 2H), 7.32 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.88 (br s, 1H), 4.93 (br d, J = 13.2 Hz, 1H), 4.67~4.61 (m, 1H), 4.49 (d, J = 6.0 Hz, 2H), 3.55~3.43 (m, 2H), 3.30~3.21 (m, 2H), 3.12 (d, J = 8.0 Hz, 1H), 2.70~2.64 (m, 1H). HPLC: 94.99% (220 nm), 94.77% (215 nm), 98.18% (254 nm). MS (ESI): C 18 H 20 Calculated mass for Cl2N6O4: 454.09, observed m / z: 455.09 [M+H]+.

[0193] compound 97 5-((3,4-Dichlorobenzyl)amino)-1-((1-methyl-1H-imidazol-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (21 mg, 51.95 μmol, 13.04% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.80 (s, 1H), 7.59~7.53 (m, 3H), 7.34~7.32 (m, 1H), 7.07 (s, 1H), 7.03 (s, 1H), 6.75 (s, 1H), 5.71 (s, 2H), 4.47 (d, J = 4.4 Hz, 2H), 3.64 (s, 3H). HPLC: 95.33% (220 nm), 94.77% (215 nm), 93.17% (254 nm). MS (ESI): C 17 H 15 Calculated mass of Cl2N7O: 403.07, measured m / z: 404.1 [M+H] + .

[0194] compound 98 tert-Butyl 3-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (26.4 mg, 54.15 μmol, 36.80% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.67 (s, 1H), 7.64 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 5.57 (s, 1H), 4.57 (d, J = 4.4 Hz, 2H), 3.71~3.68 (m, 1H), 3.57~3.53 (m, 1H), 3.46~3.41 (m, 2H), 2.33~2.30 (m, 2H), 1.39 (d, J = 8.0 Hz, 9H). HPLC: 98.32% (220 nm), 98.63% (215 nm), 96.81% (254 nm). MS (ESI): C 21 H 24Calculated mass of Cl2N6O3: 478.13, measured m / z: 479.2 [M+H] + . compound 99 Preparation of 1-(2-(2-aminoethoxy)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0195] compound 100 tert-Butyl (2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethyl)carbamate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (350 mg, 697.88 μmol, 62.42% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.09 (s, 1H), 7.62~7.52 (m, 3H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.65 (s, 1H), 4.55 (t, J = 5.2 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 3.77 (t, J = 5.6 Hz, 2H), 3.33 (t, J = 6.4 Hz, 2H), 2.98 (q, J = 6.0 Hz, 2H), 1.35 (s, 9H). HPLC: 99.17% (220 nm), 99.07% (215 nm), 99.62% (254 nm). MS (ESI): C 21 H 26 Calculated mass of Cl2N6O4: 496.14, measured m / z: 497.2 [M+H] + .

[0196] Preparation of 1-(2-(2-aminoethoxy)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] A mixture of tert-butyl N-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethyl]carbamate (compound 100) (0.1 g, 201.06 μmol, 1 equivalent) in HCl / EtOAc (20 mL) and EtOAc (5 mL) was stirred at 20° C. for 5 hours. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. Compound 1-[2-(2-aminoethoxy)ethyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (70 mg, 159.76 μmol, 79.46% yield, 98.989% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.83 (s, 3H), 7.65~7.57 (m, 3H), 7.35 (d, J = 8.4 Hz, 1H), 4.62 (t, J = 5.6 Hz, 2H), 4.53 (s, 2H), 3.85 (t, J = HPLC: 98.99 % (220 nm), 98.52 % (215 nm), 100.00 % (254 nm). MS (ESI): C 16 H 19 Calculated mass of Cl3N6O2: 396.09, measured m / z: 397.1 [M+H] + .

[0197] Compound 101 1-((1H-1,2,4-triazol-1-yl)methyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (34.7 mg, 77.16 μmol, 21.34% yield, HCl) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.75 (s, 1H), 7.99 (s, 1H), 7.74 (s, 1H), 7.61~7.57 (m, 2H), 7.35~7.32 (m, 2H), 6.74 (s, 2H), 4.52 (d, J = 4.8 Hz, 2H). HPLC: 95.10% (220 nm), 94.99% (215 nm), 100.00% (254 nm). MS (ESI): C 15 H 13 Calculated mass of Cl3N8O: 390.05, measured m / z: 391.0 [M+H] + .

[0198] Compound 102 5-((3,4-Dichlorobenzyl)amino)-1-((tetrahydrofuran-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (48.8 mg, 123.40 μmol, 52.38% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.87~7.53 (m, 4H), 7.37 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 4.57 (d, J = 4.8 Hz, 2H), 4.55~4.50 (m, 1H), 4.36 (dd, J = 5.2 Hz, HPLC: 99.70% (220 nm), 99.50% (215 nm), 99.40% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O2: 393.08, measured m / z: 394.1 [M+H] + .

[0199] compound 103 2-((1H-1,2,4-triazol-1-yl)methyl)-5-((3,4-dichlorobenzyl)amino)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (21.1 mg, 45.65 μmol, 12.62% yield, HCl) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.93~8.87 (m, 2H), 8.35 (s, 1H), 8.07 (s, 1H), 7.73~7.62 (m, 2H), 7.42 (t, J = 5.6 Hz, 1H), 6.75 (s, 2H), 4.74(s, 2H). HPLC: 92.54% (220 nm), 91.99% (215 nm), 93.12% (254 nm). MS (ESI): C 15 H 13Calculated mass of Cl3N8O: 390.05, measured m / z: 391.0 [M+H] + .

[0200] Compound 104 5-((3,4-Dichlorobenzyl)amino)-1-(2-((2-hydroxyethyl)thio)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (184.5 mg, 436.26 μmol, 52.11% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 10.47 (s, 1H), 7.68~7.47 (m, 3H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.64 (s, 1H), 4.77 (s, 1H), 4.58 (t, J = 6.8 Hz, HPLC: 97.97% (220 nm), 97.21% (215 nm), 100.00% (254 nm). MS (ESI): C 16 H 17 Calculated mass of Cl2N5O2S: 413.05, measured m / z: 414.0 [M+H] + .

[0201] compound 105 1-((1,4-Dioxan-2-yl)methyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (89.6 mg, 210.89 μmol, 57.08% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.63 (s, 3H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.77 (s, 1H), 4.57 (dd, J = 7.2 Hz, 13.6 Hz, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.37 (dd, J = 5.2 Hz, 14.0 Hz, 1H), 3.95~3.90 (m, 1H), 3.71 (s, 2H), 3.60 (s, 1H), 3.48~3.44 (m, 2H), 3.32 (dd, J = 9.6 Hz, 11.6 Hz, 1H). HPLC: 96.56% (220 nm), 95.74% (215 nm), 98.80% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O3: 409.07, measured m / z: 410.0 [M+H] + .

[0202] compound 106 5-((3,4-Dichlorobenzyl)amino)-1-(1,3-dimethoxypropan-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (68.5 mg, 158.02 μmol, 62.01% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.65~7.54 (m, 3H), 7.33 (d, J = 7.6 Hz, 1H), 6.91 (s, 1H), 5.44 (s, 1H), 4.49 (d, J = 3.6 Hz, 2H), 3.75 (t, J = HPLC: 95.10% (220 nm), 94.43% (215 nm), 97.70% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl2N5O3: 411.09, measured m / z: 412.1 [M+H] + .

[0203] Compound 107 5-((3,4-Dichlorobenzyl)amino)-1-(1,3-dihydroxypropan-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (17.5 mg, 43.55 μmol, 10.65% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.59 (d, J = 4.4 Hz, 3H), 7.33 (d, J = 6.8 Hz, 1H), 6.81 (s, 1H), 5.14~5.00 (m, 1H), 4.49 (d, J = 3.6 Hz, 2H), 3.74 (d, J = 5.6 Hz, 4H). HPLC: 95.62 % (220 nm), 95.60 % (215 nm), 95.24 % (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O3: 383.06, measured m / z: 384.1 [M+H] + .

[0204] compound 108 5-((3,4-Dichlorobenzyl)amino)-1-(2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (71.1 mg, 161.56 μmol, 32.17% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.99 (s, 1H), 7.69~7.64 (m, 2H), 7.62 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 4.62~4.56 (m, 4H), 3.81 (t, J = 5.2 Hz, 2H), 3.68 (s, 2H), 3.45~3.43 (m, 1H), 3.29~3.22 (m, 2H), 1.75~1.68 (m, 2H), 1.30~1.21 (m, 2H). HPLC: 99.60% (220 nm), 99.70% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O3: 437.10, measured m / z: 438.1 [M+H] + .

[0205] Compound 109 5-((3,4-Dichlorobenzyl)amino)-1-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (55.4 mg, 117.21 μmol, 29.49% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.58 (d, J = 4.8 Hz, 1H), 7.94~7.92 (m, 1H), 7.71 (s, 1H), 7.63~7.60 (m, 2H), 7.45~7.43 (m, 1H), 7.37~7.35 (m, 2H), 7.12 (d, J = 8.0 Hz, 1H), 5.82 (s, 2H), 4.54 (d, J = 5.2 Hz, 2H). HPLC: 92.60% (220 nm), 90.01% (215 nm), 95.32% (254 nm). MS (ESI): C 18 H 15 Calculated mass of Cl3N6O: 400.06, measured m / z: 401.0 [M+H] + .

[0206] compound 110 5-((3,4-Dichlorobenzyl)amino)-1-(2-(pyridin-3-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (131.7 mg, 287.85 μmol, 34.50% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.77 (d, J = 5.2 Hz, 1H), 8.72 (s, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.05~7.89 (m, 2H), 7.67~7.56 (m, 3H), 7.37 (d, J = 8.4 Hz, 1H), 4.79 (t, J = 6.4 Hz, 2H), 4.58 (d, J = 3.6 Hz, 2H), 3.36 (t, J = 6.4 Hz, 2H). HPLC: 98.74% (220 nm), 98.50% (215 nm), 99.11% (254 nm). MS (ESI): C 19 H 17 Calculated mass of Cl3N6O: 414.08, measured m / z: 415.0 [M+H] + .

[0207] Compound 111 5-((3,4-Dichlorobenzyl)amino)-1-(2-(pyridin-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (66.3 mg, 157.39 μmol, 45.68% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.76 (d, J = 5.2 Hz, 1H), 8.43~8.36 (m, 1H), 7.87 (d, J = 6.4 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.64~7.56 (m, 2H), 7.52 (s, 1H), 7.37~7.34 (m, 1H), 4.90 (t, J = 6.4 Hz, 2H), 4.57 (d, J = 5.2 Hz, 2H), 3.57 (s, 2H). HPLC: 98.58% (220 nm), 98.30% (215 nm), 98.93% (254 nm). MS (ESI): C 19 H 17 Calculated mass for Cl3N6O: 414.08 m / z, Found: 415.1 [M+H] + .

[0208] compound 112 5-((3,4-Dichlorobenzyl)amino)-1-(pyrazin-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (43.3 mg, 92.57 μmol, 34.74% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.56 (s, 2H), 8.49 (s, 1H), 7.68 (s, 1H), 7.65~7.58 (m, 2H), 7.46~7.30 (m, 2H), 5.83 (s, 2H), 4.54 (d, J = 4.8 Hz, 2H). HPLC: 93.80% (220 nm), 92.74% (215 nm), 99.63% (254 nm). MS (ESI): C 17 H 14 Calculated mass of Cl3N7O: 401.06, measured m / z: 402.0 [M+H] + .

[0209] compound 113 5-[(3,4-Dichlorophenyl)methylamino]-1-(3-pyridylmethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (87.3 mg, 196.51 μmol, 57.13% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.80~8.75 (m, 2H), 8.19 (d, J = 8.4 Hz, 1H), 7.88 (dd, J = 5.6 Hz, 8.0 Hz, 1H), 7.71 (s, 1H), 7.63~7.56 (m, 2H), 7.46~7.27 (m, 2H), 5.83 (s, 2H), 4.53 (d, J = 5.2 Hz, 2H). HPLC: 98.52% (220 nm), 91.50% (215 nm), 99.46% (254 nm). MS (ESI): C 18 H 15 Calculated mass of Cl3N6O: 400.06, measured m / z: 401.0 [M+H] + .

[0210] compound 114 5-((3,4-Dichlorobenzyl)amino)-1-(pyrimidin-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (54.7 mg, 128.84 μmol, 41.78% yield) as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.73 (d, J = 4.8 Hz, 2H), 7.67 (d, J = 10.0 Hz, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.45~7.35 (m, 2H), 5.85 (s, 2H), 4.57 (d, J = 4.0 Hz, 2H). HPLC: 94.74% (220 nm), 93.84% (215 nm), 98.88% (254 nm). MS (ESI): C 17 H 13 Calculated mass of Cl2N7O: 401.06, measured m / z: 402.0 [M+H] + .

[0211] compound 115 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (420 mg, 947.33 μmol, 68.28% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.61~7.52 (m, 3H), 7.31 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.79 (t, J = 4.4 Hz, 1H), 4.55 (t, J = 5.6 Hz, 2H), 4.46 (d, J = 5.6 Hz, 2H), 3.77 (s, 2H), 3.47~3.43 (m, 4H), 3.41 (d, J = 4.4 Hz, 2H), 3.34~3.30 (m, 2H). HPLC: 99.76 % (220 nm), 99.59% (215 nm), 99.72% (254 nm). MS (ESI): C 18 H 21Calculated mass of Cl2N5O4: 441.10, measured m / z: 442.1 [M+H] + .

[0212] compound 116 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (106.3 mg, 214.99 μmol, 53.25% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.63~7.55 (m, 3H), 7.33 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 6.75 (s, 1H), 4.57 (t, J = 5.6 Hz, 2H), 4.48 (d, J = 5.6 HPLC: 98.36% (220 nm), 97.32% (215 nm), 98.29% (254 nm). MS (ESI): C 20 H 25 Calculated mass of Cl2N5O5: 485.12, measured m / z: 486.1 [M+H] + .

[0213] Compound 117 5-((3,4-Dichlorobenzyl)amino)-1-(2-(pyrimidin-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (9.8 mg, 21.84 μmol, 20.14% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.70 (d, J = 4.8 Hz, 2H), 7.60~7.56 (m, 2H), 7.52~7.48 (m, 1H), 7.37~7.31 (m, 2H), 6.83 (s, 1H), 4.88 (t, J = 7.2 Hz, 2H), 4.48 (d, J = 5.6 Hz, 2H), 3.43~3.41 (m, 2H). HPLC: 92.75% (220 nm), 91.04% (215 nm), 98.20% (254 nm). MS (ESI): C 18 H 15 Calculated mass of Cl2N7O: 415.07, measured m / z: 416.0 [M+H] + .

[0214] compound 118 5-((3,4-Dichlorobenzyl)amino)-1-(oxetan-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (84.1 mg, 207.55 μmol, 24.97% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 10.75 (s, 1H), 7.66~7.53 (m, 3H), 7.32 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 6.59 (s, 1H), 4.72 (d, J = 7.6 Hz, 2H), 4.60 (dd, J = 6.0 Hz, 7.6 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 4.40 (t, J = 6.4 Hz, 2H), 3.45~3.37 (m, 1H). HPLC: 93.84% (220 nm), 88.55% (215 nm), 98.29% (254 nm). MS (ESI): C 16 H 15 Calculated mass of Cl2N5O2: 379.06, measured m / z: 380.0 [M+H] + .

[0215] Compound 119 5-((3,4-Dichlorobenzyl)amino)-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (70.4 mg, 174.59 μmol, 20.01% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.60~7.57 (m, 3H), 7.32 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H), 5.65 (t, J = 4.4 Hz, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.03~3.96 (m, 2H), 3.85~3.81 (m, 2H), 2.37~2.31 (m, 2H). HPLC: 94.30% (220 nm), 91.72% (215 nm), 90.52% (254 nm). MS (ESI): C 16 H 15Calculated mass of Cl2N5O2: 379.06, measured m / z: 380.1 [M+H] + .

[0216] compound 120 5-((3,4-Dichlorobenzyl)amino)-1-((tetrahydrofuran-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (72.6 mg, 181.68 μmol, 21.03% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.65~7.60 (m, 4H), 7.37 (d, J = 8.0 Hz, 1H), 4.57 (s, 2H), 4.46~4.40 (m, 2H), 3.74~3.71 (m, 4H), 2.75~2.72 (m, 1H), 1.88~1.84 (m, 1H), 1.67~1.57 (m, 1H). HPLC: 98.66% (220 nm), 98.29% (215 nm), 98.84% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O2: 393.08, measured m / z: 394.0 [M+H] + .

[0217] compound 121 5-((3,4-Dichlorobenzyl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (102.6 mg, 257.22 μmol, 36.39% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.60~7.56 (m, 3H), 7.32 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 6.65 (s, 1H), 5.13~4.96 (m, 1H), 4.47 (d, J = 5.6 Hz, HPLC: 98.84% (220 nm), 98.20% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O2: 393.08, measured m / z: 394.0 [M+H] + .

[0218] compound 122 5-((3,4-Dichlorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (8.0 mg, 19.89 μmol, 11.97% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ7.65~7.53 (m, 3H), 7.32 (d, J = 7.6 Hz, 1H), 6.60 (s, 1H), 5.02~4.95 (m, 1H), 4.76~4.69 (m, 1H), 4.65~4.56 (m, 1H), 4.51~4.41 (m, 3H), 4.36~4.29 (m, 1H), 2.65~2.57 (m, 2H). HPLC: 94.54% (220 nm), 93.72% (215 nm), 98.31% (254 nm). MS (ESI): C 16 H 15 Calculated mass of Cl2N5O2: 379.06, measured m / z: 380.0 [M+H] + .

[0219] compound 123 5-((3,4-Dichlorobenzyl)amino)-1-(3-hydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (27.8 mg, 75.50 μmol, 19.18% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.07 (s, 1H), 7.59~7.57 (m, 2H), 7.53 (s, 1H), 7.32 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.59~6.50 (m, 1H), 4.54~4.44 (m, 4H), 3.38 (t, J = 6.0 Hz, 2H), 1.94~1.87 (m, 2H). HPLC: 100.00% (220 nm), 83.10% (215 nm), 98.33% (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O2: 367.06, measured m / z: 368.0 [M+H] + .

[0220] compound 124 5-((3,4-Dichlorobenzyl)amino)-1-(2-(pyrazin-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (24.1 mg, 49.93 μmol, 19.19% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.51 (d, J = 2.8 Hz, 1H), 8.45 (d, J = 2.0 Hz, 1H), 8.40 (s, 1H), 7.60~7.58 (m, 2H), 7.52 (s, 1H), 7.33 (dd, J = 8.4 Hz, J = HPLC: 93.79% (220 nm), 91.00% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 16 Calculated mass of Cl3N7O: 415.07, measured m / z: 416.0 [M+H] + .

