DNA polymerase IIIC inhibitors and their use

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest to U.S. Provisional Application No. 62 / 783,754, filed on 21 December 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 for using the compositions for 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 multidrug resistance to a variety of antibiotics have emerged as a major challenge in treatment. Two Gram-positive pathogens, Staphylococcus aureus and Enterococcus faecalis / faecium, account for the majority of hospital-acquired diseases (Muto, et al.). A third organism, Streptococcus pneumoniae, is generally a community-acquired pathogen. These organisms are aerobic bacteria, meaning they grow in an oxygen-containing atmosphere.

[0003] Staphylococcus aureus is the leading cause of hospital-acquired bacteremia and skin / wound infections, and the second leading cause of hospital-acquired lower respiratory tract infections. The emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) is a serious public health concern. MRSA strains are becoming increasingly multidrug-resistant over time. In many parts of the world, MRSA infections account for the majority of sporadic community-acquired staphylococcal infections. These strains are also associated with a large number of localized (cutaneous and cutaneous structure) and invasive (bacteremia) infections.

[0004] Enterococcus faecalis and Enterococcus faecium cause hospital-acquired sepsis, endocarditis, and wound and urinary tract infections. Vancomycin-resistant phenotypes were first reported in 1987 with Enterococcus (vancomycin-resistant enterococci or VRE), years after the drug had been introduced into widespread clinical use. Today, >30% of Enterococcus faecalis infections in the ICU are VRE. There are few or no treatment options for certain diseases caused by VRE, including bloodstream infections, surgical site infections, and urinary tract infections. The incidence of VRE is approximately 20,000 cases 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, it is estimated that Streptococcus pneumoniae accounts 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 annually. The annual mortality rate from streptococcal-induced illnesses is estimated at 40,000 in the United States and 3 to 5 million worldwide. The identification of penicillin-resistant Streptococcus pneumoniae (PRSP) is increasing. The emergence and spread of drug-resistant strains of Streptococcus pneumoniae are complicating the treatment of these common infections. Anaerobic bacteria, that is, bacteria that grow in oxygen-deficient environments, are also a public health issue. Clostridium difficile is increasingly associated with disease in human patients, often as a result of treatment with certain antibiotics. The most common disease is called Clostridium difficile-associated diarrhea (CDAD).

[0006] One approach to solving the problem of multidrug-resistant bacteria involves developing effective antimicrobial agents capable of selectively attacking novel bacterial targets. The DNA pol IIIC enzyme has been shown to be critical to the replication of 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 to inhibit when discovering new Gram-positive selective antimicrobial agents.

[0007] The DNA pol IIIC enzyme is specifically required for chromosome replication in low-G:C Gram-positive organisms (both aerobic and anaerobic bacteria). Encoded by the structural gene polC, the DNA pol IIIC enzyme is one of two DNA polymerases specialized for replication that are essential in Gram-positive bacteria. PolC is absent in high-G:C bacteria, Gram-negative bacteria, and eukaryotic cells, but is tightly conserved in a broad group of Gram-positive pathogens.

[0008] Therefore, DNA pol III is essential for the replication of host chromosomes in Gram-positive bacteria with low G:C content. If its action is blocked, chromosomal DNA replication fails, and the bacterial host dies. This essential structure of pol IIIC is strongly conserved in 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). DNA pol IIIC inhibitors have shown Gram-positive antibacterial activity and in vivo protective activity, but the development has been hampered by the lack of "druggable" characteristics of the compounds, such as suitability for parenteral formulations or favorable pharmacokinetics. Therefore, there is still a need to identify compounds that can efficiently inhibit DNA pol III C and thus 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, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, and the Gram-positive anaerobic bacterium Clostridium difficile.