[0221] compound 125 5-((3,4-Difluorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (18.3 mg, 52.58 μmol, 50.62% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.00 (s, 1H), 7.59 (s, 1H), 7.42~7.33 (m, 2H), 7.18 (s, 1H), 6.53 (s, 1H), 5.03~4.93 (m, 1H), 4.79~4.69 (m, 1H), 4.66~4.57 (m, 1H), 4.48~4.42 (m, 3H), 4.36~4.30 (m, 1H), 2.63~2.58 (m, 1H), 2.42~2.37 (m, 1H). HPLC: 99.80% (220 nm), 99.76% (215 nm), 100.00% (254 nm). MS (ESI): C 16 H 15 Calculated mass of F2N5O2: 347.12, measured m / z: 348.2 [M+H] + .

[0222] compound 126 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0223] Preparation of 5-chloro-7-methoxy-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine (Step 1 in Scheme C-3) [ka] To a mixture of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (1 g, 5.42 mmol, 1 equiv.) and 1-bromo-2-methoxyethane (978.90 mg, 7.04 mmol, 661.42 μL, 1.3 equiv.) in DMF (10 mL) was added CsCO (3.53 g, 10.84 mmol, 2 equiv.) at 20 °C. The mixture was then stirred at 20 °C for 3 h. TLC showed the reaction was complete. The mixture was slowly poured into ice-water (20 mL) and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (10 mL × 4), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 12 g, elution with a 0-35% ethyl acetate / petroleum ether gradient at 40 mL / min). The eluent was removed under reduced pressure. The compound 5-chloro-7-methoxy-2-(2-methoxyethyl)pyrazolo[4,3-d]pyrimidine (0.6 g, 2.47 mmol, 45.64% yield) was obtained as a white solid. 1 H NMR (CDCl 3, 400 MHz) δ 8.05 (s, 1H), 4.71 (t, J = 5.6 Hz, 2H), 4.23 (s, 3H), 3.82 (t, J = 5.6 Hz, 2H), 3.29 (s, 3H). The compound 5-chloro-7-methoxy-1-(2-methoxyethyl)pyrazolo[4,3-d]pyrimidine (0.5 g, 2.06 mmol, 38.033% yield) was obtained as a white solid. 1 H NMR (CDCl 3, 400 MHz) δ 8.09 (s, 1H), 4.58 (t, J = 5.2 Hz, 2H), 4.23 (s, 3H), 3.85 (t, J = 5.6 Hz, 2H), 3.32 (s, 3H).

[0224] Preparation of 5-chloro-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme C-3) [ka] A solution of 5-chloro-7-methoxy-1-(2-methoxyethyl)pyrazolo[4,3-d]pyrimidine (0.6 g, 2.47 mmol, 1 equiv.) and LiOH·HO (311.25 mg, 7.42 mmol, 3 equiv.) in MeOH (5 mL) and HO (5 mL) was stirred at 20 °C for 3 h. TLC showed the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was slowly adjusted to pH 6–7 with 2 N HCl, and some solid was formed. The solid was collected after filtration. The aqueous solution was then extracted with EtOAc (20 mL × 2). The combined organic layers were washed with brine (5 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The compound 5-chloro-1-(2-methoxyethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.5 g, 2.19 mmol, 88.45% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 13.30 (s, 1H), 7.98 (s, 1H), 4.68 (t, J = 5.2 Hz, 2H), 3.75 (t, J = 5.6 Hz, 2H), 3.19 (s, 3H).

[0225] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 3 in Scheme C-3) [ka] A solution of 5-chloro-1-(2-methoxyethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (500.00 mg, 2.19 mmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (769.97 mg, 4.37 mmol, 583.31 μL, 2 equiv.) in t-BuOH (5 mL) was heated at 100 °C for 30 h. TLC indicated the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 250 mm × 50 mm 10 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 25% to 55%, 10 min). The compound 5-[(3,4-dichlorophenyl)methylamino]-1-(2-methoxyethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (510.7 mg, 1.38 mmol, 63.00% yield, 99.34% purity) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.63~7.59 (m, 3H), 7.35 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 4.59 (t, J = 5.6 Hz, 2H), 4.54 (d, J = 8.8 Hz, 2H), 3.72 (t, J = 5.2 Hz, 2H), 3.19 (s, 3H). HPLC: 99.34% (220 nm), 99.77% (215 nm), 97.94 % (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O2: 367.06, measured m / z: 368.0 [M+H] + .

[0226] compound 127 1-Benzyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (119.5 mg, 294.94 μmol, 38.44% yield) as an off-white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.62 (s, 1H), 7.60~7.57 (m, 2H), 7.34~7.24 (m, 4H), 7.21 (d, J = 6.8 Hz, 2H), 6.90 (s, 1H), 5.62 (s, 2H), 4.49 (d, J = 5.6 Hz, 2H). HPLC: 98.79% (220 nm), 98.73% (215 nm), 99.28 % (254 nm). MS (ESI): C 19 H 15 Calculated mass of Cl2N5O: 399.07, measured m / z: 400.0 [M+H] + .

[0227] compound 128 5-((3,4-Dichlorobenzyl)amino)-2-(2-methoxyethyl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (161.8 mg, 439.41 μmol, 66.98% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.85 (s, 1H), 7.61~7.59 (m, 2H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.10 (s, 1H), 4.49 (t, J = 5.6 Hz, 2H), 4.38 (t, J = 5.2 Hz, 2H), 3.80 (t, J = 6.8 Hz, 2H), 3.22 (s, 3H). HPLC: 98.12% (220 nm), 98.18% (215 nm), 100.00 % (254 nm). MS (ESI): C 15 H 15Calculated mass of Cl2N5O2: 367.06, measured m / z: 368.1 [M+H] + .

[0228] compound 129 2-Benzyl-5-((3,4-dichlorobenzyl)amino)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (49.5 mg, 123.67 μmol, 21.49% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.99 (s, 1H), 7.98 (s, 1H), 7.60~7.58 (m, 2H), 7.36~7.33 (m, 2H), 7.32~7.28 (m, 4H), 6.91 (s, 1H), 5.43 (s, 2H), 4.48 (d, J=5.6 Hz, 2H). HPLC: 98.07% (220 nm), 97.98% (215 nm), 99.17 % (254 nm). MS (ESI): C 19 H 15 Calculated mass of Cl2N5O: 399.07, measured m / z: 400.1 [M+H] + .

[0229] compound 130 5-((3,4-Dichlorobenzyl)amino)-1-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (77.2 mg, 219.18 μmol, 51.27% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.60~7.57 (m, 3H), 7.33 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 6.96 (s, 1H), 5.22~5.19 (m, 1H), 4.49 (d, J = 5.2 Hz, 2H), 1.41 (s, 6H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O: 351.07, measured m / z: 352.1 [M+H] + .

[0230] Compound 131 5-((3,4-Dichlorobenzyl)amino)-1-(5-methoxypentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (73.6 mg, 175.20 μmol, 18.97% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.60~7.56 (m, 2H), 7.55 (s, 1H), 7.33 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 6.71 (s, 1H), 4.48 (d, J = 6.0 Hz, 2H), 4.41 (t, J=6.8 Hz, 2H), 3.25 (t, J = 6.4 Hz, 2H), 3.17 (s, 3H), 1.79~1.75 (m, 2H), 1.50~1.44 (m, 2H), 1.24~1.18 (m, 2H). HPLC: 97.67% (220 nm), 97.65% (215 nm), 98.85% (254 nm). MS (ESI): C 18 H 21Calculated mass of Cl2N5O2: 409.11, measured m / z: 410.0 [M+H] + .

[0231] compound 132 5-((3,4-Dichlorobenzyl)amino)-2-isopropyl-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (48.0 mg, 136.28 μmol, 31.88% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.86 (s, 1H), 7.59~7.57 (m, 2H), 7.31 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.56~6.48 (m, 1H), 4.59~4.55 (m, 1H), 4.44 (d, J = 6.0 Hz, 2H), 1.43 (d, J = 6.8 Hz, 6H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 15 H 15 Calculated mass of Cl2N5O: 351.07, measured m / z: 352.1 [M+H] + .

[0232] compound 133 5-((3,4-Dichlorobenzyl)amino)-2-(5-methoxypentyl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (68.3 mg, 166.46 μmol, 18.03% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 10.75 (s, 1H), 7.83 (s, 1H), 7.62~7.55 (m, 2H), 7.32 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.62 (s, 1H), 4.46 (d, J = 6.0 Hz, 2H), 4.19 (t, J = 7.2 Hz, 2H), 3.27 (t, J = 6.4 Hz, 2H), 3.18 (s, 3H), 1.85~1.79 (m, 2H), 1.51~1.46 (m, 2H), 1.26~1.20 (m, 2H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 21 Calculated mass of Cl2N5O2: 409.11, measured m / z: 410.0 [M+H] + .

[0233] compound 134 1-(Cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (150 mg, 396.56 μmol, 52.58% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.63~7.59 (m, 3H), 7.35 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.54 (d, J = 4.8 Hz, 2H), 4.46 (d, J = 7.6 Hz, 2H), 2.75 (td, J = 7.6 Hz, 15.2 Hz, 1H), 1.96~1.87 (m, 2H), 1.85~1.72 (m, 4H). HPLC: 96.51% (220 nm), 96.27% (215 nm), 93.88 % (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O: 377.08, measured m / z: 378.1 [M+H] + .

[0234] compound 135 2-(Cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (141.9 mg, 375.14 μmol, 59.69% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.12~10.37 (m, 1H), 7.83 (s, 1H), 7.60~7.58 (m, 2H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.86 (s, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.23 (d, J = 7.2 Hz, 2H), 2.82~78 (m, 1H), 2.00~1.93 (m, 2H), 1.86~1.73 (m, 4H). HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00 % (254 nm).MS (ESI): C 17 H 17Calculated mass of Cl2N5O: 377.08, measured m / z: 378.1 [M+H] + .

[0235] compound 136 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-methoxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (61.5 mg, 149.17 μmol, 30.36% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 9.48 (s, 1H), 7.60~7.54 (m, 3H), 7.32 (dd, J = 2.0, 8.2 Hz, 1H), 6.76~6.59 (m, 1H), 4.56 (t, J = 5.8 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 3.78 (t, J = 5.8 Hz, 2H), 3.46 (dd, J = 3.2 Hz, 4.4 Hz, 2H), 3.34 (dd, J = 3.8 Hz, 5.8 Hz, 2H), 3.16 (s, 3H). HPLC: 98.08% (220 nm),97.73% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl2N5O3: 411.09, measured m / z: 412.1 [M+H] + .

[0236] compound 137 5-((3,4-Dichlorobenzyl)amino)-2-(2-(2-methoxyethoxy)ethyl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (117.3 mg, 284.52 μmol, 54.26% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.84 (s, 1H), 7.62~7.58 (m, 2H), 7.34 (dd, J = 2.0 Hz, 6.4 Hz, 1H), 4.49 (d, J = 5.2 Hz, 2H), 4.37 (t, J = 5.2 HPLC: 99.87% (220 nm), 99.84% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl2N5O3: 411.09, measured m / z: 412.1 [M+H] + .

[0237] compound 138 5-((3,4-Dichlorobenzyl)amino)-1-((2-methoxyethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (83.7 mg, 209.23 μmol, 41.63% yield) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.70 (s, 1H), 7.60~7.57 (m, 2H), 7.34~7.32 (m, 1H), 6.73 (s, 1H), 5.70 (s, 2H), 4.49 (d, J = 5.6 Hz, 2H), 3.58 (t, J = 4.4 Hz, 2H), 3.36 (t, J = 5.2 Hz, 2H), 3.18 (s, 3H). HPLC: 99.55% (220 nm), 99.51% (215 nm), 100% (254 nm). MS (ESI): C 16 H 17 Calculated mass of Cl2N5O3: 397.07, measured m / z: 398.1 [M+H] + .

[0238] compound 139 5-((3,4-Dichlorobenzyl)amino)-1-(pyrimidin-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (10.9 mg, 28.08 μmol, 3.88% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.99 (s, 1H), 8.94 (d, J = 4.8 Hz, 2H), 8.59 (s, 1H), 7.63~7.56 (m, 3H), 7.36 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 6.74 (s, 1H), 4.52 (d, J = 5.6 Hz, 2H). HPLC: 96.85% (220 nm), 96.29% (215 nm), 99.83% (254 nm). MS (ESI): C 16 H 11 Calculated mass of Cl2N7O: 387.04, measured m / z: 388.1 [M+H] + .

[0239] compound 140 5-((3,4-Dichlorobenzyl)amino)-2-(pyrimidin-2-yl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (8 mg, 19.56 μmol, 12.16% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 11.21 (s, 1H), 8.93 (d, J = 4.8 Hz, 2H), 7.98 (s, 1H), 7.62~7.57 (m, 3H), 7.35 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.82~6.72 (m, 1H), 4.53 (d, J = 6.0 Hz, 2H). HPLC: 94.91% (220 nm), 94.48% (215 nm), 99.09% (254 nm). MS (ESI): C 16 H 11 Calculated mass of Cl2N7O: 387.04, measured m / z: 388.0 [M+H] + .

[0240] compound 141 2-(2-(5-((3,4-Dichlorobenzyl)amino)-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)ethoxy)acetic acid was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (6.1 mg, 14.08 μmol, 5.48% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.14 (s, 1H), 10.99 (s, 1H), 8.24 (t, J = 6.0 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 1.6 Hz, 1H), 7.37 (s, 1H), 7.24~7.14 (m, 1H), 4.61 (t, J = 5.6 Hz, 2H), 4.25 (d, J = 6.0 Hz, 2H), 3.89 (s, 2H), 3.85 (t, J = 5.6 Hz, 2H). HPLC: 95.12% (220 nm), 94.13% (215 nm), 87.14% (254 nm).MS (ESI): C 16 H 15 Calculated mass of Cl2N5O4: 411.05, measured m / z: 412.0 [M+H] + .

[0241] compound 142 5-((3,4-Dichlorobenzyl)amino)-1-(oxetan-3-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (19.6 mg, 50.10 μmol, 45.42% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.73 (s, 1H), 7.59~7.57 (m, 2H), 7.32 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.64 (s, 1H), 6.11~6.04 (m, 1H), 4.95~4.89 (m, 4H), 4.47 (d, J = 6.0 Hz, 2H). HPLC: 93.61 % (220 nm), 91.82 % (215 nm), 97.14 % (254 nm). MS (ESI): C 15 H 13Calculated mass of Cl2N5O2: 365.04, measured m / z: 366.0 [M+H] + .

[0242] compound 143 5-((3,4-Dichlorobenzyl)amino)-2-(oxetan-3-yl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (16 mg, 41.96 μmol, 36.57% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.00 (s, 1H), 7.60~7.58 (m, 2H), 7.33 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.88 (s, 1H), 5.70~5.63 (m, 1H), 4.97~4.93 (m, 2H), 4.91~4.88 (m, 2H), 4.48 (d, J = 5.6 Hz, 2H). HPLC: 96.04 % (220 nm), 94.85 % (215 nm), 95.47 % (254 nm). MS (ESI): C 15 H 13 Calculated mass of Cl2N5O2: 365.04, measured m / z: 366.0 [M+H] + .

[0243] compound 144 5-((3,4-Dichlorobenzyl)amino)-1-(oxazol-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (34.8 mg, 88.81 μmol, 37.24% yield) as a white solid.1 H NMR (DMSO-d6, 400 MHz) δ 8.06 (s, 1H), 7.65 (s, 1H), 7.63~7.56 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 6.83 (s, 1H), 5.79 (s, 2H), 4.49 (d, J = 4.6 Hz, 2H). HPLC: 99.83 % (220 nm), 99.77 % (215 nm), 99.88% (254 nm). MS (ESI): C 16 H 12 Calculated mass of Cl2N6O2: 390.04, measured m / z: 391.0 [M+H] + .

[0244] compound 145 4-(5-((3,4-Dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butanoic acid was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (97.9 mg, 223.38 μmol, 38.22% yield, 90.407% purity) as a white solid. 7.9 mg of the product was carried forward. 1 H NMR (DMSO-d6, 400 MHz) δ 11.10 (s, 1H), 7.62~7.51 (m, 3H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.62 (s, 1H), 4.52~4.39 (m, 4H), 2.15 (t, J = 7.2 Hz, 2H), 2.02~1.95 (m, 2H). HPLC: 90.41 % (220 nm), 89.23 % (215 nm), 96.19% (254 nm). MS (ESI): C 16 H 15 Calculated mass of Cl2N5O3: 396.06, measured m / z: 396.2 [M+H] + .

[0245] compound 146 5-((3,4-Dichlorobenzyl)amino)-1-(4-(dimethylphosphoryl)butyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound (4.4 mg, 9.55 μmol, 9.64% yield, 96.037% purity) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.66~7.49 (m, 3H), 7.33 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 6.68 (s, 1H), 4.50~4.38 (m, 4H), 1.89~1.82 (m, 2H), 1.71~1.59 (m, 2H), 1.44~1.34 (m, 2H), 1.30 (d, J = 12.8 Hz, 6H).

[0246] compound 147 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((4-methylmorpholin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0247] compound 148 Preparation of tert-butyl 2-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)morpholine-4-carboxylate (Steps 1-3 in Scheme C-3) [ka] A mixture of tert-butyl 2-[(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]morpholine-4-carboxylate (1 g, 1.08 mmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (380.84 mg, 2.16 mmol, 288.51 μL, 2 equiv.) in t-BuOH (10 mL) was stirred at 100° C. for 12 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove t-BuOH. The residue was diluted with EtOAc (10 mL) and washed with aqueous HCl (2 N, 10 mL × 6). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The compound tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]morpholine-4-carboxylate (1 g, crude) was obtained as a white solid. The residue (200 mg) was purified by preparative HPLC (neutral condition column: Welch Xtimate C18 150 mm × 25 mm 5 μm; mobile phase: [water (10 mM NH4HCO3)-MeCN]; B%: 50% to 70%, 10.5 min). The solvent was removed by lyophilization. The compound tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]morpholine-4-carboxylate (23.4 mg, 45.88 μmol, 4.24% yield, 99.87% purity) was obtained as a white solid and used further. 1 H NMR (DMSO-d 6,400 MHz) δ 11.03 (s, 1H), 7.60~7.57 (m, 3H), 7.33 (d, J = 8.0 Hz, 1H), 6.60 (s, 1H), 4.58~4.55 (m, 1H), 4.48~4.41 (m, 3H), 3.78~3.75 (m, 2H), 3.67~3.62 (m, 2H), 3.31~3.28 (m, 1H), 2.88 (s, 1H), 2.67 (s, 1H), 1.36 (s, 9H). HPLC: 99.87 % (220 nm), 99.88 % (215 nm), 100.00 % (254 nm). MS (ESI): C 22 H 26 Calculated mass for Cl2N6O4: 508.14, measured m / z: 509.2 [M+H] + .

[0248] compound 149 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(morpholin-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] A solution of tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]morpholine-4-carboxylate (200 mg, 392.63 μmol, 1 equiv.) in HCl / EtOAc (2 mL) and EtOAc (1 mL) was stirred at 25°C for 2 h. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove EtOAc. The residue was purified by preparative HPLC (HCl condition column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 50%, 8 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-(morpholin-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (35.4 mg, 79.42 μmol, 20.23% yield, 100% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 9.33 (s, 2H), 7.67 (s, 1H), 7.63~7.59 (m, 2H), 7.36 (d, J = 8.0 Hz, 1H), 4.67~4.62 (m, 1H), 4.55~4.48 (m, 3H), 4.18~4.17 (m, 1H), 3.92~3.89 (m, 1H), 3.65 (t, J = 12.0 Hz, 1H), 3.21~3.12 (m, 2H), 2.90~2.87 (m, 2H). HPLC: 100.00 % (220 nm), 100.00% (215 nm), 100.00% (254 nm).MS (ESI): C 17 H 19 Calculated mass of Cl3N6O2: 408.09, measured m / z: 409.1 [M+H] + .

[0249] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((4-methylmorpholin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-(morpholin-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (400 mg, 897.40 μmol, 1 equiv., HCl) in MeOH (5 mL) was added HCHO (218.48 mg, 2.69 mmol, 200.44 μL, 37% purity, 3 equiv.) at 0° C. The mixture was stirred at 0° C. for 10 minutes. Then, AcOH (5.39 mg, 89.74 μmol, 5.13 μL, 0.1 equiv.) and NaBHCN (451.16 mg, 7.18 mmol, 8 equiv.) were added at 0° C. The mixture was stirred at 25° C. for 12 hours. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl condition: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 45%, 10 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[(4-methylmorpholin-2-yl)methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (49.1 mg, 106.69 μmol, 11.89% yield, 99.9% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 11.33 (s, 1H), 8.37 (s, 1H), 7.74 (s, 1H), 7.68 (d, J = 1.2 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 8.4 Hz, 1.2 Hz, 1H), 4.67~4.62 (m, 3H), 4.57~4.53 (m, 1H), 4.27~4.26 (m, 1H), 3.98~3.94 (m, 1H), 3.74 (t, J = 12.0 Hz, 1H), 3.39 (d, J = 12.0 Hz, 1H), 3.30 (d, J = 12.0 Hz, 1H), 2.98~2.90 (m, 2H), 2.74 (m, 3H). HPLC: 99.90 % (220 nm), 99.85 % (215 nm), 99.57 % (254 nm). MS (ESI): C 18 H 21 Calculated mass of Cl3N6O2: 422.10, measured m / z: 423.0 [M+H] + .

[0250] compound 150 Preparation of 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-1-isonicotinoylpyrrolidine-2-carboxylic acid hydrochloride

[0251] Preparation of 1-(tert-butoxycarbonyl)-4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pyrrolidine-2-carboxylic acid (Steps 1-3 in Scheme C-3) [ka]

[0252] A solution of 1-tert-butoxycarbonyl-4-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)pyrrolidine-2-carboxylic acid (520 mg, 1.35 mmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (477.05 mg, 2.71 mmol, 361.40 μL, 2 equiv.) in 2-methylbutan-2-ol (3 mL) was stirred at 130 °C for 10 h. LCMS and HPLC indicated the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 30% to 50%, 12 min). The mixture was dried under lyophilization to give 1-(tert-butoxycarbonyl)-4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pyrrolidine-2-carboxylic acid (140 mg, 267.50 μmol, 19.74% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.62 (s, 1H), 7.59~7.53 (m, 2H), 7.30 (d, J = 8.8 Hz, 1H), 5.67~5.52 (m, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.37~4.30 (m, 1H), 3.78~3.74 (m, 1H), 3.63~3.55 (m, 1H), 2.82~2.69 (m, 1H), 2.38~2.31 (m, 1H), 1.39~1.25 (m, 9H).

[0253] Compound 151 Preparation of 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pyrrolidine-2-carboxylic acid hydrochloride (Step 1 in Scheme C-3) [ka] To a solution of 1-tert-butoxycarbonyl-4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]pyrrolidine-2-carboxylic acid (140 mg, 267.50 μmol, 1 equiv.) in EtOAc (3 mL) was added HCl / EtOAc (4 M, 3 mL) at 0°C. The mixture was stirred at 25°C for 4 hours. LCMS and HPLC indicated the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 50%, 12 min). The mixture was dried under lyophilization to give 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pyrrolidine-2-carboxylic acid hydrochloride (120 mg, 249.49 μmol, 93.27% yield, 95.577% purity, HCl) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 10.02 (s, 1H), 9.13 (s, 1H), 7.72 (s, 1H), 7.64~7.53 (m, 2H), 7.33 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 6.95 (s, 1H), 5.79 HPLC: 95.58% (220 nm), 94.08% (215 nm), 94.72% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl3N6O3: 422.07, measured m / z: 423.1 [M+H] + .

[0254] Preparation of 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-1-isonicotinoylpyrrolidine-2-carboxylic acid hydrochloride (Step 2 in Scheme C-3) [ka] To a solution of 4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]pyrrolidine-2-carboxylic acid (70 mg, 152.27 μmol, 1 equiv., HCl) in DCM (3 mL) was added TEA (61.63 mg, 609.07 μmol, 84.78 μL, 4 equiv.). Pyridine-4-carbonyl chloride (70.48 mg, 395.90 μmol, 2.6 equiv., HCl) was then added portionwise to the mixture at 0° C. The mixture was stirred at 25° C. for 3 hours. LCMS indicated the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (Phenomenex Luna C18 150 mm × 30 mm 5 μm column; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 40%, 12 min). The mixture was lyophilized to dryness to give 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-1-isonicotinoylpyrrolidine-2-carboxylic acid hydrochloride (25.0 mg, 43.68 μmol, 28.69% yield, 98.683% purity, HCl) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.89 (d, J = 6.4 Hz, 2H), 7.92 (s, 1H), 7.88~7.73 (m, 2H), 7.72~7.66 (m, 1H), 7.65~7.59 (m, 2H), 7.42~7.33 (m, 1H), 5.75~5.64 (m, 1H), 4.72 (t, J = 8.0 Hz, 1H), 4.64~4.57 (m, 2H), 4.08 (s, 1H), 3.79~3.78 (m, 1H), 2.86~2.80 (m, 1H), 2.60 (d, J = 6.4 Hz, 1H). HPLC: 98.68% (220 MS (ESI): C 23 H 20 Calculated mass for Cl3N7O4: 527.09, measured m / z: 528.1 [M+H] + .

[0255] compound 152 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0256] compound 153 tert-Butyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)piperidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)piperidine-1-carboxylate (33.7 mg, 65.68 μmol, 46.48% yield, 96.163% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.59~7.57 (m, 3H), 7.32 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 6.76 (s, 1H), 5.02~4.98 (m, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.05 (br d, J = 13.2 Hz, 2H), 2.92~2.89 (m, 2H), 1.92~1.85 (m, 4H), 1.42 (s, 9H). HPLC: 96.16% (220 nm), 96.40% (215 nm), 100.00% (254 nm). MS (ESI): C 22 H 26 Calculated mass of Cl2N6O3: 492.14, measured m / z: 493.2 [M+H] + .

[0257] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka] The compound 5-((3,4-dichlorobenzyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (1 g, 2.33 mmol, 39.64% yield, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.96 (s, 1H), 8.74 (s, 1H), 7.65 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.8 Hz, 1H), 7.33~7.29 (m, 1H), 5.14 (s, 1H), 4.52 (d, J = 5.2 Hz, 2H), 3.41-3.37 (m, 2H), 3.14~3.10 (m, 2H), 2.24~2.10 (m, 4H). HPLC: 96.39% (220 nm), 96.35% (215 nm), 96.48% (254 nm). MS (ESI): C 17 H 18Calculated mass of Cl2N6O: 392.09, measured m / z: 393.1 [M+H] + .

[0258] compound 154 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(piperidin-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0259] tert-Butyl 3-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl 3-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (430 mg, crude) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.60 (d, J = 2.4 Hz, 2H), 7.58 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 4.50 (d, J = 5.2 Hz, 2H), 4.32 (d, J = 7.2 Hz, 2H), 3.69 (s, 2H), 2.89~2.68 (m, 1H), 2.60~2.53 (m, 1H), 1.98 (s, 2H), 1.60 (d, J = 10.0 Hz, 3H), 1.30 (br s, 9H). 5-((3,4-Dichlorobenzyl)amino)-1-(piperidin-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 1 in Scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(piperidin-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (500 mg, crude) as a white solid. 80 mg (crude) was purified by preparative HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 45%, 10 min) to obtain 20.9 mg of pure product, which was used for further purification. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.86 (d, J = 6.8 Hz, 1H), 8.55 (br s, 1H), 7.62 (t, J = 13.2 Hz, 3H), 7.37 (d, J = 8.4 Hz, 1H), 4.54 (d, J = 5.2 Hz, 2H), 4.43~4.39 (m, 2H), 3.19~3.02 (m, 2H), 2.81~2.67 (m, 2H), 2.31~2.26 (m, 1H), 1.77 (d, J = 12.8 Hz, 1H), 1.66~1.58 (m, 2H), 1.27~1.18 (m, 1H). HPLC: 99.10% (220 nm), 98.78% (215 nm), 98.43% (254 nm). MS (ESI): C 18 H 21 Calculated mass of Cl3N6O: 406.11, Measured m / z: 407.11 [M+H] + .

[0260] compound 155 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(pyrrolidin-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0261] tert-Butyl 3-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl 3-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]pyrrolidine-1-carboxylate (220 mg, 445.90 μmol, 43.82% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.10 (s, 1H), 7.63~7.50 (m, 3H), 7.33 (d, J = 8.4 Hz, 1H), 6.56 (t, J = 6.0 Hz, 1H), 4.51~4.38 (m, 4H), 3.31~3.28 (m, 2H), 3.28~3.23 (m, 1H), 3.19 (s, 1H), 3.04 (br s, 1H), 1.80 (br s, 1H), 1.58 (d, J = 4.0 Hz, 1H), 1.37 (s, 9H).

[0262] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(pyrrolidin-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] To a solution of tert-butyl 3-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]pyrrolidine-1-carboxylate (compound 165) (220 mg, 445.90 μmol, 1 equiv.) in 2 mL of EtOAc was added HCl / EtOAc (4 M, 2 mL, 17.94 equiv.) at 0 °C. The mixture was stirred at 25 °C for 4 h. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 35%, 12 min). The mixture was lyophilized to give 5-[(3,4-dichlorophenyl)methylamino]-1-(pyrrolidin-3-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (180 mg, 418.87 μmol, 93.94% yield, HCl) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 9.06 (br s, 2H), 7.67~7.57 (m, 3H), 7.34 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 4.60~4.41 (m, 4H), 3.18 (br s, 2H), 3.09~3.07 (m, HPLC: 99.50% (220 nm), 99.24% (215 nm), 99.65% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl3N6O: 392.09, measured m / z: 393.1 [M+H] + .

[0263] compound 156 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-3-ylsulfonyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] To a mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-(4-piperidyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (80 mg, 186.16 μmol, 1 equiv., HCl) and TEA (75.35 mg, 744.65 μmol, 103.65 μL, 4 equiv.) in DCM (3 mL) was added pyridine-3-sulfonyl chloride (33.06 mg, 186.16 μmol, 8.53 μL, 1 equiv.) dropwise at 0° C. The mixture was then stirred at 20° C. for 1 h. LC-MS showed the reaction was complete. The mixture was quenched with ice water (5 mL), and the organic layer was separated. The aqueous solution was extracted with DCM (5 mL × 4). The combined organic layers were washed with brine (2 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 mm x 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 45% to 70%, 10 min). The eluate was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(3-pyridylsulfonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (26.7 mg, 46.07 μmol, yield 24.75%, purity 98.51%, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.96 (s, 1H), 8.92 (d, J = 4.0 Hz, 1H), 8.23~8.20 (m, 1H), 7.74~7.71 (m, 1H), 7.61~7.58 (m, 3H), 7.33 (dd, J = 2.0 Hz, HPLC: 98.51% (220 nm), 98.36% (215 nm), 95.74% (254 nm). MS (ESI): C22 H 22 Calculated mass of Cl3N7O3S: 533.08 m / z Measured: 354.2 [M+H] + .

[0264] compound 157 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(1-(2-methoxyethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka]

[0265] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-(4-piperidyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one hydrochloride (60 mg, 139.62 μmol, 1 equiv., HCl) and 1-bromo-2-methoxyethane (58.22 mg, 418.87 μmol, 39.34 μL, 3 equiv.) in CHCN (1 mL) was added KCO (57.89 mg, 418.87 μmol, 3 equiv.). The mixture was stirred at 80 °C for 16 h. LCMS and HPLC showed the reaction was complete. The mixture was filtered. The filtrate was purified by preparative HPLC (Phenomenex Luna C18 100 mm × 30 mm 5 μm column; mobile phase: [water (0.05% HCl)-MeCN]; B%: 20%–40%, 10 min). The mixture was lyophilized to give 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(2-methoxyethyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (18.6 mg, 40.51 μmol, 29.01% yield, 98.295% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 10.19 (s, 1H), 7.65 (s, 1H), 7.61~7.56 (m, 2H), 7.48 (s, 1H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 5.13~5.06 (m, 1H), 4.53 (d, J = 5.2 Hz, 2H), 3.73~3.67 (m, 2H), 3.59 (d, J = 12.0 Hz, 2H), 3.40 (s, 1H), 3.30 (s, 3H), 3.28 (d, J = 5.6 Hz, 2H), 3.20 (s, 1H), 2.41~2.31 (m, 2H), 2.16~2.13 (m, 2H). HPLC: 98.30% (220 nm), 98.30% (215 nm), 98.14% (254 nm). MS (ESI): C 20 H 25 Calculated mass of Cl3N6O2: 486.11, measured m / z: 451.2 [M+H] + .

[0266] compound 158 5-((3,4-Dichlorobenzyl)amino)-1-(2-morpholino-2-oxoethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholino-2-oxoethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (16.8 mg, 37.65 μmol, 17.24% yield, 98.005% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.64~7.59 (m, 4H), 7.36 (d, J = 6.8, 1H), 5.38 (s, 2H), 5.57 (s, 2H), 3.65~3.56 (m, 4H), 3.42 (s, 4H). HPLC: 98.01% (220 nm), 97.95% (215 nm), 98.09% (254 nm). MS (ESI): C 18 H 18 Calculated mass of Cl2N6O3: 436.08, measured m / z: 437.1 [M+H] + .

[0267] compound 159 5-((3,4-Dichlorobenzyl)amino)-1-((1-methyl-5-oxopyrrolidin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-((1-methyl-5-oxopyrrolidin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (56.2 mg, 132.45 μmol, 30.33% yield, 99.285% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.13 (s, 1H), 7.72~7.66 (m, 2H), 7.63 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 4.64 (d, J = 3.6 Hz, 2H), 4.50 (d, J = 7.2 Hz, 2H), 3.37~3.33 (m, 1H), 3.18~3.14 (m, 1H), 2.92~2.80 (m, 1H), 2.67 (s, 3H), 2.35~2.29 (m, 1H), 2.12~2.09 (m, 1H). HPLC: 99.29% (220 nm), 99.21% (215 nm), 98.28% (254 nm). MS (ESI): C 18 H 18 Calculated mass of Cl2N6O2: 420.09, measured m / z: 421.1 [M+H] + .

[0268] compound 160 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-morpholinoethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-morpholinoethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (54.9 mg, 105.69 μmol, 17.32% yield, 96.991% purity, HCl) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 10.50 (s, 1H), 7.65~7.58 (m, 3H), 7.36 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 4.63 (t, J = 5.2 Hz, 2H), 4.56 (d, J = 4.8 Hz, 2H), 3.87~3.81 (m, 4H), 3.75 (t, J = 4.4 Hz, 2H), 3.66 (t, J = 12.0 Hz, 2H), 3.27~3.20 (m, 4H), 3.01~2.92 (m, 2H). HPLC: 96.99% (220 nm), 94.02% (215 nm), 99.79% (254 nm).MS (ESI): C 20 H 25 Calculated mass of Cl3N6O3: 466.13, measured m / z: 467.1 [M+H] + .

[0269] compound 161

[0270] 5-((3,4-Dichlorobenzyl)amino)-1-(1-(pyridin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride (40.9 mg, 85.66 μmol, 14.17% yield, 98.510% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.02 (s, 1H), 8.00~7.97 (m, 1H), 7.61 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.34 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 6.94 (t, J = 6.8 Hz, 1H), 5.28~5.16 (m, 1H), 4.53 (d, J = 5.2 Hz, 2H), 4.38 (d, J = 13.6 Hz, 2H), 3.50~3.37 (m, 2H), 2.15~2.04 (m, 4H). HPLC: 98.51% (220 nm), 98.10% (215 nm), 99.43% (254 nm). MS (ESI): C 22 H 23 Calculated mass of Cl4N7O: 469.12, measured m / z: 470.1 [M+H] + .

[0271] compound 162 5-((3,4-Dichlorobenzyl)amino)-1-(1-(pyridin-4-yl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(4-pyridyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (17.6 mg, 37.42 μmol, 10.31% yield, 95.01% purity, HCl) as a pale yellow solid. 1 H NMR (DMSO-d 6,400 MHz) δ 13.55 (s 1H), 8.26~8.25 (m, 2H), 7.63~7.59 (m, 3H), 7.50 (s 1H), 7.35 (d, J = 6.8 Hz, 1H), 7.27 (d, J = 7.2 Hz, 2H), 5.29~5.23 (m, 1H), 4.55 (d, J = 5.2 Hz, 2H), 4.35 (d, J = 14.0 Hz, 2H), 3.48~3.39 (m, 2H), 2.14~2.02 (m, 4H). HPLC: 95.01 % (220 nm), 90.40% (215 nm), 92.03% (254 nm). MS (ESI): C 22 H 23 Calculated mass of Cl4N7O: 469.12, measured m / z: 470.1 [M+H] + .

[0272] compound 163 5-((3,4-Dichlorobenzyl)amino)-1-(1-(pyridin-3-yl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-3-yl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride (70.0 mg, 144.42 μmol, 25.01% yield, 97.038% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.53 (d, J = 2.8 Hz, 1H), 8.20~8.13 (m, 2H), 8.11~7.88 (m, 1H), 7.87~7.83 (m, 1H), 7.67 (s, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 5.20~5.12 (m, 1H), 4.62 (d, J = 4.4 Hz, 2H), 4.14 (d, J = 12.8 Hz, 2H), 3.26~3.16 (m, 2H), 2.13~2.01 (m, 4H). HPLC: 97.04% (220 nm), 96.74% (215 nm), 98.80% (254 nm). MS (ESI): C 22 H 23 Calculated mass of Cl4N7O: 469.12, measured m / z: 470.1 [M+H] + .