[0010] In one aspect, the present invention provides a compound of the formula shown below: [Chemical formula] (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 cycloalkyl, Each of R4, R6 and R7 is independently H or C 1~6 alkyl, R5 is OH or C 1~6 alkyl, CH(R8R9), Each of R8 and R9 is independently H, halo or C 1~6 alkyl, W is H, halo, substituted or unsubstituted C 1~6 alkyl, substituted or unsubstituted C 3~8Cycloalkyl, substituted, or unsubstituted C 2~8 Heterocycline, substituted or unsubstituted C 6~14 Aryl, substituted, or unsubstituted C 1~10 Heteroaryl, NH2, CN, OR 10 , SR 11 COR 12 , OCOR 13 , NR 14 COR 15 , NR 16 R 17 , NR 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 of these independently represents H, substituted or unsubstituted C. 1~6 Alkyl, substituted, or unsubstituted C 3~8 Cycloalkyl, substituted, or unsubstituted C 2~8 Heterocycline, substituted or unsubstituted C 6~14 Aryl, substituted, or unsubstituted C 1~10 It is a heteroaryl, m is 1-5, o is 0-4, p is 0-4, q is 0-4. R2 is H, halo, CN, substituted or unsubstituted C 1~6 Alkyl, CO2R 21 CONR 23 R 24 , substitution or non-substitution C 2~8 Heterocyclyl or substituted or unsubstituted C 1~10 It is a heteroaryl, R3 consists of F, Cl, Br, I, and C. 1~6 alkyl, OH, CN, C 1~6 -C substituted with one or more substituents selected from the group consisting of alkyl, CF3, CHF2, CF3CH2, OCH3, and OCF3 6~14Aryl or C 1~10 It is a heteroaryl, R0 is H, CH2OPO(OH)2, CH2OCONHCH2(CH2) t OPO(OH)2, CH2OCOCH2(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), and Y is a direct bond or oxygen. s is either 0 or 1, t is 1, 2, or 3. R 28 and R 29 Each is independently a hydrogen atom or a hydrolyzable ester group, and R 28 If R is hydrogen, 29 is -P(O)OR 32 Ure 33 It could be, R 30 and R 31 Each of these is independently hydrogen or a C1-4 alkyl group. R 32 and R 33 Each is characterized by a compound having (independently a hydrogen atom 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] The specific compounds of the above formula are described herein. The present invention encompasses all enantiomers, racemics, tautomers, and diastereoisomers of the compounds described herein, as well as mixtures thereof. The present invention is further characterized by a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier.

[0012] In another aspect, the present invention features formulations of compounds of formula I that are suitable, for example, for coating the surfaces of medical devices as described herein. In such formulations, the compounds of the present invention can be mixed with a suitable biocompatible coating agent, or can be covalently or otherwise bonded to the coating agent (e.g., electrostatically or as a ligand).

[0013] In another aspect, the present invention features a method for inhibiting bacterial growth, comprising the step of bringing a region where bacteria tend to grow (e.g., a habitat or surface, such as that of a medical device) into contact with a compound of formula I. The present invention also features a method for treating an animal with respect to a Gram-positive bacterial infection, comprising the step of administering to the animal a therapeutically effective amount of the compound of formula I.

[0014] In various aspects of the present invention, the compounds of the present invention are useful for treating or preventing infectious diseases, or for 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 microorganisms that produce DNA pol IIIC enzymes.

[0015] Detailed description of the invention The features and other details of the present invention are described in more detail below. The detailed embodiments described herein are for illustrative purposes only and should not be understood as limitations of the invention. The principal features of the present invention can be used in various embodiments without departing from the scope of the invention.

[0016] definition The term "alkyl" is preferably defined as a branched or unbranched saturated acyclic hydrocarbon group 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; l-ethyl-1-methylpropyl; l-ethyl-2-methylpropyl; and hexyl. Alkyl groups may be unsubstituted or substituted as described herein.

[0017] The term "cycloalkyl" is defined as a monocyclic or bicyclic structure having only carbon atoms in one or more rings, each ring preferably having 3 to 8 members. Exemplary cycloalkyl groups include cyclopropyl; cyclobutyl; cyclopentyl; and cyclohexyl. Cycloalkyl groups may 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 contain an aromatic ring. Each ring preferably contains 2 to 8 carbon atoms and 1 to 4 oxygen, nitrogen, and / or sulfur atoms. Examples include azilidinyl, azetidinyl, morpholinyl, oxazolidinyl, oxezolinyl, oxecanyl, oxepanyl, oxyranyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, and tetrahydrothiopyranyl. The heterocyclyl group may 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 6 to 14 carbon atoms. Examples include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl. The aryl group may 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, prinyl, pyrazinyl, pyridadinyl, pyridinyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thienyl, triazinyl, and triazolyl. The heteroaryl group may be unsubstituted or substituted as described herein.

[0021] The term "halo" is defined as fluoro, bromo, chloro, or iodine. 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 "aryl amino" is defined as -NHR where R is an aryl group. The term "alkyl thio" is defined as -SR where R is an alkyl group. The term "aryl thio" is defined as -SR where R is an aryl group. The term "quaternary amino" is defined as -NRR'R'' where R, R' and R'' are, independently, alkyl, aryl, heteroaryl and heterocyclyl + as defined.

[0022] The term "substituted" means that one or more hydrogen atoms of a group or part of a group are replaced by substituents including, but not limited to, C 1~6 alkoxy, C 6~14 aryloxy, sulfhydryl (-SH), C 1~6 alkyl thio, C 6~14 aryl thio, amino (-NH2), C 1~6 alkyl amino, C 6~14 aryl amino, disubstituted amino, quaternary amino, hydroxyl (-OH), carboxyl (-COOR), halo, cyano (-CN), azide (-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 [[ID=?]]alkylsulfonyl, (SO2)O-C 1~6 alkyl, -(SO2)O-C 3~8 cycloalkyl, -(SO2)O-C 3~8 cycloalkyl, -(SO2)-C 6~14 aryl, -(SO2)O-C 6~14 aryl, -(SO2)-C 1~10 heteroaryl, -(SO2)O-C 1~10 heteroaryl, -(SO2)-C 2~8 heterocyclyl and -(SO2)O-C [[ID=?]] 2~8 [[ID=?]]heterocyclyl and is defined as being replaced by substituents including, but not limited to, those described above.