[0273] compound 164 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((2-hydroxyethyl)sulfonyl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethylsulfanyl)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 241.37 μmol, 1 equiv.) (Steps 1–3 in Scheme C-3), sodium periodate (206.50 mg, 965.46 μmol, 53.50 μL, 4 equiv.), and ruthenium trichloride (5.01 mg, 24.14 μmol, 1.61 μL, 0.1 equiv.) in THF (2.5 mL) and HO (2.5 mL) was stirred at 50°C for 3 h. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (Welch Xtimate C18 100 mm × 25 mm 3 μm column; mobile phase: [water (0.1% TFA)-MeCN]; B%: 20%–55%, 12 min). The aqueous solution was lyophilized to afford 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethylsulfonyl)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (41 mg, 91.03 μmol, 37.72% yield, 99.093% purity) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.62 (s, 1H), 7.59~7.58 (m, 2H), 7.32 (dd, J = 1.6 Hz, 6.8 Hz, 1H), 6.75 (s, 1H), 4.86 (t, J = 6.8 Hz, 2H), 4.48 (d, J = HPLC: 99.09% (220 nm), 98.89% (215 nm), 100.00% (254 nm). MS (ESI): C 16 H 17 Calculated mass of Cl2N5O4S: 445.04, measured m / z: 446.0 [M+H] + .

[0274] compound 165 Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(methylsulfonyl)acetamide

[0275] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)acetic acid [ka] To a solution of 2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)acetic acid (0.32 g, 1.40 mmol, 1 equiv.) in t-BuOH (3 mL) was added (3,4-dichlorophenyl)methanamine (369.66 mg, 2.10 mmol, 280.04 μL, 1.5 equiv.). The mixture was stirred at 100°C for 24 hours. LCMS showed the reaction was complete. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 20% to 45%, 12 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by lyophilization. The compound 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetic acid (50 mg, 135.81 μmol, yield 9.70%) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.62~7.58 (m, 3H), 7.35~7.33 (m, 1H), 6.94 (s, 1H), 5.16 (s, 2H), 4.51 (d, J = 5.2 Hz, 2H).

[0276] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(methylsulfonyl)acetamide [ka]

[0277] To a solution of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]acetic acid (40 mg, 108.64 μmol, 1 equiv.) (Steps 1–3 in Scheme C-3) in DCM (1 mL) was added methanesulfonamide (20.67 mg, 217.29 μmol, 2 equiv.), DMAP (6.64 mg, 54.32 μmol, 0.5 equiv.), and DCC (22.42 mg, 108.64 μmol, 21.98 μL, 1 equiv.). The mixture was stirred at 40° C. under N for 10 h. LCMS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 150 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 20% to 45%, 12 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(methylsulfonyl)acetamide (21.1 mg, 46.89 μmol, yield 43.16%, purity 98.960%) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 12.25 (s, 1H), 7.66~7.58 (m, 3H), 7.35 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H), 5.24 (s, 2H), 4.53 (s, 2H), 3.24 (s, 3H). HPLC: 98.96% (220 nm), 98.99% (215 nm), 98.78% (254 nm). MS (ESI): C 15 H 14Calculated mass of Cl2N6O4S: 444.02, measured m / z: 445.0 [M+H] + .

[0278] compound 166 tert-Butyl 4-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethyl]piperazine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl 4-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethyl]piperazine-1-carboxylate (20 mg, 34.80 μmol, 9.29% yield, 98.58% purity) as a white solid which was used further. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.00 (s, 1H), 7.58~7.56 (m, 3H), 7.33~7.31 (m, 1H), 6.56 (s, 1H), 4.56 (t, J = 5.2 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 3.75 (t, J = 5.2 Hz, 2H), 3.44 (t, J = 5.2 Hz, 2H), 3.20 (s, 4H), 2.35 (t, J = 5.2 Hz, 2H), 2.22 (t, J = 4.8 Hz, 4H), 1.38 (s, 9H). HPLC: 98.58% (220 nm), 98.17% (215 nm), 98.39 % (254 nm). MS (ESI): C 25 H 33 Calculated mass for Cl2N7O4: 565.20, measured m / z: 566.2 [M+H] + .

[0279] compound 167 Preparation of tert-butyl 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-(piperazin-1-yl)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-piperazin-1-ylethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (43.3 mg, 86.11 μmol, 24.39% yield, HCl) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 9.52 (s, 1H), 7.62~7.59 (m, 3H), 7.36~7.34 (m, 1H), 7.20 (s, 1H), 4.63 (t, J = 5.2 Hz, 2H), 4.47 (d, J = 5.2 Hz, 2H), 3.86 (t, J = 5.2 Hz, 2H), 3.78 (t, J = 3.2 Hz, 2H), 3.64~3.53 (m, 8H), 3.24~3.17 (m, 2H). HPLC: 99.61 % (220 nm), 99.52 % (215 nm), 100.00% (254 nm). MS (ESI): C 20 H 27 Calculated mass of Cl4N7O2: 465.14, measured m / z: 466.1 [M+H] + .

[0280] compound 168 Preparation of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-hydroxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one

[0281] 5-[(3,4-Dichlorophenyl)methylamino]-1-[2-(3-tetrahydropyran-2-yloxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-tetrahydropyran-2-yloxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (180 mg, crude) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.02 (s, 1H), 7.59~7.56 (m, 3H), 7.32 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.74 (s, 1H), 4.55 (t, J = 5.6 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 4.41~4.40 (m, 1H), 3.75 (t, J = 5.6 Hz, 2H), 3.66~3.60 (m, 2H), 3.54~3.49 (m, 2H), 3.24~3.18 (m, 2H), 1.65~1.60 (m, 2H), 1.59~1.50 (m, 2H), 1.45~1.34 (m, 4H).

[0282] Preparation of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-hydroxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-tetrahydropyran-2-yloxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (180 mg, 362.62 μmol, 1 equiv.) in MeOH (1 mL) and HCl / MeOH (5 mL, 4 M) was stirred at 25°C for 1 h. HPLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 mm × 30 mm 3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 20% to 50%, 10.5 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-hydroxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (48.5 mg, 116.16 μmol, 32.03% yield, 98.74% purity) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.02 (s, 1H), 7.59~7.56 (m, 3H), 7.32 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.58 (s, 1H), 4.55 (t, J = 5.6 Hz, 2H), 4.47 (d, J = HPLC: 98.74% (220 nm), 98.25% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl2N5O3: 411.09, measured m / z: 412.0 [M+H] + .

[0283] compound 169 Preparation of 5-[(3,4-dichlorophenyl)methylamino]-1-[3-(2-hydroxyethoxy)propyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one

[0284] 1-[3-(2-benzyloxyethoxy)propyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 1-[3-(2-benzyloxyethoxy)propyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (180 mg, 358.29 μmol, 44.82% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.62~7.54 (m, 4H), 7.33~7.29 (m, 5H), 6.63 (s, 1H), 4.49~4.46 (m, 4H), 3.53~3.49 (m, 2H), 3.39~3.36 (m, 6H), 2.01~1.98 (m, 2H).

[0285] Preparation of 5-[(3,4-dichlorophenyl)methylamino]-1-[3-(2-hydroxyethoxy)propyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one [ka] To a solution of 1-[3-(2-benzyloxyethoxy)propyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidin-7-one (150 mg, 298.57 μmol, 1 equiv.) in 10 mL of EtOAc was added 10% Pd / C (5 mg) under a N atmosphere. The suspension was degassed and purged with H three times. The mixture was stirred at 25 °C under H (15 psi) for 12 h. LC-MS showed the reaction was complete. The reaction mixture was filtered to remove insoluble material and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 mm × 30 mm 3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 20% to 50%, 10.5 min). The solvent was removed by lyophilization to give the compound 5-[(3,4-dichlorophenyl)methylamino]-1-[3-(2-hydroxyethoxy)propyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (6.5 mg, 15.46 μmol, yield 5.18%, purity 98.03%) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.06 (s, 1H), 7.59~7.55 (m, 3H), 7.32 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.56 (s, 1H), 4.54 (t, J = 5.6 Hz, 1H), 4.49~4.45 (m, 4H), 3.48~3.44 (m, 2H), 3.34~3.30 (m, 4H), 2.01~1.97 (m, 2H). HPLC: 98.03% (220 nm), 97.71% (215 nm), 96.81% (254 nm). MS (ESI): C 17 H 19 Calculated mass of Cl2N5O3: 411.09, measured m / z: 412.2 [M+H] + .

[0286] compound 170 5-((3,4-Dichlorobenzyl)amino)-1-(2-(pyridin-3-yloxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-pyridyloxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (202 mg, 431.32 μmol, 69.90% yield, 99.873% purity, HCl) as a white solid. 59.0 mg was used for further analysis. 1 H NMR (DMSO-d6, 400 MHz) δ 8.65 (d, J = 2.4 Hz, 1H), 8.49 (d, J = 5.4 Hz, 1H), 8.12 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.92 (dd, J = 5.6 Hz, 8.8 Hz, 1H), 7.72 (s, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 4.88 (t, J = 4.8 Hz, 2H), 4.74~4.58 (m, 4H). HPLC: 99.87% (220 nm), 99.85% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 17 Calculated mass of Cl3N6O2: 430.07, measured m / z: 431.1 [M+H] + .

[0287] Compound 171 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((2-(hydroxymethyl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0288] Preparation of methyl 4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)oxazole-2-carboxylate and methyl 4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)oxazole-2-carboxylate (Step 1 in Scheme C-3) [ka] To a solution of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.4 g, 2.17 mmol, 1 equiv.) in THF (5 mL) was added PPh3 (852.59 mg, 3.25 mmol, 1.5 equiv.) and methyl 4-(hydroxymethyl)oxazole-2-carboxylate (408.59 mg, 2.60 mmol, 1.2 equiv.). DIAD (657.30 mg, 3.25 mmol, 632.02 μL, 1.5 equiv.) was then added dropwise at 0°C. The mixture was stirred at 25°C for 12 h. TLC (PE: EtOAc = 1:1) showed the reaction was complete. The reaction mixture was quenched with HO (5 mL), and some white solid was formed. The solid was collected after filtration. The solid was washed with EtOAc (2 mL) and concentrated under reduced pressure. A mixture of compounds methyl 4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)oxazole-2-carboxylate (0.45 g, 1.39 mmol, 64.15% yield) and methyl 4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)oxazole-2-carboxylate was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.40 (s, 1H), 8.29 (s, 1H), 5.71 (s, 2H), 4.16 (s, 3H), 3.84 (s, 3H). 1 H NMR (DMSO-d 6, 400 MHz) δ8.73 (s, 1H), 8.50 (s, 1H), 5.72 (s, 2H), 4.11 (s, 3H), 3.86 (s, 3H).

[0289] Preparation of 4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)oxazol-2-yl)methanol and (4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)oxazol-2-yl)methanol [ka] To a mixture of methyl 4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]oxazole-2-carboxylate (0.36 g, 1.11 mmol, 1 equiv.) and methyl 4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)methyl]oxazole-2-carboxylate (1.11 mmol, 1 equiv.) in MeOH (2 mL) was added NaBH (167.97 mg, 4.44 mmol, 4 equiv.) in portions at 0° C. The mixture was stirred at 25° C. for 13 hours. Then, NaBH (167.97 mg, 4.44 mmol, 4 equiv.) was added again in portions at 0° C. The mixture was stirred at 25° C. for 12 hours. TLC showed the reaction was complete. The reaction mixture was quenched with HO (10 mL) and then extracted with EtOAc (15 mL × 3). The combined organic layers were washed with brine (10 mL × 1), dried over NaSO, filtered, and concentrated under reduced pressure. A mixture of the compounds (4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)oxazol-2-yl)methanol (0.27 g, 913.15 μmol, 82.27% yield) and (4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)oxazol-2-yl)methanol was obtained as a pale yellow oil. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.26 (s, 1H), 8.06 (s, 1H), 5.60 (s, 2H), 5.40 (d, J=6.0 Hz, 2H), 4.17 (s, 3H). 1 H NMR (DMSO-d 6,400 MHz) δ 8.18 (s, 1H), 8.01 (s, 1H), 5.64 (s, 2H), 5.44 (d, J=6.4 Hz, 2H), 4.11 (s, 3H).

[0290] Preparation of 5-chloro-1-((2-(hydroxymethyl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and 5-chloro-2-((2-(hydroxymethyl)oxazol-4-yl)methyl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme C-3) [ka]

[0291] To a mixture of [4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]oxazol-2-yl]methanol (0.27 g, 913.15 μmol, 1 equiv.) and [4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)methyl]oxazol-2-yl]methanol (913.15 μmol, 1 equiv.) in HO (2 mL) and MeOH (4 mL) was added LiOH·HO (114.95 mg, 2.74 mmol, 3 equiv.). The mixture was stirred at 25 °C for 10 h. TLC indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH = 6 with HCl (3 N). The mixture was then extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (6 mL × 1), dried over NaSO, filtered, and concentrated under reduced pressure to give a mixture of 5-chloro-1-((2-(hydroxymethyl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (0.25 g, crude) and 5-chloro-2-((2-(hydroxymethyl)oxazol-4-yl)methyl)-2H-pyrazolo[4,3-d]pyrimidin-7(6H)-one as a pale yellow oil. 1 H NMR (DMSO-d 6,400 MHz) δ 7.96 (s, 1H), 7.95 (s, 1H), 5.62 (s, 2H), 4.41 (d, J=6.4 Hz, 2H). 1 H NMR (DMSO-d 6, 400 MHz) δ 8.38 (s, 1H), 8.15 (s, 1H), 5.45 (s, 2H), 4.44 (d, J=6.4 Hz, 2H).

[0292] Preparation of (5-((3,4-dichlorobenzyl)amino)-1-((2-(hydroxymethyl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one) (Step 3 in Scheme C-3) [ka] To a mixture of 5-chloro-2-[[2-(hydroxymethyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (781.10 μmol, 1 equiv.) and 5-chloro-1-[[2-(hydroxymethyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.22 g, 781.10 μmol, 1 equiv.) in 2-methylbutan-2-ol (5 mL) was added (3,4-dichlorophenyl)methanamine (275.01 mg, 1.56 mmol, 208.34 μL, 2 equiv.). The mixture was stirred at 130° C. for 10 hours. LCMS and HPLC indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA condition column: Phenomenex Luna C18 150 × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 20% to 35%, 12 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-2-[[2-(hydroxymethyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (33.1 mg, 78.58 μmol, 10.06% yield) was obtained as a white solid. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(hydroxymethyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (30.5 mg, 68.96 μmol, 8.83% yield, 95.236% purity) was obtained as a pale yellow solid, 20.1 mg of which was used further. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.90 (s, 1H), 7.59~7.57 (m, 3H), 7.32 (d, J = 6.4 Hz, 1H), 6.70 (s, 2H), 5.51 (s, 2H), 4.47 (d, J = 6.0 Hz, 2H), 4.41 (s, 2H). HPLC: 95.24% (220 nm), 94.04% (215 nm), 97.02% (254 nm). MS (ESI): C 17 H 14Calculated mass of Cl2N6O3: 420.05, measured m / z: 421.0 [M+H] + .

[0293] compound 172 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((2-(1-methylpiperidin-3-yl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0294] tert-Butyl 3-[4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]oxazol-2-yl]piperidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl 3-[4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]oxazol-2-yl]piperidine-1-carboxylate (220 mg, 382.97 μmol, 97.97% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.82 (s, 1H), 7.61~7.54 (m, 4H), 7.34~7.30 (m, 1H), 6.66 (s, 1H), 5.50 (s, 2H), 4.47 (d, J = 6.0 Hz, 2H), 3.90~3.72 (m, 2H), 2.91~2.85 (m, 3H), 2.03~1.99 (m, 1H), 1.70~1.66 (m, 2H), 1.46~1.40 (m, 1H), 1.35 (s, 9H).

[0295] compound 173 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((2-(piperidin-3-yl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] A mixture of tert-butyl 3-[4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]oxazol-2-yl]piperidine-1-carboxylate (40 mg, 69.63 μmol, 1 equiv.) in EtOAc (1 mL) and HCl / EtOAc (4 M, 3 mL) was stirred at 25°C for 2 h. HPLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The filtrate was purified by preparative HPLC (HCl condition column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 10% to 40%, 12 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(3-piperidyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (17 mg, 30.86 μmol, 44.33% yield, 92.74% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.93 (s, 2H), 7.92 (s, 1H), 7.62~7.59 (m, 3H), 7.35 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 5.52 (s, 2H), 4.53 (d, J = 5.2 Hz, 2H), 3.37~3.20 (m, 3H), 3.09~3.00 (m, 1H), 2.91~2.83 (m, 1H), 2.09~2.05 (m, 1H), 1.83~1.62 (m, 3H). HPLC: 92.74 % (220 nm), 88.05 % (215 nm), 90.43 % (254 nm). MS (ESI): C 21 H 22Calculated mass of Cl3N7O2: 473.11, measured m / z: 474.1 [M+H] + .

[0296] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((2-(1-methylpiperidin-3-yl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka]

[0297] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(3-piperidyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (130 mg, 254.50 μmol, 1 equiv., HCl) in MeOH (2 mL) was added formaldehyde (61.97 mg, 763.50 μmol, 56.85 μL, 37% purity, 3 equiv.) at 0° C. The mixture was stirred at 0° C. for 10 min. Then, AcOH (1.53 mg, 25.45 μmol, 1.46 μL, 0.1 equiv.) and NaBHCN (79.96 mg, 1.27 mmol, 5 equiv.) were added at 0° C. The mixture was stirred at 25° C. for 10 h. LC-MS showed the reaction was complete. The reaction mixture was quenched with HO (2 mL) at 0°C and then concentrated under reduced pressure. The aqueous solution was extracted with EtOAc (2 mL x 3). The combined organic layers were washed with brine (2 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl condition column: Phenomenex Luna C18 150 mm x 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 10% to 40%, 12 min). The solvent was removed by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(1-methyl-3-piperidyl)oxazol-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (50.6 mg, 93.65 μmol, 36.80% yield, 97.14% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 10.54 (s, 1H), 7.97 (s, 1H), 7.71 (s, 1H), 7.66~7.64 (m, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.36 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 5.53 (s, 2H), 4.57 (d, J = 5.2 Hz, 2H), 3.40~3.33 (m, 3H), 3.13~3.04 (m, 1H), 2.91~2.87 (m, 1H), 2.75 (d, J = 4.8 Hz, 3H), 2.11~2.08 (m, 1H), 1.89~1.83 (m, 2H), 1.54~1.49 (m, 1H). HPLC: 97.14 % (220 nm), 95.64 % (215 nm), 96.18 % (254 nm). MS (ESI): C 22 H 24 Calculated mass of Cl3N7O2: 487.13, measured m / z: 488.1 [M+H] + .

[0298] compound 174 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((6-(hydroxymethyl)pyridin-3-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0299] Preparation of methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylate (Step 1 in Scheme C-3) [ka]

[0300] To a solution of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (400 mg, 2.17 mmol, 1 equiv.), methyl 5-(2-hydroxyethoxy)pyridine-2-carboxylate (555.51 mg, 2.82 mmol, 1.3 equiv.), and PPh3 (852.59 mg, 3.25 mmol, 1.5 equiv.) in THF (5 mL) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 5 h. TLC showed the reaction was complete. The reaction mixture was quenched with HO (2 mL) and extracted with EtOAc (2 mL × 3). The combined organic layers were washed with brine (2 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 12 g, elution with 0–100% ethyl acetate / methanol at 45 mL / min). The eluent was removed under reduced pressure. The compound methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylate (580 mg, 1.59 mmol, 73.58% yield) was obtained as a white solid. The compound methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)ethoxy]pyridine-2-carboxylate (280 mg, 769.75 μmol, 35.52% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.30 (s, 1H), 8.25 (d, J = 2.8 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.46 (dd, J = 8.8 Hz, 2.8 Hz, 1H), 4.93 (t, J = 5.2 Hz, 2H), 4.63 (t, J = 5.2 Hz, 2H), 4.07 (s, 3H), 3.83 (s, 3H).

[0301] Preparation of 5-[2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylic acid (Step 2 in Scheme C-3) [ka] To a solution of methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylate (100 mg, 274.91 μmol, 1 equiv.) in MeOH (1 mL) and HO (2 mL) was added LiOH·HO (34.61 mg, 824.73 μmol, 3 equiv.). The mixture was stirred at 25 °C for 2 h. TLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH = 6 with 3 N HCl, resulting in the formation of some white solid. The solid was collected after filtration and concentrated under reduced pressure. Compound 5-[2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylic acid (95 mg, crude) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.25 (d, J = 2.8 Hz, 1H), 8.00~7.96 (m, 2H), 7.47 (dd, J = 8.8 Hz, 2.8 Hz, 1H), 4.94 (t, J = 5.2 Hz, 2H), 4.61 (t, J = 5.2 Hz, 2H).

[0302] compound 175 Preparation of 5-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)picolinic acid hydrochloride (Step 3 in Scheme C-3) [ka] A mixture of 5-[2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylic acid (90 mg, 268.09 μmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (94.39 mg, 536.19 μmol, 71.51 μL, 2 equiv.) in 2-methyl-2-butanol (2 mL) was stirred at 140 °C for 4 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15% to 45%, 12 min). The solvent was removed by lyophilization. The compound 5-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]pyridine-2-carboxylic acid (45.1 mg, 86.74 μmol, 32.35% yield, 98.42% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.26 (d, J = 2.4 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H), 7.62~7.59 (m, 2H), 7.48 (dd, J = 8.4 Hz, 2.8 Hz, 1H), 7.36 (s, 1H), 7.35 (d, J = 8.0 Hz, 1H), 4.85 (t, J = 5.2 Hz, 2H), 4.58 (t, J = 5.2 Hz, 2H), 4.54 (d, J = 4.4 Hz, 2H). HPLC: 98.42 % (220 nm), 97.74% (215 nm), 99.67% (254 nm). MS (ESI): C 20 H 17 Calculated mass of Cl3N6O4: 474.06, measured m / z: 475.0 [M+H] + .

[0303] Preparation of [5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]-2-pyridyl]methanol [ka]

[0304] To a solution of methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]pyridine-2-carboxylate (150 mg, 412.37 μmol, 1 equiv.) in THF (1 mL) was added DIBAL-H (1 M, 2.06 mL, 5 equiv.) dropwise at 0° C. The mixture was then stirred at 25° C. for 6 h. TLC showed the reaction was complete. The reaction mixture was quenched with HO (2 mL) and extracted with EtOAc (2 mL × 3). The combined organic layers were washed with brine (2 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The compound [5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]-2-pyridyl]methanol (130 mg, 387.20 μmol, yield 93.90%) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.30 (s, 1H), 8.05~8.04 (m, 1H), 7.32~7.31 (m, 2H), 5.27 (t, J = 5.6 Hz, 1H), 4.89 (t, J = 5.2 Hz, 2H), 4.51 (t, J = 5.2 Hz, 2H), 4.45 (d, J = 6.0 Hz, 2H), 4.07 (s, 3H).

[0305] Preparation of 5-chloro-1-[2-[[6-(hydroxymethyl)-3-pyridyl]oxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (Step 2 in Scheme C-3) [ka] To a solution of [5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy]-2-pyridyl]methanol (130 mg, 387.20 μmol, 1 equiv.) in MeOH (1 mL) and HO (1 mL) was added LiOH·HO (48.74 mg, 1.16 mmol, 3 equiv.). The mixture was stirred at 25 °C for 2 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH = 6 with 3 N HCl and extracted with EtOAc (3 mL × 3). The combined organic layers were washed with brine (3 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The compound 5-chloro-1-[2-[[6-(hydroxymethyl)-3-pyridyl]oxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (100 mg, 310.83 μmol, 80.28% yield) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.08~8.07 (m, 1H), 8.00 (s, 1H), 7.32 (d, J = 2.0 Hz, 2H), 5.27 (s, 1H), 4.90 (t, J = 5.2 Hz, 2H), 4.49 (t, J = 5.2 Hz, 2H), 4.46 (d, J = 4.0 Hz, 2H). 5-((3,4-Dichlorobenzyl)amino)-1-(2-((6-(hydroxymethyl)pyridin-3-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Step 3 in Scheme C-3. [ka]

[0306] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-[[6-(hydroxymethyl)-3-pyridyl]oxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (82.6 mg, 162.01 μmol, 52.12% yield, 97.63% purity, HCl) as a white solid. 1H NMR (DMSO-d 6, 400 MHz) δ 8.40 (d, J = 2.8 Hz, 1H), 8.02 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.66~7.59 (m, 4H), 7.35 (dd, J = 8.0 Hz, HPLC: 97.63 % (220 nm), 96.85 % (215 nm), 97.96 % (254 nm).MS (ESI): C 20 H 19 Calculated mass of Cl3N6O3: 460.08, measured m / z: 461.1 [M+H] + .

[0307] compound 176 5-((3,4-Dichlorobenzyl)amino)-1-((1-(2-methoxyethyl)-5-oxopyrrolidin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-((1-(2-methoxyethyl)-5-oxopyrrolidin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (306 mg, 642.75 μmol, 52.34% yield, 97.743% purity) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.62~7.59 (m, 3H), 7.34 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H), 4.50~4.46 (m, 4H), 3.39~3.36 (m, 3H), 3.30~3.29 (m, 2H), 3.28~3.19 (m, 4H), 2.75 (s, 1H), 2.34 (t, J = 7.6 Hz, 1H), 2.11 (dd, J = 6.0 Hz, 16.8 Hz, 1H). HPLC: 97.74% (220 nm), 97.24% (215 nm), 99.81% (254 nm). MS (ESI): C 20 H 22 Calculated mass of Cl2N6O3: 464.11, measured m / z: 465.1 [M+H] + .

[0308] compound 177 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((1-(2-hydroxyethyl)-5-oxopyrrolidin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka]

[0309] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[[1-(2-methoxyethyl)-5-oxo-pyrrolidin-3-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (50 mg, 107.45 μmol, 1 equiv.) in DCM (2 mL) was added BBr3 (80.76 mg, 322.35 μmol, 31.06 μL, 3 equiv.) dropwise at 0° C. The mixture was stirred at 0° C. for 1 hour. LCMS and HPLC showed the reaction was complete. Ice water (2 mL) was added to the mixture. The mixture was extracted with EtOAc (5 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (Phenomenex Luna C18 150 mm × 30 mm 5 μm column; mobile phase: [water (0.05% HCl)-MeCN]; B%: 20% to 50%, 12 min). The mixture was dried under lyophilization to give 5-((3,4-dichlorobenzyl)amino)-1-((1-(2-hydroxyethyl)-5-oxopyrrolidin-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (13.2 mg, 29.25 μmol, 27.22% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.63~7.53 (m, 3H), 7.32 (d, J = 10.0 Hz, 1H), 7.07~6.90 (m, 1H), 4.52~4.40 (m, 4H), 3.23~3.21 (m, 1H), 3.21~3.17 (m, 2H), 3.17~3.07 (m, 2H), 3.03~2.90 (m, 1H), 2.82 (d, J = 6.4 Hz, 1H), 2.30~2.24 (m, 1H), 2.08 (dd, J = 6.0 Hz, 16.8 Hz, 1H). HPLC:97.85% (220 nm), 97.63% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 20 Calculated mass of Cl2N6O3: 450.10 m / z Found: 451.1 [M+H] + .

[0310] compound 178 5-((3,4-Dichlorobenzyl)amino)-1-(2-(oxetan-3-yloxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(oxetan-3-yloxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (83.6 mg, 192.47 μmol, 52.10% yield, 94.451% purity) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.67~7.60 (m, 3H), 7.37 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H), 4.60~4.54 (m, 6H), 4.54~4.49 (m, 1H), 4.28~4.25 (m, 2H), 3.76 (t, J = 5.6 Hz, 2H). HPLC: 94.45% (220 nm), 94.06% (215 nm), 95.57% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O3: 409.07, measured m / z: 410.0 [M+H] + .

[0311] Compound 179 5-((3,4-Dichlorobenzyl)amino)-1-(3-(3-hydroxycyclobutoxy)propyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[3-(3-hydroxycyclobutoxy)propyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (88.5 mg, 200.74 μmol, 37.48% yield, 99.42% purity) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.05 (s, 1H), 7.59~7.57 (m, 2H), 7.54 (s, 1H), 7.32 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.61 (s, 1H), 4.97 (d, J = 6.8 Hz, 1H), 4.48~4.43 (m, 4H), 3.69~3.60 (m, 1H), 3.42~3.37 (m, 1H), 3.21 (t, J = 6.0 Hz, 2H), 2.48~2.43 (m, 2H), 1.99~1.93 (m, 2H), 1.70~1.62 (m, 2H). HPLC: 99.42% (220 nm), 99.21% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O3: 437.10, measured m / z: 438.1 [M+H] + .

[0312] compound 180 5-((3,4-Dichlorobenzyl)amino)-1-(1-(pyridin-4-yl)pyrrolidin-3-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-4-yl)pyrrolidin-3-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride (12.5 mg, 27.30 μmol, 18.91% yield, 99.647% purity) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ8.30~8.19 (m, 2H), 7.68~7.55 (m, 3H), 7.33 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 6.90 (dd, J = 6.4 Hz, J = 14.8 Hz, 2H), 5.85 (s, 1H), 4.51 (d, J=5.6 Hz, 2H), 4.07~3.97 (m, 1H), 3.96~3.89 (m, 1H), 3.72 (d, J = 6.8 Hz, 2H), 2.62~2.55 (m, 2H). HPLC:99.65% (220 nm), 99.59% (215 nm), 99.68% (254 nm). MS (ESI): C 21 H 21 Calculated mass for Cl4N7O: 455.10 m / z, Found: 456.1 [M+H] + .

[0313] Compound 181 5-((3,4-Dichlorobenzyl)amino)-1-(2-(pyridin-3-ylmethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-pyridylmethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (183.7 mg, 378.49 μmol, 55.10% yield, 99.26% purity, HCl) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ8.83 (d, J = 5.6 Hz, 1H), 8.73 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.31 (s, 1H), 8.01~7.98 (m, 1H), 7.69 (s, 1H), 7.68 (d, HPLC: 99.26 % (220 nm), 99.19% (215 nm), 99.61% (254 nm). MS (ESI): C 20 H 19 Calculated mass of Cl3N6O2: 444.09, measured m / z: 445.1 [M+H] + .

[0314] compound 182 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((1-(pyridin-2-yl)pyrrolidin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride

[0315] tert-Butyl 2-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]pyrrolidine-1-carboxylate (0.13 g, 263.49 μmol, 23.31% yield) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.66~7.61 (m, 3H), 7.37 (s, 1H), 4.58 (s, 2H), 4.45~4.43 (m, 2H), 4.17~4.15 (m, 1H), 3.35~3.20 (m, 2H), 1.76~1.73 (m, 4H), 1.23 (m, 9H).

[0316] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(pyrrolidin-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] To a solution of tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]pyrrolidine-1-carboxylate (0.13 g, 263.49 μmol, 1 equiv.) in EtOAc (1 mL) was added HCl / EtOAc (2 mL, 4N). The mixture was stirred at 25° C. for 12 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-(pyrrolidin-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, crude) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 9.35 (s, 1H), 8.89 (s, 1H), 7.72 (s, 1H), 7.62~7.60 (m, 3H), 7.37~7.34 (m, 1H), 4.83~4.78 (m, 1H), 4.73~4.59 (m, 1H), 4.55 (s, 2H), 3.94~3.90 (m, 1H), 3.31~3.14 (m, 2H), 1.99~1.86 (m, 4H).

[0317] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((1-(pyridin-2-yl)pyrrolidin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-(pyrrolidin-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.1 g, 232.70 μmol, 1 equiv., HCl) in DMF (1 mL) was added 2-fluoropyridine (45.19 mg, 465.41 μmol, 39.99 μL, 2 equiv.) and KCO (96.49 mg, 698.11 μmol, 3 equiv.). The mixture was stirred at 100° C. for 12 hours. LCMS and HPLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl condition: Phenomenex Luna C18 150 × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 15% to 40%, 12 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[1-(2-pyridyl)pyrrolidin-2-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (37.0 mg, 72.55 μmol, 31.18% yield, 99.374% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.07 (s, 3H), 7.69~7.63 (m, 3H), 7.37 (s, 1H), 7.23 (s, 1H), 6.93 (s, 1H), 4.59 (s, 5H), 3.64 (s, 1H), 3.43 (s, 1H), 1.97 (s, 3H), 1.76 (s, 1H). HPLC: 99.37% (220 nm), 99.41% (215 nm), 99.66% (254 nm). MS (ESI): C 22 H 23 Calculated mass of Cl4N7O: 469.12, measured m / z: 470.1 [M+H] + .

[0318] compound 183 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-(dimethylamino)-3-methoxypropan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride

[0319] tert-Butyl (2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)-3-methoxypropyl)(methyl)carbamate was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound tert-butyl (2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)-3-methoxypropyl)(methyl)carbamate (0.13 g, 234.04 μmol, 54.07% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.60~7.58 (m, 3H), 7.34 (d, J = 8.0 Hz, 1H), 4.55~4.52 (m, 4H), 3.90~3.77 (m, 1H), 3.56~3.51 (m, 1H), 3.25~3.22 (m, 3H), 3.21 (s, 3H), 3.18~3.00 (m, 2H), 2.64 (s, 3H), 1.34 (s, 9H).

[0320] compound 184 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(pyrrolidin-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] A solution of tert-butyl N-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]-3-methoxy-propyl]-N-methyl-carbamate (0.13 g, 234.04 μmol, 1 equiv.) in HCl / EtOAc (2 mL) was stirred at 25° C. for 10 h. LCMS showed the reaction was complete. The reaction mixture was filtered under reduced pressure. Some pale yellow solid was formed. The solid was collected after filtration. The compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-methoxy-3-(methylamino)propan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (28.5 mg, 55.75 μmol, 23.82% yield, 96.203% purity, HCl) was obtained as a pale yellow solid and used subsequently. The compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-methoxy-3-(methylamino)propan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (80 mg, HCl) was obtained as a pale yellow solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.68 (s, 1H), 8.45 (s, 1H), 7.63~7.59 (m, 3H), 7.40 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 4.67~4.59 (m, 2H), 4.53 (d, J = 5.6 Hz, 2H), 3.96~3.92 (m, 2H), 3.80 (s, 1H), 3.32 (d, J = 4.4 Hz, 2H), 3.19 (s, 3H), 3.04~3.02 (m, 1H), 3.93~3.90 (m, 1H), 2.55 (s, 3H). HPLC: 96.20% (220 nm), 96.13% (215 nm), 95.56% (254 nm). MS (ESI): C 19 H 25 Calculated mass of Cl3N6O3: 454.13, measured m / z: 455.1 [M+H] + .

[0321] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-(dimethylamino)-3-methoxypropan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka]

[0322] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-[1-(methoxymethyl)-2-(methylamino)ethoxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (80 mg, 175.69 μmol, 1 equiv.) in MeOH (1 mL) was added formaldehyde (42.77 mg, 527.08 μmol, 39.24 μL, 3 equiv.) and AcOH (1.06 mg, 17.57 μmol, 1.00 μL, 0.1 equiv.) at 0°C. NaBHCN (33.12 mg, 527.08 μmol, 3 equiv.) was then added portionwise at 0°C. The mixture was stirred at 25°C for 3 h. LCMS and HPLC indicated the reaction was complete. HO (1 mL) was added to the reaction mixture. The mixture was filtered under reduced pressure. The filtrate was purified by preparative HPLC (HCl condition: Phenomenex Luna C18 150 × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 15% to 45%, 12 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-(dimethylamino)-3-methoxypropan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (40.1 mg, 79.28 μmol, 45.12% yield, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 9.54 (s, 1H), 7.61~7.59 (m, 3H), 7.33 (d, J = 7.6 Hz, 1H), 7.18 (s, 1H), 4.62~4.60 (m, 2H), 4.50 (d, J = 4.8 Hz, 2H), 4.00~3.93 (m, 1H), 3.92 (s, 2H), 3.34~3.29 (m, 2H), 3.20 (s, 3H), 3.14~3.13 (m, 2H), 3.71 (s, 6H). HPLC: 99.40% (220 nm), 99.24% (215 nm), 99.04% (254 nm). MS (ESI): C 20 H 27 Calculated mass of Cl3N6O3: 468.14, measured m / z: 469.1 [M+H] + .

[0323] compound 185 5-((3,4-Dichlorobenzyl)amino)-1-(2-(2-methoxy-1-(pyridin-3-yl)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-methoxy-1-(pyridin-3-yl)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (98.1 mg, 185.02 μmol, 38.07% yield, 99.172% purity, HCl) as a white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.80 (d, J = 5.2 Hz, 1H), 8.69 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.96~7.94 (m, 1H), 7.78 (s, 1H), 7.65~7.63 (m, 3H), 7.39~7.37 (m, 1H), 4.78 (t, J = 4.0 Hz, 1H), 4.68~4.63 (m, 1H), 4.59~4.58 (m, 2H), 4.56~4.54 (m, 1H), 3.94~3.93 (m, 1H), 3.84~3.82 (m, 1H), 4.54~4.44 (m, 2H), 3.17 (s, 3H). HPLC: 99.17% (220 nm), 99.03% (215 nm), 100.00% (254 nm). MS (ESI): C 22 H 23 Calculated mass of Cl3N6O3: 488.11, measured m / z: 489.1 [M+H] + .

[0324] compound 186 Preparation of methyl 2-(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethoxy)acetate

[0325] Preparation of 2-(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethoxy)acetaldehyde [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-[2-(2-hydroxyethoxy)ethoxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (0.6 g, 1.36 mmol, 1 equiv.) (Steps 1–3 in Scheme C-3) in DCM (30 mL) was added DMP (2.30 g, 5.43 mmol, 1.68 mL, 4 equiv.) portionwise at 0°C. The mixture was stirred at 25°C for 32 h. TLC (ethyl acetate:methanol = 10:1, Rf = 0.58) indicated the reaction was complete. The reaction mixture was quenched with HO (10 mL) at 0°C. The organic layer was separated, and the aqueous solution was then extracted with DCM (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The compound 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethoxy]acetaldehyde (230 mg, crude) was obtained as a yellow oil.