[0023] Furthermore, alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl groups are C 6~14 Ariel, C 3~8 Cycloalkyl, C 1~10 Heteroaryl or C 2~8 The group may be substituted with a heterocyclyl group. Cycloalkyl, heteroaryl, and heterocyclyl groups may also be substituted with alkyl groups. Substituents may be substituted with halogens, trifluoromethyl, hydroxyl, or carboxyl, as described for the parent group. As used herein, the terms “administer” or “to administer” mean a method of giving an animal one or more unit doses of an antimicrobial pharmaceutical composition (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 microorganisms involved, and the severity of the actual microbial infection.

[0024] "Animals" means any animals susceptible to Gram-positive bacterial infections. For example, animals may include humans, dogs, cats, pigs, cattle, horses, goats, chickens, turkeys, sheep, rats, mice, and rabbits, as well as other animals kept for commercial purposes or as pets. The term "animals susceptible to microbial infections" is defined as animals that are at increased risk of contracting microbial infections compared to the general population. Examples of such animals include those who have recently undergone surgical procedures, or humans with immunodeficiency, such as those diagnosed with AIDS (Acquired Immunodeficiency Syndrome), or those with grafts requiring immunosuppressant drugs. Such animals may be identified using methods known to those skilled in the art.

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

[0026] The term "inhibition" is defined as reducing the cell growth rate of a microbial organism 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 in vitro growth assays, using standard liquid culture techniques. Compounds that exhibit inhibition of colony formation at minimum inhibitory concentrations (MICs) 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 which microorganisms may be present or present, or on which the presence of microorganisms is desired to be prevented. 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 an invasion of a host animal by pathogenic microorganisms. For example, an infection can include an overgrowth of microorganisms that normally exist within or on the animal, or the growth of microorganisms that do not normally exist within or on the animal. More generally, a microbial infection can be any situation where the presence of a population (singular or plural) of microorganisms causes damage to the host animal. Thus, an animal is "suffering from" a microbial infection when an excessive amount of a population of microorganisms is present within or on the animal, or when the presence of a population (singular or plural) of microorganisms causes damage to the cells or other tissues of the animal. 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 classification of bacteria.

[0029] "Pharmaceutically acceptable salts" means 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, although not pharmaceutically acceptable per se, can be useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include those of alkali metals (e.g., sodium or potassium), alkaline earth metals (e.g., magnesium), ammonium, and NR4 + (where R is C 1~4 alkyl) salts. Preferred salts include hydrochloride, hydrobromide, sulfate, mesylate, maleate, tartrate, and fumarate salts. The following reference to a compound according to the invention includes compounds of the general formula shown, as well as their pharmaceutically acceptable salts.

[0030] "Prevention" of microbial growth or infection is defined as the application of the compound of the present invention to prevent microbial growth or infection. The amount of the compound of the present invention required to prevent microbial growth can be determined, for example, by an in vitro growth assay, for example, by standard liquid culture techniques. The amount of the compound of the present invention required to prevent microbial infection can be determined, for example, by an in vivo assay, for example, by determining the amount of the compound that must be administered to prevent infection in research animals, for example, guinea pigs after inoculation with microorganisms. Generally, compounds that show prevention at suitable concentrations, for example <100 μg / ml, more preferably <10 μg / ml, are useful for further testing as therapeutic agents.

[0031] The term "treatment" is defined as the medical management of a patient with the intention of resulting in the cure, remission, or prevention of a disease, condition, or disorder. This term encompasses both active treatment, i.e., treatment aimed specifically at improving the disease, condition, or disorder, and causal treatment, i.e., treatment aimed at eliminating the cause of the disease, condition, or disorder. Furthermore, this term encompasses palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment aimed at preventing the disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment aimed at improving the disease, condition, or disorder. The term "treatment" also encompasses symptomatic treatment, i.e., treatment targeting the systemic symptoms of the disease, condition, or disorder. The term "therapeutic dose" is defined as the amount administered to an animal in need of it that alleviates at least some of the symptoms of a bacterial infection.

[0032] Regarding prevention, the "therapeutic dose" is the amount administered to animals susceptible to bacterial infections that helps inhibit such infections or otherwise reduce their likelihood.

[0033] Details of one or more aspects of the present invention are described in the accompanying description below. Other features, purposes, and advantages of the present invention will become apparent from the description and claims.