[0326] compound 187 Preparation of 2-(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethoxy)acetic acid) [ka] To a solution of 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethoxy]acetaldehyde (230 mg, 522.40 μmol, 1 equiv.) and 2-methylbut-2-ene (732.74 mg, 10.45 mmol, 1.11 mL, 20 equiv.) in HO (1 mL), THF (1 mL), and t-BuOH (1 mL) was added sodium chlorite (51.97 mg, 574.63 μmol, 1.1 equiv.) in portions at 0°C. The mixture was stirred at 20°C for 3 h. LC-MS indicated the reaction was nearly complete. The reaction mixture was quenched with HO (5 mL) at 20°C and then extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 25% to 40%, 10 min). The aqueous solution was lyophilized. The compound 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethoxy]acetic acid (31 mg, 65.58 μmol, 12.55% yield, 96.527% purity) was obtained as a brown solid. 10.8 mg was used for further analysis. The desired compound (31 mg, 65.58 μmol, 12.55% yield) was obtained as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.66~7.61 (m, 3H), 7.46 (s, 1H), 7.37 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 6.75 (s, 1H), 5.55 (s, 1H), 4.86 (s, 1H), 4.73 (d, J = 16.4 Hz, 1H), 4.62 (t, J = 5.6 Hz, 2H), 4.45 (d, J = 16.4 Hz, 1H), 3.83 (t, J = 5.6 Hz, 2H), 3.40 (t, J = 4.4 Hz, 4H). HPLC: 96.53% (220 nm), 95.61% (215 nm), 94.88% (254 nm). MS (ESI): C 18 H 19 Calculated mass of Cl2N5O5: 455.08, measured m / z: 456.1 [M+H] + .

[0327] Preparation of methyl 2-(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethoxy)acetate [ka]

[0328] To a solution of 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethoxy]acetic acid (20 mg, 43.83 μmol, 1 equiv.) in MeOH (10 mL) was added SOCl2 (26.07 mg, 219.16 μmol, 15.90 μL, 5 equiv.) dropwise at 0 °C. The mixture was stirred at 25 °C for 5 h. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Xtimate C18 100 mm × 30 mm 3 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 25% to 50%, 10 min). The aqueous solution was lyophilized to give methyl 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]ethoxy]acetate (3.6 mg, 7.37 μmol, 16.81% yield, 96.244% purity) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.61~7.56 (m, 3H), 7.33 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 6.70 (s, 1H), 4.62~4.53 (m, 2H), 4.48 (d, J = 5.6 Hz, HPLC: 96.24% (220 nm), 96.15% (215 nm), 96.33% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O5: 469.09, measured m / z: 470.1 [M+H] + .

[0329] compound 188 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((1,1-dioxidetetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0330] 5-((3,4-Dichlorobenzyl)amino)-1-(2-((tetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((tetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (72 mg, 156.23 μmol, 27.32% yield, 98.591% purity) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.60~7.56 (m, 3H), 7.33 (d, J = 8.4 Hz, 1H), 6.68 (s, 1H), 4.55 (t, J = 5.6 Hz, 2H), 4.48 (d, J = 5.6 Hz, 2H), 3.78 (t, J = 5.6 Hz, 2H), 3.27 (t, J = 8.0 Hz, 1H), 2.61~2.55 (m, 2H), 2.40~2.37 (m, 2H), 1.93~1.86 (m, 2H), 1.54~1.46 (m, 2H). HPLC: 98.59% (220 nm), 98.28% (215 nm), 94.36% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O2S: 453.08 m / z, Found: 454.1 [M+H] + .

[0331] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((1,1-dioxidetetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka] To a mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-(2-tetrahydrothiopyran-4-yloxyethyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one (30 mg, 66.03 μmol, 1 equiv.) in THF (3 mL) and HO (3 mL), RuCl (1.37 mg, 6.60 μmol, 0.44 μL, 0.1 equiv.) was added, followed by NaIO (56.49 mg, 264.10 μmol, 14.63 μL, 4 equiv.) at 0 °C. The mixture was stirred at 50 °C for 16 h. LC-MS and HPLC showed the reaction was complete. The mixture was poured into HO (5 mL) and then extracted with EtOAc (5 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 20% to 50%, 10 min). The mixture was concentrated under reduced pressure to give 5-((3,4-dichlorobenzyl)amino)-1-(2-((1,1-dioxidetetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (8.6 mg, 16.87 μmol, yield 25.55%, purity 95.423%) as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ7.61~7.56 (m, 3H), 7.33 (d, J = 8.4 Hz, 1H), 6.73 (s, 1H), 4.59 (t, J = 5.2 Hz, 2H), 4.48 (d, J = 5.6 Hz, 2H), 3.79 (t, J = 5.2 Hz, 2H), 3.61~3.60 (m, 1H), 2.94~2.84 (m, 4H), 1.95 (d, J = 4.4 Hz, 4H). HPLC: 95.42% (220 nm), 93.82% (215 nm), 94.82% (254 nm). MS (ESI): C19H 21 Calculated mass of Cl2N5O4S: 485.07 m / z Found: 486.0 [M+H] + .

[0332] compound 189 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(3,4-dihydroxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0333] compound 190 5-((3,4-Dichlorobenzyl)amino)-1-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[(3,4-dimethoxyphenyl)methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (40 mg, 86.90 μmol, 36.44% yield, 100.00% purity) as a white solid, 5.8 mg of which was used further. 1H NMR (DMSO-d6, 400 MHz) δ 7.60~7.54 (m, 3H), 7.32 (dd, J = 2.0, 8.4 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.74 (dd, J HPLC: 100.00% (220 nm), 100.00% (215 nm), 100.00% (254 nm). MS (ESI): C 21 H 19 Calculated mass of Cl2N5O3: 459.09, measured m / z: 460.0 [M+H] + .

[0334] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(3,4-dihydroxybenzyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[(3,4-dimethoxyphenyl)methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (30 mg, 65.17 μmol, 1 equiv.) in DCM (1 mL) was added BBr3 (65.31 mg, 260.69 μmol, 25.12 μL, 4 equiv.) dropwise at 0 °C. The mixture was stirred at 25 °C for 1 h. LC-MS showed the reaction was complete. The reaction mixture was quenched with HO (0.1 mL) at 0 °C and then concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 20% to 45%, 10 min). The solution was lyophilized to give 5-[(3,4-dichlorophenyl)methylamino]-1-[(3,4-dihydroxyphenyl)methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (7.2 mg, 15.48 μmol, 23.75% yield, 92.908% purity) as a gray solid. The desired compound (7.2 mg, 15.48 μmol, 23.75% yield) was obtained as a gray solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.88 (s, 1H), 7.58 (d, J = 7.6 Hz, 3H), 7.58 (d, J = 7.2 Hz, 1H), 6.72~6.60 (m, 3H), 6.58~6.52 (m, 1H), 5.41 (s, 2H), 4.47 (d, J = 6.0 Hz, 2H). HPLC: 92.91% (220 nm), 91.72% (215 nm), 91.74% (254 nm). MS (ESI): C 19 H 15 Calculated mass of Cl2N5O3: 431.06, measured m / z: 432.0 [M+H] + .

[0335] Compound 191 Preparation of (E)-2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)acetaldehyde oxime

[0336] Preparation of 2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]acetaldehyde [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (70 mg, 175.77 μmol, 1 equiv.) in DCM (10 mL) was added DMP (134.19 mg, 316.39 μmol, 97.95 μL, 1.8 equiv.) portionwise at 0 °C. The mixture was stirred at 25 °C for 15 h. LCMS showed the reaction was complete. The mixture was quenched with H2O (5 mL), and the organic solvent was removed under reduced pressure. The aqueous solution was extracted with EtOAc (5 mL × 5). The combined organic layers were washed with saturated NaHCO3 (4 mL × 1) and brine (4 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure. The compound 2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]acetaldehyde (60 mg, 151.43 μmol, 86.15% yield) was obtained as a white solid.

[0337] Preparation of (E)-2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)acetaldehyde oxime [ka] To a solution of 2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]acetaldehyde (50 mg, 126.19 μmol, 1 equiv.) in EtOH (2 mL) at 0 °C was added NHOH·HCl (17.54 mg, 252.38 μmol, 2 equiv.) and TEA (31.92 mg, 315.48 μmol, 43.91 μL, 2.5 equiv.). The mixture was stirred at 25 °C for 10 h. LCMS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (neutral condition column: Phenomenex Gemini-NX C18 75 mm × 30 mm 3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 25% to 50%, 10.5 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by lyophilization. The compound (1E)-2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]ethoxy]acetaldehyde oxime (15.0 mg, 35.10 μmol, yield 27.81%, purity 96.223%) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.09 (s, 0.5H), 11.01 (s, 1H), 10.91 (s, 0.5H), 7.59~7.57 (m, 3H), 7.32 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 6.65 (t, J = 8.0 Hz, 1H), 6.58 (s, 1H), 4.58 (t, J = 6.4 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 4.18 (d, J = 4.0 Hz, 1H), 3.94 (d, J = 6.4 Hz, 1H), 3.83~3.77 (m, 1H), 3.34 (s, 1H). HPLC: 96.22% (220 nm), 94.69% (215 nm), 93.56% (254 nm). MS (ESI): C 16 H 16Calculated mass of Cl2N6O3: 410.07, measured m / z: 411.0 [M+H] + .

[0338] compound 192 Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0339] Preparation of (E)-1-(4-bromobut-2-en-1-yl)-5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (Step 1 in Scheme C-3) [ka] To a solution of (E)-1,4-dibromobut-2-ene (2.90 g, 13.54 mmol, 5 equiv.) in DMF (5 mL) was added CsCO (1.77 g, 5.42 mmol, 2 equiv.). Then, 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.5 g, 2.71 mmol, 1 equiv.) in DMF was added dropwise at 0 °C. The mixture was stirred at 25 °C for 1 h. LCMS showed the reaction was complete. H0 (10 mL) was added. The reaction mixture was extracted with EtOAc (15 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 12 g, elution with a 0-50% ethyl acetate / petroleum ether gradient at 50 mL / min) to give the compound (E)-1-(4-bromobut-2-en-1-yl)-5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.4 g, 1.26 mmol, 46.50% yield) as a pale yellow solid. 1 H NMR (DMSO-d 6,400 MHz) δ 8.27 (s, 1H), 6.09-6.01 (m, 1H), 5.82-5.73 (m, 1H), 5.16 (d, J = 5.6 Hz, 2H), 4.16 (s, 3H), 4.11 (d, J = 7.6 Hz, 2H). The compound 2-[(E)-4-bromobut-2-enyl]-5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine (0.28 g, 881.70 μmol, 32.55% yield) was obtained as a white solid.

[0340] Preparation of (E)-5-chloro-7-methoxy-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine [ka] To a solution of 1-[(E)-4-bromobut-2-enyl]-5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine (0.12 g, 377.87 μmol, 1 equiv.) in MeOH (1.5 mL) was added NaOMe (30.62 mg, 566.81 μmol, 1.5 equiv.). The mixture was stirred at 25° C. for 12 hours. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The compound (E)-5-chloro-7-methoxy-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine (0.12 g, crude) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.26 (s, 1H), 5.90~5.84 (m, 1H), 5.63~5.56 (m, 1H), 5.14 (d, J=5.6Hz, 2H), 4.15 (s, 3H), 3.83 (d, J=4.0 Hz, 2H), 3.17 (s, 3H).

[0341] Preparation of (E)-5-chloro-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme C-3) [ka] To a solution of 5-chloro-7-methoxy-1-[(E)-4-methoxybut-2-enyl]pyrazolo[4,3-d]pyrimidine (0.12 g, 446.60 μmol, 1 equiv.) in HO (1 mL) and THF (1 mL) was added LiOH·HO (56.22 mg, 1.34 mmol, 3 equiv.). The mixture was stirred at 25 °C for 10 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove THF. The mixture was then adjusted to pH 5 with HCl (2 N) and extracted with EtOAc (10 mL × 5). The combined organic layers were washed with brine (5 mL × 1), dried over NaSO, filtered, and concentrated under reduced pressure. The compound (E)-5-chloro-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (70 mg, crude) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 13.30 (s, 1H), 7.98 (s, 1H), 5.88-5.82 (m, 1H), 5.61-5.55 (m, 1H), 5.13 (d, J = 4.8 Hz, 2H), 3.82 (d, J = 4.4 Hz, 2H), 3.18 (s, 3H).

[0342] Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 3 in Scheme C-3) [ka] To a solution of 5-chloro-1-[(E)-4-methoxybut-2-enyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (70 mg, 274.86 μmol, 1 equiv.) in t-BuOH (2 mL) was added (3,4-dichlorophenyl)methanamine (96.78 mg, 549.73 μmol, 73.31 μL, 2 equiv.). The mixture was stirred at 100°C for 12 h. LC-MS and HPLC indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions: column: Nano-micro Kromasil C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30% to 45%, 10 min). The compound (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (54.8 mg, 133.12 μmol, 48.43% yield, 95.77% purity) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.07 (s, 1H), 7.59~7.57 (m, 3H), 7.32 (d, J = 10.0 Hz, 1H), 6.60 (s, 1H), 5.85~5.80 (m, 1H), 5.55~5.51 (m, 1H), 5.03 (d, J = 5.2 Hz, 2H), 4.47 (d, J = 5.6 Hz, 2H), 3.81 (d, J = 5.6 Hz, 2H), 3.17 (s, 3H). HPLC: 95.77% (220 nm), 95.00% (215 nm), 96.86% (254 nm). MS (ESI): C 17 H 17 Calculated mass of Cl2N5O2: 393.08, measured m / z: 394.1 [M+H] + .

[0343] compound 193 Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0344] Preparation of sodium 2-methoxyethanolate [ka] To a solution of NaH (683.36 mg, 17.08 mmol, 2.05 μL, 60% purity, 1.3 equivalents) in CH3-THF (10 mL) was added dropwise 2-methoxyethanol (1 g, 13.14 mmol, 1.04 mL, 1 equivalent) at 0 °C. The mixture was stirred at 0 °C for 2 hours. TLC showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. Compound sodium 2-methoxyethanolate (0.4 g, 4.08 mmol, 31.03% yield) was obtained as a white solid.

[0345] Preparation of (E)-5-chloro-7-(2-methoxyethoxy)-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine [ka] To a solution of 2-methoxyethoxysodium (74.12 mg, 755.74 μmol, 1.5 equiv.) in 2-methoxyethanol (2 mL) was added 1-[(E)-4-bromobut-2-enyl]-5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine (0.16 g, 503.83 μmol, 1 equiv.). The mixture was stirred at 25 °C for 1 h. LC-MS indicated the reaction was complete. HO (5 mL) was added. The reaction mixture was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (5 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 4 g, eluting with a 0–60% ethyl acetate / petroleum ether gradient at 36 mL / min). The eluate was then concentrated under reduced pressure. The compound (E)-5-chloro-7-(2-methoxyethoxy)-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine (70 mg, 196.19 μmol, yield 38.94%) was obtained as a yellow oil. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.26 (s, 1H), 5.90~5.85 (m, 1H), 5.72~5.67 (m, 1H), 5.13 (d, J = 5.6 Hz, 2H), 4.68 (dd, J = 6.4 Hz, 4.4Hz, 2H), 3.91 (d, J = 4.8 Hz, 2H), 3.78 (d, J = 2.0 Hz, 2H), 3.44~3.40 (m, 4H), 3.39 (s, 3H), 3.20 (s, 3H).

[0346] Preparation of (E)-5-chloro-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme C-3) [ka]

[0347] To a solution of 5-chloro-7-(2-methoxyethoxy)-1-[(E)-4-(2-methoxyethoxy)but-2-enyl]pyrazolo[4,3-d]pyrimidine (70 mg, 196.19 μmol, 1 equiv.) in HO (1 mL) and THF (1 mL) was added LiOH·HO (24.70 mg, 588.56 μmol, 3 equiv.). The mixture was stirred at 25 °C for 10 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove THF. The mixture was adjusted to pH = 5 with HCl (2 N) and then extracted with EtOAc (10 mL × 5). The combined organic layers were washed with brine (5 mL × 1), dried over NaSO, filtered, and concentrated under reduced pressure. The compound (E)-5-chloro-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (60 mg, crude) was obtained as a pale yellow oil. 1 H NMR (DMSO-d 6, 400 MHz) δ 13.30 (s, 1H), 7.98 (s, 1H), 5.89~5.83 (m, 1H), 5.61~5.54 (m, 1H), 4.13 (d, J = 5.6Hz, 2H), 3.90 (d, J = 5.2 Hz, 2H), 3.46~3.41 (m, 4H), 3.21(s, 3H).

[0348] Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 3 in Scheme C-3) [ka] To a solution of 5-chloro-1-[(E)-4-(2-methoxyethoxy)but-2-enyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (60 mg, 200.85 μmol, 1 equiv.) in t-BuOH (2 mL) was added (3,4-dichlorophenyl)methanamine (70.72 mg, 401.71 μmol, 53.57 μL, 2 equiv.). The mixture was stirred at 100°C for 12 hours. LC-MS and HPLC indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions: column: Boston Prime C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30% to 55%, 10 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by lyophilization to obtain the compound (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-(2-methoxyethoxy)but-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (59.8 mg, 136.43 μmol, yield 67.93%) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.60~7.57 (m, 3H), 7.33 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.74 (s, 1H), 5.87~5.79 (m, 1H), 5.56~5.50 (m, 1H), 4.03 (d, J = HPLC: 99.52% (220 nm), 99.29% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O3: 437.10, measured m / z: 438.1 [M+H] + .

[0349] Compound 194 Preparation of ethyl 4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-4-carboxylate hydrochloride

[0350] 1-tert-Butyl 4-ethyl 4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-1,4-dicarboxylate was prepared according to the procedure described herein as Steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 1-tert-butyl 4-ethyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-1,4-dicarboxylate (0.22 g, 379.65 μmol, 66.80% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.59~7.57 (m, 3H), 7.32 (d, J = 8.4 Hz, 1H), 8.82 (s, 1H), 4.61 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H), 4.04 (q, J = 6.8 Hz, 2H), 3.76~3.73 (m, 2H), 2.78~2.74 (m, 2H). 1.85~1.81 (m, 2H), 1.45~1.37 (m, 11H), 1.12 (t, J = 6.8 Hz, 3H).

[0351] Preparation of tert-butyl 4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-4-(hydroxymethyl)piperidine-1-carboxylate [ka] To a solution of O1-tert-butyl O4-ethyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-1,4-dicarboxylate (0.1 g, 172.57 μmol, 1 equiv.) in THF (3 mL) was added LiBH4 (15.04 mg, 690.28 μmol, 4 equiv.) at 0 °C. The mixture was then stirred at 55 °C for 6 h. LCMS and TLC showed the reaction was complete. The mixture was quenched with ice water (2 mL), and the organic solvent was removed under reduced pressure. The aqueous solution was extracted with EtOAc (5 mL × 4). The combined organic layers were washed with brine (2 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give tert-butyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]-4-(hydroxymethyl)piperidine-1-carboxylate (90 mg, 167.46 μmol, 97.04% yield) as a white solid. 1 H NMR (DMSO-d 6, 400MHz) δ 7.61~7.30 (m, 3H), 7.30 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H), 4.47 (s, 2H), 3.40~3.30 (m, 4H). 2H), 1.88~1.94 (m, 2H), 1.38~1.26 (m, 11H).