[0034] In one embodiment, the present invention relates to formula I [ka] Regarding compounds by [company name]. In some embodiments, R1 may be H, methyl, other substituted or unsubstituted alkyl, cyclic, or heterocyclyl groups, as illustrated below. [ka]

[0035] In some embodiments, R1 may be a substituted or unsubstituted aryl or heteroaryl group, 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 , substitution or non-substitution C 2~8 Heterocyclyl or substituted or unsubstituted C 1~10 It is a heteroaryl compound.

[0036] In some embodiments, R3 may be a substituted or unsubstituted aryl or heteroaryl group, as illustrated below. [ka]

[0037] antibacterial compounds Preferred compounds are: 5-((3,4-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 1-Allyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)butylacetate, 1-(cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-[(3,4-cyclolophenyl)methylamino]-1-phenyl-6H-pyrazolo[4,3-d]pyrimidine-7-one, 1-Cyclopropyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, Cyclopentyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, Cetyl 5-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)pentanoate, 5-[(3,4-cyclolophenyl)methylamino]-1-(4-pyridyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one, 3-Chloro-5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-cyclolobenzyl)amino)-3-fluoro-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 3-Chloro-5-((3,4-dichlorobenzyl)amino)-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 1-(2-(4-acetylpiperazine-1-yl)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((4-methylmorpholine-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(3-methylpicolinoyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-difluorobenzyl)amino)-3-fluoro-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((4-chloro-3-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1,3-dimethyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholinoethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(4-hydroxybutyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one), 5-((3,4-dichlorobenzyl)amino)-1-(thiazole-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((tetrahydrofuran-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(3-hydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyrazine-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-isopropyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(5-methoxypentyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-((2-methoxyethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-ylsulfonyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, Ethyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)pentanoate, Isopropyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)pentanoate, Ethyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)butanoate, Methyl 3-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)propanoate, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-2-ylmethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-ylmethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-4-ylmethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride, 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(oxazol-4-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one, 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(thiazole-2-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one, 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methoxypicolinoyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methylpicolinoyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one, 3-Chloro-5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one, 5-((3,4-difluorobenzyl)amino)-3-fluoro-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 3-Chloro-5-((3,4-dichlorobenzyl)amino)-1-(1-nicotinoylpiperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, Ammonium (2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy)ethoxy)methylphosphate (5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-7-oxo-1H-pyrazolo[4,3-d]pyrimidine-6(7H)-yl)methyl dihydrogen phosphate, or include their optical isomers, isotopic isomers, prodrugs, or pharmaceutically acceptable salts thereof.

[0038] Synthesis method The following examples are provided for illustrative purposes of the present invention, but are not intended to limit the scope or spirit of the invention. The compounds of the present invention include those specifically disclosed in this specification and below, and can be prepared as described in the following scheme. For example, the compounds of formula I can be prepared as described in the following scheme, and it is known to those skilled in the art that fragments and combinations thereof can be made. In some schemes provided herein, compounds may be indicated in parentheses. Those skilled in the art will recognize that compounds indicated in parentheses indicate 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 equivalents) in H2SO4 (119.84 g, 1.22 mol, 65.13 mL, 4.67 equivalents), 2-methylpyrazole-3-carboxylic acid (33 g, 261.67 mmol, 1 equivalent) was gradually added at 20°C–25°C. The mixture was stirred at 30°C–40°C for 1 hour, then at 75°C–80°C for 5 hours. TLC showed that the starting material had been 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. After filtration, the solid was collected 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-nitropyrazole-3-carboxylic acid (37 g, 216.23 mmol, yield 82.64%) was obtained as a white solid. 1 1H 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-nitropyrazole-3-carboxylic acid (35 g, 204.55 mmol, 1 equivalent) and DMF (149.51 mg, 2.05 mmol, 157.38 μL, 0.01 equivalent) in SOCl2 (150 mL) was stirred at 85°C for 1 hour. TLC showed that 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-nitropyrazole-3-carbonyl chloride (38 g, 200.47 mmol, yield 98.01%) was obtained as a colorless oil.

[0042] Preparation of 1-methyl-4-nitro-1H-pyrazole-5-carboxamide (Step 3 in Scheme A) [ka] 2-methyl-4-nitropyrazole-3-carbonyl chloride (38 g, 200.47 mmol, 1 equivalent) was added dropwise to NH3·H2O (150 mL) at 0°C. The mixture was stirred at 25°C for 1 hour. TLC and LC-MS showed that the reaction was complete. Some solid was produced. After filtration, the solid was collected. The aqueous solution was extracted with ELISA (80 μL × 5). The combined organic layers were washed with brine (50 mL × 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The combined crude product was used in the next step without further purification. 2-methyl-4-nitropyrazole-3-carboxamide (32 g, 188.10 mmol, yield 93.83%) was obtained as a pale yellow solid. 1 1H 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-nitropyrazole-3-carboxamide (32 g, 188.10 mmol, 1 equivalent) and 10% Pd / C (3 g) in EtOH (600 mL) was stirred at 25°C under H2 (45 psi) for 5 hours. TLC showed that no starting material remained and one major novel spot with greater polarity was detected. After filtration, the filtrate was concentrated under reduced pressure. The crude was used in the next step without further purification. 4-amino-2-methylpyrazole-3-carboxamide (23 g, 164.12 mmol, yield 87.25%) was obtained as a purple solid. 1 1H 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-methylpyrazole-3-carboxamide (23 g, 164.12 mmol, 1 equivalent) in CH3CN (500 mL), CDI (34.60 g, 213.35 mmol, 1.3 equivalents) was gradually added over 1 hour at 100°C. The mixture was then heated under N2 at 100°C for 12 hours. A gray solid was formed. LC-MS showed that no starting material remained. Several novel peaks were shown on LC-MS, and approximately 80% of the desired compound was detected. After filtration at 90°C, the solid was collected. The crude 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, yield 91.69%) was obtained as a gray solid. 1 1H 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 equivalent) in POCl3 (239.96 g, 1.56 mol, 145.43 mL, 10 equivalents), DBU (142.95 g, 938.98 mmol, 141.53 mL, 6 equivalents) was added dropwise at 50°C under N2. The mixture was stirred at 85°C for 12 hours. LC-MS showed that no starting material remained. The mixture was poured into ice water (1 L) and then extracted with SiO2 (200 mL x 6). The combined organic layers were washed with saturated NaHCO3 to pH=7 and brine (100 mL x 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 80 g, 150 mL / min, eluate with a 0-20% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure to obtain 5,7-dichloro-1-methyl-pyrazolo[4,3-d]pyrimidine (14 g, 68.96 mmol, yield 44.06%) as a pale yellow oil. 1 1H 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]pyrimidine-7(6H)-one (Step 7 in Scheme A) [ka] A solution of 5,7-dichloro-1-methyl-pyrazolo[4,3-d]pyrimidine (14 g, 68.96 mmol, 1 equivalent) in dioxane (140 mL) and H2O (100 mL) was added dropwise at 0°C to a solution of NaOH (2.76 g, 68.96 mmol, 1 equivalent) in H2O (20 mL). The mixture was then stirred at 100°C for 6 hours. TLC and LC-MS showed 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 x 2) to recover the starting material. The aqueous solution was then diluted to pH=5 with 2N HCl. A white solid was formed. After filtration, the solid was collected 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]pyrimidine-7-one (12.7 g, 68.80 mmol, 99.78% yield) was obtained as a white solid. 1 1H 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]pyrimidine-7-one (541.76 μmol, 1 equivalent), R3(CH2) nA solution of NH2 (1.63 mmol, 3 equivalents) and a base (no base, or TEA or DIEA) or TFA in a solvent (t-BuOH, i-PrOH, or NMP) (6 mL / mmol) was heated at (100°C to 160°C) for a set time (4 to 20 hours). LC-MS and HPLC showed that the reaction was complete. The reaction mixture was quenched with H2O and extracted with SiO2. The combined organic layers were washed with brine, dehydrated with Na2SO4, 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)Waters Xbridge 150mm×25mm 5μm;d)Nano-micro Kromasil C18 100mm×30mm 5μm;e)Boston Prime C18 150mm×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 NH4HCO3)-MeCN], B%: 5%~50%, 20 min; d) [Water (0.04% NH3·H2O + 10 mM NH4HCO3)-MeCN], B%: 15%~60%, 10.5 min; e) [Water (10 mM NH4HCO3)-MeCN], B%: 1%~25%, 10 min. The aqueous solutions were freeze-dried to obtain the desired products.

[0048] compound 1 Preparation of 5-((4-chloro-3-fluorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 8 in Scheme A) [ka] A solution of 5-chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.1 g, 541.76 μmol, 1 equivalent) and (4-chloro-3-fluorophenyl)methaneamine (259.38 mg, 1.63 mmol, 198.39 μL, 3 equivalents) in t-BuOH (3 mL) was heated at 100 °C for 16 hours. LC-MS and HPLC showed that 5-chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one was completely consumed, and a single major peak with the desired mass was detected. The reaction mixture was quenched with H2O (5 mL) at 25 °C and then extracted with ELISA (10 mL × 3). The combined organic layers were washed with brine (10 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge 150 mm × 25 mm 5 μm; mobile phase: [water (10 mM NH4HCO3)-MeCN]; B%: 25%~45%, 20 min). The aqueous solution was lyophilized to obtain 5-[(4-chloro-3-fluorophenyl)methylamino]-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (71.8 mg, 233.34 μmol, yield 43.07%) 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 ClFN5O: 307.06 m / z, measured value: 308.0 [M+H] + .

[0049] compound 2 5-((3,4-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (0.8 g, 2.45 mmol, yield 64.54%) was obtained as a grayish-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 m / z, measured value: 324.0 m / z [M+H] + .