[0352] Compound 195 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((4-(hydroxymethyl)piperidin-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride [ka] To a mixture of tert-butyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]-4-(hydroxymethyl)piperidine-1-carboxylate (0.13 g, 241.89 μmol, 1 equiv.) in EtOAc (4 mL), HCl / EtOAc (4 M, 6.05 mL, 100 equiv.) was added, and the mixture was stirred at 20 °C for 2 h. LCMS showed the reaction was complete. Some white solid was formed. After filtration, the solid was collected. The solid was purified by preparative HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 10% to 35%, 12 min). The eluent was removed under reduced pressure. The compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[4-(hydroxymethyl)-4-piperidyl]methyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one (102 mg, 210.83 μmol, 87.16% yield, 97.93% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.70 (s, 1H), 8.51 (s, 1H), 7.95 (s, 1H), 7.71 (s, 1H), 7.66~7.61 (m, 2H), 7.38 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 4.59 (d, J = 4.4 Hz, 2H), 4.50 (s, 2H), 3.37 (s, 2H). 3.08~3.03 (m, 4H), 1.68~1.64 (m, 2H), 1.51~1.45 (m, 2H). HPLC: 97.93% (220 nm), 97.47% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 23 Calculated mass of Cl3N6O2: 436.12 m / z Found: 437.1 [M+H] + .

[0353] Preparation of 1-(tert-butoxycarbonyl)-4-((5-chloro-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-4-carboxylic acid (Step 2 in Scheme C-3) [ka]

[0354] A solution of O1-tert-butyl O4-ethyl 4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]piperidine-1,4-dicarboxylate (0.1 g, 220.30 μmol, 1 equiv.) and LiOH·HO (46.22 mg, 1.10 mmol, 5 equiv.) in MeOH (1 mL), THF (1 mL), and HO (1 mL) was stirred at 20 °C for 3 h. NaOH (17.62 mg, 440.61 μmol, 2 equiv.) was then added, and the mixture was stirred at 70 °C for 5 h. TLC showed the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was slowly adjusted to pH 6–7 with 2 N HCl, which produced some solid. The solid was collected after filtration. The aqueous solution was then extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (5 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give 1-tert-butoxycarbonyl-4-[(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]piperidine-4-carboxylic acid (80 mg, 194.25 μmol, 88.17% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.94 (s, 1H), 4.71 (s, 2H), 3.76~3.72 (m, 2H), 2.80~2.79 (m, 2H), 1.91~1.84 (m, 2H), 1.40~1.35 (m, 11H).

[0355] Preparation of 1-(tert-butoxycarbonyl)-4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-4-carboxylic acid (Step 3 in Scheme C-3) [ka] A solution of (3,4-dichlorophenyl)methanamine (68.39 mg, 388.50 μmol, 51.81 μL, 2 equiv.) and 1-tert-butoxycarbonyl-4-[(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]piperidine-4-carboxylic acid (80 mg, 194.25 μmol, 1 equiv.) in t-BuOH (2 mL) was heated at 100° C. for 30 h. LCMS showed the reaction was nearly complete. The solvent was removed under reduced pressure. The compound 1-tert-butoxycarbonyl-4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-4-carboxylic acid (0.13 g, crude) was obtained as an off-white solid. MS (ESI): C 24 H 28 Calculated mass of Cl2N6O5: 550.15 m / z Found: 495.0 [M+H-Boc] + .

[0356] compound 196 Preparation of 4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-4-carboxylic acid hydrochloride [ka] To a mixture of 1-tert-butoxycarbonyl-4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-4-carboxylic acid (0.11 g, 199.48 μmol, 1 equiv.) in EtOAc (5 mL), HCl / EtOAc (4 M, 4.99 mL, 100 equiv.) was added, and the mixture was stirred at 20 °C for 2 h. LCMS showed the reaction was complete. Some solid was formed. After filtration, the solid was collected. The solid was purified by preparative HPLC (column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 10% to 40%, 12 min). The eluate was dried under lyophilization. The compound 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-4-carboxylic acid (40.2 mg, 81.00 μmol, 40.60% yield, 98.28% purity, HCl) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.77~8.75 (m, 1H), 8.44~8.42 (m, 1H), 7.65 (s, 1H), 7.61~7.59 (m, 2H), 7.25 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.33~7.32 HPLC: 98.28% (220 nm), 98.41% (215 nm), 95.91% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl3N6O3: 450.10 m / z Measured: 451.0 [M+H] + .

[0357] Preparation of ethyl 4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)piperidine-4-carboxylate hydrochloride [ka] To a mixture of O1-tert-butyl O4-ethyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-1,4-dicarboxylate (30 mg, 51.77 μmol, 1 equiv.) in EtOAc (1 mL), HCl / EtOAc (4 M, 1.29 mL, 100 equiv.) was added, and the mixture was stirred at 20° C. for 2 h. TLC showed the reaction was complete. Some white solid was formed. After filtration, the solid was collected. The solid was concentrated under reduced pressure to give ethyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]methyl]piperidine-4-carboxylate (15.6 mg, 29.46 μmol, 56.90% yield, 97.41% purity, HCl) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.77~8.74 (m, 1H), 8.52~8.48 (m, 1H), 7.63 (s, 1H), 7.60~7.58 (m, 2H), 7.33 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.09~7.07 (m, 1H), 4.65 (s, 2H), 4.49 (d, J = 5.6 Hz, 2H), 4.07 (q, J = 7.2 Hz, 2H), 3.27~3.23 (m, 2H), 2.77~2.67 (m, 2H), 2.02~1.99 (m, 2H), 1.79~1.76 (m, 2H), 1.15 (t, J = 7.2 Hz, 3H). HPLC: 97.41% (220 nm), 96.42% (215 nm), 100.00% (254 nm). MS (ESI): C 21 H 25Calculated mass of Cl3N6O3: 478.13 m / z Measured: 479.1 [M+H] + .

[0358] Compound 197 Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)acetamide

[0359] Preparation of 2-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)acetamide (Step 1 in Scheme C-3) [ka] To a solution of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (700 mg, 3.79 mmol, 1 equiv.) and 2-chloroacetamide (709.26 mg, 7.58 mmol, 2 equiv.) in DMF (5 mL) was added KCO (1.05 g, 7.58 mmol, 2 equiv.). The mixture was stirred at 70 °C for 10 h. TLC showed the reaction was complete. The mixture was poured into HO (7 mL), and the mixture was adjusted to pH = 7 with HCl (2 M). The mixture was extracted with DCM and i-PrOH (3:1, 10 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a mixture of 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)acetamide (900 mg, 3.72 mmol, 98.21% yield) and 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)acetamide as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.26 (s, 1H), 5.12 (s, 2H), 4.11 (s, 3H).

[0360] Preparation of 2-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)acetamide [ka] To a mixture of 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)acetamide (392 mg, 1.62 mmol, 1 equiv.) and 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)acetamide (1.00 equiv.) in DMF (5 mL) was added dropwise DIEA (1.05 g, 8.11 mmol, 1.41 mL, 5 equiv.) and SEM-Cl (1.03 g, 6.16 mmol, 1.09 mL, 3.8 equiv.) at 0 °C. The mixture was then stirred at 40 °C for 10 h. TLC showed the reaction was complete. The mixture was poured into HO (5 mL) and adjusted to pH = 7 with HCl (3 M) at 0 °C. The mixture was extracted with EtOAc (8 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 4 g, eluting with a 0–55% ethyl acetate / petroleum ether gradient at 40 mL / min). The eluate was concentrated under reduced pressure to give 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (30 mg, 80.67 μmol, 4.97% yield) and 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (60 mg, 161.34 μmol, 9.94% yield) as white solids. 1 H NMR (DMSO-d 6, 400 MHz) δ 8.29 (s, 1H), 5.21 (s, 2H), 4.53 (d, J = 6.8 Hz, 2H), 4.11 (s, 3H), 3.44 (t, J = 8.4 Hz, 2H), 1.24 (s, 2H), -0.02 (s, 9H).

[0361] Preparation of 2-(5-chloro-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)acetamide (Step 2 in Scheme C-3) [ka]

[0362] To a solution of 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (75 mg, 201.67 μmol, 1 equiv.) in MeOH (1 mL) and HO (1 mL) was added LiOH·HO (25.39 mg, 605.01 μmol, 3 equiv.). The mixture was stirred at 25 °C for 10 h. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure, and then the aqueous solution was adjusted to pH = 7 with HCl (2 M). The aqueous solution was extracted with EtOAc (3 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give 2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (72 mg, 201.19 μmol, 99.76% yield) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.99 (s, 1H), 5.19 (s, 2H), 4.52 (d, J = 6.4 Hz, 2H), 3.49~3.41 (m, 2H), 0.84~0.82 (m, 2H), -0.02 (s, 9H).

[0363] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)acetamide (Step 3 in Scheme C-3) [ka] A solution of 2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (50 mg, 139.72 μmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (49.19 mg, 279.43 μmol, 37.27 μL, 2 equiv.) in t-BuOH (2 mL) was stirred at 110 °C for 10 h. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Xtimate C18 100 mm × 25 mm 3 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 40% to 60%, 12 min). The mixture was dried under lyophilization. The residue was purified twice by preparative HPLC (Phenomenex Luna C18 150 mm × 30 mm 5 μm column; mobile phase: [water (0.1% TFA)-MeCN]; B%: 45%–65%, 12 min). The mixture was lyophilized to dryness to give 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-N-(2-trimethylsilylethoxymethyl)acetamide (5.4 mg, 10.20 μmol, yield 7.30%, purity 94.00%) as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 11.08 (s, 1H), 8.71 (t, J = 6.0 Hz, 1H), 7.58~7.55 (m, 3H), 7.31 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.64 (s, 1H), 5.06 (s, HPLC: 94.00% (220 nm), 92.65% (215 nm), 76.77% (254 nm). MS (ESI): C 20 H 26 Calculated mass of Cl2N6O3Si: 496.12, measured m / z: 497.1 [M+H] + .

[0364] Compound 198 Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N,2,2-trimethylpentanamide

[0365] Compound 199 5-(5-((3,4-Dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-2,2-dimethylpentanoic acid was prepared according to the procedure described herein as steps 1-3 in Scheme C-3. [ka] The procedure afforded the desired compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-2,2-dimethyl-pentanoic acid (93.4 mg, 212.99 μmol, 42.42% yield, 99.953% purity) as a white solid, 26.3 mg of which was used further. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.65~7.51 (m, 3H), 7.33 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H), 4.48 (d, J = 4.4 Hz, 2H), 4.39 (s, 2H), 1.72 (s, 2H), 1.44~1.33 (m, 2H), 1.03 (s, 6H). HPLC: 99.95% (220 nm), 99.94% (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O3: 437.10, measured m / z: 438.1 [M+H] + .

[0366] Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N,2,2-trimethylpentanamide [ka] A mixture of 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-2,2-dimethylpentanoic acid (30 mg, 68.45 μmol, 1 equiv.), methanamine hydrochloride (9.24 mg, 136.89 μmol, 2 equiv.), EDCI (15.75 mg, 82.13 μmol, 1.2 equiv.), HOBt (1.85 mg, 13.69 μmol, 0.2 equiv.), and DIEA (26.54 mg, 205.34 μmol, 35.77 μL, 3 equiv.) in DMF (1 mL) was stirred at 20° C. for 16 h. LC-MS indicated the reaction was complete. After filtration, the filtrate was purified by preparative HPLC (Phenomenex Luna C18 150 mm x 30 mm 5 μm column; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15%-35%, 12 min). The aqueous solution was lyophilized to give the compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-N,2,2-trimethylpentanamide (12.7 mg, 27.80 μmol, 40.62% yield, 98.800% purity) as an off-white solid. 1 H NMR (DMSO-d 6,400 MHz) δ 7.64 (d, J = 1.2 Hz, 1H), 7.61 (t, J = 3.6 Hz, 2H), 7.36 (d, J = 6.8 Hz, 2H), 4.56 (d, J = 4.4 Hz, 2H), 4.37 (t, J = 6.8 Hz, HPLC: 98.80% (220 nm), 98.35% (215 nm), 98.34% (254 nm). MS (ESI): C 20 H 24 Calculated mass of Cl2N6O2: 450.13, measured m / z: 451.1 [M+H] + .

[0367] compound 200 Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-methyl-2-phenylpentanamide

[0368] Preparation of diethyl 2-(3-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)propyl)-2-phenylmalonate (Step 1 in Scheme C-3) [ka] A mixture of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (300 mg, 1.63 mmol, 1 equiv.), diethyl 2-(3-bromopropyl)-2-phenylpropanedioate (870.92 mg, 2.44 mmol, 470.47 μL, 1.5 equiv.), and CsCO (1.06 g, 3.25 mmol, 2 equiv.) in DMF (3 mL) was stirred at 25 °C for 5 h. TLC showed the reaction was complete. The reaction mixture was quenched with HO (5 mL) and extracted with EtOAc (5 mL × 3). The combined organic layers were washed with brine (5 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 4 g, elution with a 0-40% ethyl acetate / petroleum ether gradient at 65 mL / min). The eluent was removed under reduced pressure. The compound diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)propyl]-2-phenyl-propanedioate (450 mg, 976.33 μmol, 60.07% yield) was obtained as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) δ 7.99 (s, 1H), 7.27 (s, 5H), 4.51 (t, J = 6.8 Hz, 2H), 4.22–4.16 (m, 7H), 2.29–2.25 (m, 2H), 1.88–1.84 (m, 2H), 1.25–1.18 (m, 6H). The compound diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-2-yl)propyl]-2-phenyl-propanedioate (270 mg, 585.80 μmol, 36.04% yield) was obtained as a colorless oil. 1 H NMR (CDCl 3, 400 MHz) δ 7.94 (s, 1H), 7.32~7.27 (m, 5H), 4.40 (t, J = 7.2 Hz, 2H), 4.24~4.20 (m, 7H), 2.29~2.24 (m, 2H), 2.00~1.96 (m, 2H), 1.23~1.20 (m, 6H).

[0369] Preparation of 5-(5-chloro-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-phenylpentanoic acid [ka] A mixture of diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)propyl]-2-phenyl-propanedioate (200 mg, 433.92 μmol, 1 equiv.) and NaOH (104.13 mg, 2.60 mmol, 6 equiv.) in MeOH (1 mL) and HO (1 mL) was stirred at 80° C. for 3 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH = 6 with 3 N HCl and extracted with EtOAc (6 mL × 3). The combined organic layers were washed with brine (6 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The compound 5-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-phenyl-pentanoic acid (175 mg, crude) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 13.27 (s, 1H), 12.25 (s, 1H), 7.94 (s, 1H), 7.29~7.20 (m, 5H), 4.51 (t, J = 6.4 Hz, 2H), 3.49 (t, J = 7.6 Hz, 1H), 1.90~1.58 (m, 4H).

[0370] Compound 201 Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-phenylpentanoic acid (Step 3 in Scheme C-3) [ka]

[0371] A mixture of 5-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-phenylpentanoic acid (175 mg, 504.66 μmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (177.68 mg, 1.01 mmol, 134.61 μL, 2 equiv.) in 2-methyl-2-butanol (3 mL) was stirred at 140 °C for 4 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 45% to 60%, 10 min). The solvent was removed by lyophilization. The compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-2-phenyl-pentanoic acid (131.2 mg, 264.99 μmol, 52.51% yield, 98.23% purity) was obtained as a white solid, 31.2 mg of which was used further. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.60~7.56 (m, 3H), 7.34~7.20 (m, 6H), 6.82 (s, 1H), 4.48 (d, J = 5.2 Hz, 2H), 4.42 (t, J = 6.0 Hz, 2H), 3.49 (t, J = 8.0 HPLC: 98.23 % (220 nm), 97.82 % (215 nm), 100.00% (254 nm). MS (ESI): C 23 H 21 Calculated mass of Cl2N5O3: 485.10, measured m / z: 486.1 [M+H] + .

[0372] Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-N-methyl-2-phenylpentanamide [ka] A mixture of 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-2-phenylpentanoic acid (30 mg, 61.68 μmol, 1 equiv.), methanamine hydrochloride (8.33 mg, 123.37 μmol, 2 equiv.), DIEA (23.92 mg, 185.05 μmol, 32.23 μL, 3 equiv.), HOBt (1.67 mg, 12.34 μmol, 0.2 equiv.), and EDCI (14.19 mg, 74.02 μmol, 1.2 equiv.) in DMF (0.5 mL) was stirred at 25 °C for 12 h. LC-MS indicated the reaction was complete. The reaction mixture was filtered to remove insoluble material. The filtrate was purified by preparative HPLC (HCl condition: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.04% HCl)-MeCN]; B%: 25% to 50%, 12 min). The solvent was removed by lyophilization. The compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-N-methyl-2-phenyl-pentanamide (13.8 mg, 27.59 μmol, 44.73% yield, 99.85% purity) was obtained as a white solid. 1 H NMR (DMSO-d 6, 400 MHz) δ 7.94 (s, 1H), 7.78 (s, 1H), 7.65~7.60 (m, 3H), 7.37 (d, J = 7.2 Hz, 1H), 7.24~7.19 (m, 5H), 4.57 (s, 2H), 4.42 (s, HPLC: 99.85 % (220 nm), 99.93 % (215 nm), 99.21% (254 nm). MS (ESI): C 24 H 24Calculated mass of Cl2N6O2: 498.13, measured m / z: 499.1 [M+H] + .

[0373] compound 202 Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-methylpentanoic acid

[0374] Diethyl 2-(3-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)propyl)-2-methylmalonate was prepared according to the procedure described herein as Step 1 in Scheme C-3. [ka] A mixture of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.3 g, 1.63 mmol, 1 equiv.), diethyl 2-(3-bromopropyl)-2-methyl-propanedioate (623.65 mg, 2.11 mmol, 201.22 μL, 1.3 equiv.), and CsCO (1.06 g, 3.25 mmol, 2 equiv.) in DMF (2 mL) was stirred at 25 °C for 4 h. TLC showed the reaction was complete. The reaction mixture was quenched with HO (15 mL) at 20 °C and then extracted with EtOAc (15 mL × 3). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 12 g, elution with a 0-30% ethyl acetate / petroleum ether gradient at 35 mL / min). The eluent was removed under reduced pressure. The compound diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)propyl]-2-methyl-propanedioate (440 mg, 1.10 mmol, 67.88% yield) was obtained as a white solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.25 (s, 1H), 4.51 (t, J = 5.6 Hz, 2H), 4.16 (s, 3H), 4.05 (q, J = 7.2 Hz, 4H), 1.73 (s, 4H), 1.27 (s, 3H), 1.09 (t, J = 6.8 Hz, 6H).