[0050] compound 3 5-(benzylamino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded the desired compound (70.8 mg, 271.41 μmol, 41.75% yield) as a grayish-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 N5O: 255.11 m / z; Measured value: 256.1 m / z [M+H] + .

[0051] compound 4 1-Methyl-5-((pyridine-2-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 N6O: 256.11 m / z; Measured value: 257.2 m / z [M+H] + .

[0052] compound 5 1-Methyl-5-(3-pyridylmethylamino)-6H-pyrazolo[4,3-d]pyrimidine-7-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 N6O: 256.11 m / z; measured mass: 257.1 m / z [M+H] + .

[0053] compound 6 1-Methyl-5-((pyridine-4-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 N6O: 256.11 m / z; measured mass: 257.1 m / z [M+H] + .

[0054] compound 7 5-((3,4-difluorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (0.1288 g, 442.22 μmol, 81.63%) was obtained 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 m / z; Measured mass: 292.1 m / z [M+H] + .

[0055] compound 8 5-((3,4-dichlorophenethyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (0.119 g, 351.87 μmol, 98.20%) was obtained 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 m / z; Measured mass: 338.1 m / z [M+H] + .

[0056] compound 9 5-((3,4-dichlorophenyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (46.3 mg, 149.29 μmol, yield 27.56%) was obtained 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 m / z; Measured mass: 310.0 m / z [M+H] + .

[0057] compound 10 1-Methyl-5-(phenylamino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 N5O: 241.10 m / z; Measured value: 242.1 m / z [M+H] + .

[0058] compound 11 1-Methyl-5-((pyrimidine-2-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (31 mg, 120.50 μmol, yield 22.24%) was obtained 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 N7O: 257.10 m / z; Measured mass: 258.1 m / z [M+H] + .

[0059] compound 12 1-Methyl-5-((pyrazine-2-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (30.6 mg, 118.95 μmol, yield 21.96%) was obtained 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 N7O: 257.10 m / z; Measured mass: 258.1 m / z [M+H] + .

[0060] compound 13 5-(((1H-indazole-5-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (56.6 mg, 191.13 μmol, 35.28% yield) was obtained as a white solid according to the procedure. 1 1H 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 N7O: 295.12 m / z; Measured mass: 296.1 m / z [M+H] + .

[0061] compound 14 5-(((1H-indole-5-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 N6O: 294.12 m / z; Measured value: 295.1 m / z [M+H] + .

[0062] compound 15 1-Methyl-5-((thiazole-4-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (66.9 mg, 255.06 μmol, yield 58.85%) was obtained 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 N6OS mass calculation value: 262.06 m / z, measured value: 263.0 [M+H] + .

[0063] compound 16 5-(((1H-pyrazole-3-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 m / z; Measured value: 246.1 [M+H] + .

[0064] compound 17 5-(((2H-1,2,3-triazol-4-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (55.9 mg, 227.03 μmol, yield 41.91%) was obtained as a white solid according to the procedure. 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 N8O: 246.10 m / z; Measured mass: 247.1 m / z [M+H] + .

[0065] compound 18 5-((benzo[d]thiazole-2-ylmethyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 N6OS mass calculation value: 312.08 m / z, measured value: 313.1 [M+H] + .

[0066] compound 19 5-(((1H-benzo[d]imidazole-5-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded the desired compound (44.5 mg, 149.58 μmol, yield 22.01%) 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 N7O: 295.12 m / z; Measured mass: 296.2 m / z [M+H] + .

[0067] compound 20 1-Methyl-5-((thiazole-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (19.7 mg, 75.11 μmol, yield 13.86%) was obtained 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 N6OS mass calculation value: 262.06 m / z, measured value: 263.0 [M+H] + .

[0068] compound 21 1-Methyl-5-((oxazol-5-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (39.5 mg, 160.42 μmol, 29.61% yield) was obtained as a white solid according to the procedure. 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 m / z; Measured value: 247.1 m / z [M+H] + .

[0069] compound 22 5-(((1H-imidazole-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (26.4 mg, 107.27 μmol, yield 19.80%) was obtained as a white solid according to the procedure. 1 1H 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 N7O: 245.10 m / z; Measured mass: 246.1 m / z [M+H] + .

[0070] compound 23 1-Methyl-5-((pyridazin-3-ylmethyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] Following the procedure, the desired compound (4.9 mg, 19.05 μmol, yield 3.52%) was obtained as a light brown solid. 1 1H 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 N7O: 257.10 m / z; Measured mass: 258.1 m / z [M+H] + .

[0071] compound 24 5-((3-chloro-4-fluorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded the desired compound (139 mg, 451.72 μmol, 83.38% yield) as a white solid. 1 1H 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 ClFN5O: 307.06 m / z, measured value: 308.0 [M+H] + .

[0072] compound 25 5-((4-chlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded the desired compound (23.1 mg, 79.73 μmol, 14.72% yield) as a grayish-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 ClN5O: 289.07 m / z, measured value: 290.1 ​​[M+H] + .

[0073] compound 26 5-((3-chlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (157.9 mg, 391.09 μmol, yield 72.19%, TFA) was obtained as a white solid according to the procedure. 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 ClN5O: 289.07 m / z, measured value: 290.1 ​​[M+H] + .

[0074] compound 27 5-((2-chlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 ClN5O: 289.07 m / z, measured value: 290.1 ​​[M+H] + .

[0075] compound 28 5-((2,4-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded the desired compound (92.6 mg, 276.14 μmol, 50.97% yield) as a grayish-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 m / z, measured value: 324.0 m / z [M+H] + .

[0076] compound 29 5-((2,3-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 m / z, measured value: 324.0 m / z [M+H] + .

[0077] compound 30 5-((2,6-dichlorobenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (26.5 mg, 81.75 μmol, yield 15.09%) was obtained as a white solid according to the procedure. 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 m / z, measured value: 324.0 m / z [M+H] + .

[0078] compound 31 5-((3-chloro-4-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 ClN5O: 303.09 m / z; Measured mass: 304.0 m / z [M+H] + .

[0079] compound 32 5-((3,4-dimethoxybenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 m / z, measured value: 316.1 m / z [M+H] + .

[0080] compound 33 5-((3,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (117 mg, 412.95 μmol, yield 84.69%) was obtained as a white solid by following the procedure. 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 N5O: 283.14 m / z; Measured value: 284.1 m / z [M+H] + .

[0081] compound 34 5-((4-chloro-3-methoxybenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 m / z, measured value: 320.1 m / z [M+H] + .

[0082] compound 35 5-((4-chloro-3-methylbenzyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded the desired compound (102.3 mg, 336.79 μmol, 69.07% yield) as a pale 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 ClN5O: 303.09 m / z; Measured mass: 304.1 m / z [M+H] + .

[0083] compound 36 5-(((4,5-dichloropyridine-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one-2,2,2-trifluoroacetate was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (35.8 mg, 110.10 μmol, yield 33.87%) was obtained as a white solid according to the procedure. 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 m / z, measured value: 325.0 m / z [M+H] + .

[0084] compound 37 5-(((4,5-dichloro-1H-imidazole-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (23.2 mg, 73.85 μmol, yield 16.41%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 314.0 m / z [M+H] + .

[0085] compound 38 5-(((4-chloro-5-ethyl-1H-imidazole-2-yl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 8 in scheme A. [ka] The desired compound (8.4 mg, 27.01 μmol, yield 9.97%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 308.1 m / z [M+H] + .