[0375] Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-methylpentanoic acid (Step 2 in Scheme C-3) [ka] A mixture of diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)propyl]-2-methyl-propanedioate (380 mg, 952.76 μmol, 1 equiv.) and NaOH (266.77 mg, 6.67 mmol, 7 equiv.) in HO (2 mL) and MeOH (2 mL) was stirred at 100° C. for 3 h. LC-MS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH = 5 with HCl (3 M) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (5 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The compound 2-[3-(5-chloro-7-hydroxy-pyrazolo[4,3-d]pyrimidin-2-yl)propyl]-2-methyl-propanedioic acid (230 mg, 699.71 μmol, 73.44% yield) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 13.27 (s, 1H), 12.66 (s, 1H), 7.96 (s, 1H), 4.50 (t, J = 6.4 Hz, 2H), 1.80~1.69 (m, 2H), 1.68~1.60 (m, 2H), 1.20 (s, 3H).

[0376] Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)-2-methylpentanoic acid [ka]

[0377] A mixture of 2-[3-(5-chloro-7-hydroxy-pyrazolo[4,3-d]pyrimidin-2-yl)propyl]-2-methyl-propanedioic acid (170 mg, 517.18 μmol, 1 equiv.) and (3,4-dichlorophenyl)methanamine (182.09 mg, 1.03 mmol, 137.95 μL, 2 equiv.) in 2-methylbutan-2-ol (3 mL) was stirred at 140 °C for 16 h. LC-MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30% to 50%, 10 min). The aqueous solution was removed by lyophilization to give the compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]-2-methyl-pentanoic acid (80 mg, 187.63 μmol, 36.28% yield, 99.510% purity) as a white solid, 30.0 mg of which was used for further processing. 1 H NMR (DMSO-d6, 400 MHz) δ 7.65~7.55 (m, 3H), 7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.84 (s, 1H), 4.49 (d, J = 5.6 Hz, 2H), 4.41 (t, J = 7.2 HPLC: 99.51% (220 nm), 99.27% ​​(215 nm), 100.00% (254 nm).MS (ESI): C 18 H 19Calculated mass of Cl2N5O3: 423.09, measured m / z: 424.1 [M+H] + .

[0378] compound 203 Preparation of ethyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate [ka] To a solution of 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]pentanoic acid (60 mg, 146.25 μmol, 1 equiv.) (Steps 1–3 in Scheme C-3) in EtOH (2 mL) was added SOCl (87.00 mg, 731.25 μmol, 53.05 μL, 5 equiv.) dropwise at 0° C. The mixture was then stirred at 80° C. for 10 h. LCMS indicated the reaction was complete. Some white solid was formed. The solid was collected after filtration and then concentrated under reduced pressure. The compound ethyl 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]pentanoate (57.1 mg, 127.37 μmol, 87.09% yield, 97.77% purity) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.62~7.59 (m, 3H), 7.35 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.26 (s, 1H), 4.52 (d, J = 5.2 Hz, 2H), 4.43 (t, J = 6.8 HPLC: 97.77% (220 nm), 97.72% (215 nm), 98.01% (254 nm). MS (ESI): C 19 H21 Calculated mass of Cl2N5O3: 437.10 m / z Measured: 438.0 [M+H] + .

[0379] compound 204 Preparation of isopropyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate [ka]

[0380] To a solution of 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]pentanoic acid (40 mg, 97.50 μmol, 1 equiv.) (Steps 1–3 in Scheme C-3) in i-PrOH (1 mL) was added SOCl (58.00 mg, 487.50 μmol, 35.36 μL, 5 equiv.) dropwise at 0° C. The mixture was then stirred at 80° C. for 20 h. LCMS indicated the reaction was complete. Some white solid was formed. The solid was collected after filtration and then concentrated under reduced pressure. The compound isopropyl 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]pentanoate (23.9 mg, 51.15 μmol, yield 52.46%, purity 96.81%) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.61~7.59 (m, 3H), 7.33 (d, J = 2.0 Hz, 8.0 Hz, 1H), 4.85~4.82 (m, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.42 (t, J = HPLC: 96.81% (220 nm), 96.76% (215 nm), 94.56% (254 nm). MS (ESI): C20 H 23 Calculated mass of Cl2N5O3: 451.12 m / z Found: 452.1 [M+H] + .

[0381] compound 205 Preparation of ethyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butanoate [ka] To a solution of 4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]butanoic acid (45 mg, 113.57 μmol, 1 equiv.) (Steps 1–3 in Scheme C-3) in EtOH (5 mL) was added SOCl2 (67.56 mg, 567.86 μmol, 41.19 μL, 5 equiv.) at 0 °C. The mixture was stirred at 80 °C for 16 h. LC-MS showed the reaction was complete. The reaction mixture was filtered to remove insoluble materials and concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30%–60%, 10 min). The aqueous solution was lyophilized to give compound ethyl 4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]butanoate (15.6 mg, 36.65 μmol, 32.27% yield, 99.67% purity) as a pale yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.66~7.59 (m, 3H), 7.37 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 4.57 (d, J = 4.8 Hz, 2H), 4.46 (t, J = 6.8 Hz, 2H), 3.99 (d, J = 7.2 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 2.07~2.00 (m, 2H), 1.14 (t, J = 6.8 Hz, 3H). HPLC: 99.67 % (220 nm), 99.65 % (215 nm), 100.00% (254 nm). MS (ESI): C 18 H 19 Calculated mass of Cl2N5O3: 423.09, measured m / z: 424.1 [M+H] + .

[0382] compound 206 Preparation of isopropyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butanoate [ka]

[0383] To a solution of 4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]butanoic acid (Steps 1–3 in Scheme C-3) (45 mg, 113.57 μmol, 1 equiv.) in i-PrOH (5 mL) was added SOCl2 (67.56 mg, 567.86 μmol, 41.19 μL, 5 equiv.) at 0°C. The mixture was stirred at 80°C for 16 h. LC-MS showed the reaction was complete. The reaction mixture was filtered to remove insoluble material and concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 35%–55%, 10 min). The aqueous solution was lyophilized to obtain compound isopropyl 4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidin-1-yl]butanoate (17.0 mg, 38.79 μmol, yield 34.15%, purity 100.00%) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.67~7.58 (m, 3H), 7.36 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 4.86~4.80 (m, 1H), 4.55 (d, J = 5.6 Hz, 2H), 4.46 (t, J = HPLC: 100.00 % (220 nm), 100.00 % (215 nm), 100.00% (254 nm). MS (ESI): C 19 H 21 Calculated mass of Cl2N5O3: 437.10, measured m / z: 438.1 [M+H] + .

[0384] compound 207 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(((2R,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one

[0385] Preparation of ((2R,5R)-5-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-1,4-dioxan-2-yl)methanol and ((2S,5R)-5-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidin-1-yl)methyl)-1,4-dioxan-2-yl)methanol (Step 1 in Scheme C-3) [ka] To a solution of ((2S,5R)-5-(iodomethyl)-1,4-dioxan-2-yl)methanol (838.82 mg, 3.25 mmol, 2 equiv.) and ((2R,5R)-5-(iodomethyl)-1,4-dioxan-2-yl)methanol and 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.3 g, 1.63 mmol, 1 equiv.) in acetone (1 mL) was added CsCO (1.59 g, 4.88 mmol, 3 equiv.). The mixture was stirred at 70 °C for 16 h. LCMS showed the reaction was complete. The reaction mixture was filtered to remove insoluble material, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 mm × 30 mm 8 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 10% to 30%, 10 min). The aqueous solution was lyophilized to give the compound [(2S,5R)-5-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]-1,4-dioxan-2-yl]methanol (40 mg, 127.10 μmol, 7.82% yield) as an off-white solid. 1H NMR (CDCl3, 400 MHz) δ 8.08 (s, 1H), 5.05 (dd, J = 8.4 Hz, 14.0 Hz, 1H), 4.62 (dd, J = 5.6 Hz, 14.4 Hz, 1H), 4.26–4.24 (m, 3H), 4.21–4.10 (m, 1H), 3.97–3.56 (m, 8H). The compound [(2R,5R)-5-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]-1,4-dioxan-2-yl]methanol (40 mg, 127.10 μmol, 7.82% yield) was obtained as an off-white solid. 1 H NMR (CDCl3, 400 MHz) δ 8.03~8.01 (m, 1H), 4.60~4.55 (m, 1H), 4.49~4.44 (m, 1H), 4.23~4.18 (m, 3H), 3.95~3.93 (m, 1H), 3.81~3.72 (m, 2H), 3.56~3.35 (m, 6H).

[0386] Preparation of 5-chloro-1-(((2R,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (Step 2 in Scheme C-3) [ka]

[0387] To a solution of [(2R,5R)-5-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)methyl]-1,4-dioxan-2-yl]methanol (40 mg, 127.10 μmol, 1 equiv.) in MeOH (2 mL) and HO (2 mL) was added LiOH·HO (16.00 mg, 381.29 μmol, 3 equiv.). The mixture was stirred at 25 °C for 3 h. TLC showed the reaction was comple...

Claims

1. Formula I 【Chemistry 1】 wherein A and B are independently N or CH; n is 0 to 3; R １ is (CH ２ ) ｍ - {(V) ｏ - (CH ２ ) ｐ } ｑ -W, V is CH ２ , CH=CH, C≡C, CO, O, S, SO, SO ２ , N.R. ４ , CHR ５ ,OC(O),(O)CO,CONR ６ , N.R. ７ CO, SO ２ NH, NHSO ２ ; C ３～８ is cycloalkyl, R ４ , R ６ and R ７ each independently represents H or C １～６ is alkyl, R ５ is OH or C １～６ Alkyl, CH(R ８ R ９ ) and R ８ and R ９ each independently represents H, halo, or C １～６ is alkyl, W is H, halo, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C ３～８ Cycloalkyl, substituted or unsubstituted C ２～８ Heterocyclyl, substituted or unsubstituted C ６～１４ Aryl, substituted or unsubstituted C １～１０ Heteroaryl, NH ２ , C.N., O.R. １０ , S.R. １１ , C.O.R. １２ , O.C.O.R. １３ , N.R. １４ COR １５ , N.R. １６ R １７ , N.R. １８ (CO)NHR １９ , CH(CO ２ R ２０ ) ２ , CO ２ R ２１ , NHSO ２ R ２２ , C.O.R. ２３ R ２４ , C.H. ２ CO ２ R ２５ , S(O)R ２６ or S(O ２ ) R ２７ and R １０ ~R ２７ each independently represents H, substituted or unsubstituted C １～６ Alkyl, substituted or unsubstituted C ３～８ Cycloalkyl, substituted or unsubstituted C ２～８ Heterocyclyl, substituted or unsubstituted C ６～１４ Aryl, substituted or unsubstituted C １～１０ is heteroaryl, m is 1 to 5, o is 0 to 4, p is 0 to 4, and q is 0 to 4; R ２ is H, halo, CN, substituted or unsubstituted C １～６ Alkyl, CO ２ R ２１ , C.O.R. ２３ R ２４ , substituted or unsubstituted C ２～８ Heterocyclyl or substituted or unsubstituted C １～１０ is heteroaryl, R ３ are Cl, F, Br, I, OH, CN, C １～６ Alkyl, CF ３ , CHF ２ , C.F. ３ CH ２ and OCF ３ C substituted with one or more substituents selected from the group ６～１４ Aryl or C １～１０ is heteroaryl, R ０ is H, CH ２ OPO (OH) ２ , C.H. ２ OCONHCH ２ (CH ２ ) ｔ OPO (OH) ２ , C.H. ２ OCOCH ２ (CH ２ ) ｔ OPO (OH) ２、 COO (CH ２ ) ｔ OPO (OH) ２ , C.H. ２ OPO(OH)OPO(OH) ２ or (CR ３０ R ３１ O)s-X-Y-(CR ３０ R ３１ ) ｔ -OPO(OR ２８ ) (OR ２９ ) and X is a direct bond or (C=O), Y is a direct bond or oxygen, s is 0 or 1; t is 1, 2, or 3; R ２８ and R ２９ are each independently hydrogen or a hydrolyzable ester group, R ２８ is hydrogen, R ２９ -P(O)OR ３２ OR ３３ It can be, R ３０ and R ３１ are each independently hydrogen or C1-4 alkyl; R ３２ and R ３３ are each independently hydrogen or a hydrolyzable ester group), or an optical isomer thereof, an isotopic isomer thereof, a prodrug or a pharmaceutically acceptable salt thereof.

2. A and B are independently N or CH; n is 0 to 3; R １ But (CH2) ｍ - {(V) ｏ - (CH ２ ) ｐ } ｑ -W, V is CH ２ , CO, O, OC(O) or (O)CO; W is H, halo, substituted or unsubstituted C1-6 alkyl, NH ２ , CN or OR １０ and R １０ is H or substituted or unsubstituted C １～６ is alkyl, m is 1 to 5, o is 0 to 4, p is 0 to 4, and q is 0 to 4; R ２ is H, halo, CN, substituted or unsubstituted C １～６ Alkyl, CO ２ R ２１ , C.O.R. ２３ R ２４ , substituted or unsubstituted C ２～８ Heterocyclyl or substituted or unsubstituted C １～１０ is heteroaryl, R ３ Cl, F, Br, I, OH, CN, C １～６ Alkyl, CF ３ , CHF ２ , C.F. ３ CH ２ and OCF ３ C substituted with one or more substituents selected from the group ６～１４ Aryl or C １～１０ is heteroaryl, R ０ is H, or an optical isomer thereof, an isotopic isomer thereof, a prodrug or a pharmaceutically acceptable salt thereof.

3. A and B are N; n is 0 to 1; R １ However, (CH ２ ) ｍ - {(V) ｏ - (CH ２ ) ｐ } ｑ -W, V is CO, O, OC(O) or (O)CO; W is H, halo, NH ２ , CN or OR １０ and R １０ is H or substituted or unsubstituted C １～６ is alkyl, m is 1 to 5, o is 0 to 1, p is 0 to 4, and q is 0 to 4; R ２ is H or halo or CN, R ３ But Cl, F, CF ３ , CHF ２ , C.F. ３ CH ２ and OCF ３ C substituted with one or more substituents selected from the group ６～１４ Aryl or C １～１０ is heteroaryl, R ０ is H, 3. The compound according to claim 2, or an optical isomer thereof, an isotopic isomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

4. A and B are N; n is 1, R １ But (CH2) ｍ - {(V) ｏ - (CH ２ ) ｐ } ｑ -W, V is CO, O, OC(O) or (O)CO; W is H, halo, NH ２ , CN or OR １０ and R １０ is H or substituted or unsubstituted C １～６ is alkyl, m is 1 or 2, o is 1, p is 1 or 2, and q is 0 or 1; R ２ is H or halo or CN, R ３ But Cl, F, CF ３ , CHF ２ , C.F. ３ CH ２ and OCF ３ C substituted with one or more substituents selected from the group ６～１４ Aryl or C １～１０ is heteroaryl, R ０ is H, 4. The compound according to claim 3, or an optical isomer thereof, an isotopic isomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

5. A and B are N; n is 1, R １ But (CH2) ｍ - {(V) ｏ - (CH ２ ) ｐ } ｑ -W, V is O, OC(O) or (O)CO; W is H, halo, NH ２ OR １０ and R １０ is H or substituted or unsubstituted C １～６ is alkyl, m is 1 or 2, o is 1, p is 1 or 2, and q is 0 or 1; R ２ is H or halo or CN, R ３ But Cl, F, CF ３ , CHF ２ , C.F. ３ CH ２ and OCF ３ C substituted with one or more substituents selected from the group ６～１４ Aryl or C １～１０ is heteroaryl, R ０ is H, 5. The compound according to claim 4, or an optical isomer thereof, an isotopic isomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

6. A and B are N; n is 1, R １ But (CH2) ｍ - {(V) ｏ - (CH ２ ) ｐ } ｑ -W, V is O, W is H, halo, NH ２ OR １０ and R １０ is H, m is 2, o is 1, p is 2, and q is 1; R ２ is H or halo or CN, R ３ But Cl, F, CF ３ , CHF ２ , C.F. ３ CH ２ and OCF ３ C substituted with one or more substituents selected from the group ６～１４ Aryl or C １～１０ is heteroaryl, R ０ is H, 6. The compound according to claim 5, or an optical isomer thereof, an isotopic isomer thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition comprising a compound of claim 1 and at least one pharmaceutically acceptable carrier.

8. 10. A method for inhibiting the growth of Gram-positive bacteria, comprising contacting an environment with an effective amount of a compound of claim 1.

9. The method of claim 8 , wherein the surface is a surface of a medical device.

10. 10. A method of treating a gram-positive bacterial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of claim 1.

11. 10. A surface coating comprising the compound of claim 1 and a coating agent, wherein the coating agent is capable of adhering the compound to a habitat.

12. 5-((3,4-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 1-allyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butyl acetate, 1-(cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-[(3,4-cyclophenyl)methylamino]-1-phenyl-6H-pyrazolo[4,3-d]pyrimidin-7-one, 1-cyclopropyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, cyclopentyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, cetyl 5-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate, 5-[(3,4-cyclophenyl)methylamino]-1-(4-pyridyl)-6H-pyrazolo[4,3-d]pyrimidin-7-one, 3-chloro-5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-cyclolobenzyl)amino)-3-fluoro-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 3-chloro-5-((3,4-dichlorobenzyl)amino)-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 1-(2-(4-acetylpiperazin-1-yl)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((4-methylmorpholin-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(3-methylpicolinoyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-difluorobenzyl)amino)-3-fluoro-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((4-chloro-3-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1,3-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholinoethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(4-hydroxybutyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one), 5-((3,4-dichlorobenzyl)amino)-1-(thiazol-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((tetrahydrofuran-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(3-hydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyrazin-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(5-methoxypentyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((2-methoxyethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-3-ylsulfonyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybut-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, ethyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate, isopropyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)pentanoate, ethyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)butanoate, methyl 3-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)propanoate, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridin-4-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one dihydrochloride, 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(oxazole-4-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(thiazole-2-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methoxypicolinoyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methylpicolinoyl)piperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 3-chloro-5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidin-7-one, 5-((3,4-difluorobenzyl)amino)-3-fluoro-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 3-chloro-5-((3,4-dichlorobenzyl)amino)-1-(1-nicotinoylpiperidin-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one hydrochloride, ammonium (2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-1-yl)ethoxy)ethoxy)methyl phosphate, (5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-7-oxo-1H-pyrazolo[4,3-d]pyrimidin-6(7H)-yl)methyl dihydrogen phosphate and pharmaceutically acceptable salts thereof.

13. 13. A pharmaceutical composition comprising a compound of claim 12 and at least one pharmaceutically acceptable carrier.

14. 13. A method for inhibiting the growth of Gram-positive bacteria, comprising contacting an environment with an effective amount of a compound of claim 12.

15. The method of claim 14 , wherein the surface is a surface of a medical device.

16. 13. A method of treating a gram-positive bacterial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of claim 12.

17. 13. A surface coating comprising the compound of claim 12 and a coating agent, wherein the coating agent is capable of adhering the compound to a habitat.

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