[0086] compound 39 5-((1,3-dihydroisobenzofuran-5-yl)amino)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 8 in scheme A. [ka] The procedure yielded 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 m / z; Measured mass: 284.1 m / z [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]pyrimidine-7(6H)-one (Step 1 in Scheme B-1) [ka] HBF4 (677.22 mg, 7.71 mmol, 480.30 μL, 2.5 equivalents) was added to a mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (1 g, 3.08 mmol, 1 equivalent) and NIS (694.03 mg, 3.08 mmol, 1 equivalent) in MeCN (8 mL). The mixture was stirred at 100°C for 4 hours. LC-MS showed that the reaction was complete. A small amount of yellow solid was produced. After filtration, the mixture was washed with H2O (8 mL) and SiO (8 mL) and the solid was collected. Compound 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (730 mg, 1.62 mmol, yield 52.58%) was obtained as a yellow solid. 1 1H 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] 5-[(3,4-dichlorophenyl)methylamino]-3-iodo-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (300 mg, 666.58 μmol, 1 equivalent) and TEA (269.80 mg, 2.67 mmol, 371.12 μL, 4 equivalents) were dissolved in MeOH (10 mL) and DMF (3 mL) and Pd(dppf)Cl2 (97.55 mg, 133.32 μmol, 0.2 equivalents) under an N2 atmosphere. The suspension was degassed and purged three times with CO. The mixture was stirred at 80°C under CO (50 psi) for 5 hours. LC-MS showed that 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, 4g, 65mL / min, eluate with a 0-100% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure. 50 mg of the obtained crude product was further purified by preparative HPLC (column: Nano-micro Kromasil C18 100mm × 30mm 8μm; 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 next. 1 1H 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 m / z, measured value: 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 equivalent) and LiOH·H2O (2.35 mmol, 3 equivalents) in MeOH (2 mL) and H2O (2 mL) (or in MeNH2 (2 M, 15.7 mmol, 7.85 mL, 20 equivalents in EtOH)) was stirred at 25°C for a set time (1 to 12 hours). LC-MS showed that 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%~65%, 10 min). The solvent was removed by freeze-drying to obtain 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] Following the procedure, the desired compound (14.1 mg, 37.32 μmol, yield 4.75%, purity 97.44%) was obtained as a light brown solid. 1 1H 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 m / z, measured value: 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 equivalent) in MeNH2 (2 M in EtOH, 1.31 mL, 20 equivalents) was stirred at 25°C for 12 hours. LC-MS indicated that 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%~55%, 10 min). The solvent was removed by lyophilization. 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 1H 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 m / z; Measured mass: 381.1 m / z [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] Following the procedure, the desired compound (3.3 mg, 7.80 μmol, yield 5.96%) was obtained as a brown solid. 1 1H 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 m / z; Measured mass: 395.1 m / z [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]pyrimidine-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]pyrimidine-7-one (0.2 g, 444.38 μmol, 1 equivalent), 2,4,6-trimethyl-1,3,5,2,4,6-trioxatrivolinane (334.71 mg, 1.33 mmol, 372.73 μL, 50% in THF, 3 equivalents), Pd(dppf)Cl2 (48.77 mg, 66.66 μmol, 0.15 equivalents), and K2CO3 (184.26 mg, 1.33 mmol, 3 equivalents) in dioxane (10 mL) was stirred at 120 °C under N2 for 10 hours. LC-MS showed that 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%~50%, 10 min) to obtain 5-[(3,4-dichlorophenyl)methylamino]-1,3-dimethyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (22.4 mg, 66.23 μmol, yield 14.90%) as a grayish-white solid. 1 1H 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 m / z; Measured mass: 338.2 m / z [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-carbonitrili (Step 1 in Scheme B-3) [ka] A mixture of 3-bromo-5-[(3,4-dichlorophenyl)methylamino]-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (50 mg, 124.05 μmol, 1 equivalent) and CuCN (12.22 mg, 136.46 μmol, 29.81 μL, 1.1 equivalents) in DMSO (1 mL) was stirred at 160°C for 4 hours. LC-MS indicated that the reaction was complete. The reaction mixture was filtered to remove insoluble matter. The filtrate was first purified by preparative HPLC (column: Welch Xtimate C18 100mm×25mm 3μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 30%~60%, 12 min), and then secondly purified by preparative HPLC (column: Welch Xtimate C18 150mm×25mm 5μm; mobile phase: [water (10mM NH4HCO3)-MeCN]; B%: 40%~65%, 10.5 min). The solvent was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile (5.3 mg, 14.75 μmol, yield 11.89%, purity 97.20%) was obtained as a white solid. 1 1H 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 m / z, measured value: 349.0 m / z [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]pyrimidine-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]pyrimidine-7-one (0.3 g, 666.58 μmol, 1 equivalent), 2,3-dihydrofuran (233.60 mg, 3.33 mmol, 252.00 μL, 5 equivalents), AcOK (163.54 mg, 1.67 mmol, 2.5 equivalents), Pd(OAc)2 (29.93 mg, 133.32 μmol, 0.2 equivalents), and PPh3 (17.48 mg, 66.66 μmol, 0.1 equivalent) in DMF (1 mL) was degassed, purged three times with N2, and then stirred at 100°C under an N2 atmosphere for 12 hours. LC-MS and HPLC showed that the reaction was complete. The reaction mixture was cooled to room temperature and quenched with H2O (5 mL) at 0°C. The mixture was extracted with ELISA (8 mL x 3). The combined organic layer was washed with brine (5 mL x 1), dehydrated with Na2SO4, 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]pyrimidine-7-one (0.6 g, crude) was obtained as a pale yellow solid. 0.15 g of the crude was further purified by preparative HPLC (column: Xtimate C18 100 mm x 30 mm 3 μm; mobile phase: [water (0.04% HCl)-MeCN]; B%: 40%~55%, 10 min). MeCN was removed under reduced pressure at 30°C. The aqueous solution was dehydrated by freeze-drying to obtain 5-[(3,4-dichlorophenyl)methylamino]-3-(2,5-dihydrofuran-2-yl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (24.9 mg, 62.01 μmol, yield 9.30%, purity 97.685%) as a pale yellow solid, which was then used. 1 1H 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 m / z; Measured mass: 392.1 m / z [M+H] + .

[0100] compound 47 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-methyl-3-(tetrahydrofuran-2-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 2 in Scheme B-4) [ka] 5-[(3,4-dichlorophenyl)methylamino]-3-(2,5-dihydrofuran-2-yl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.3 g, 229.45 μmol, 1 equivalent) was dissolved in EtOH (50 mL) and THF (50 mL), to which Rh(PPh3)3Cl (21.23 mg, 22.95 μmol, 0.1 equivalent) was added. The suspension was degassed and purged three times with H2. The mixture was stirred at 30°C under H2 (20 psi) for 16 hours. LC-MS and HPLC showed that 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%~55%, 12 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-methyl-3-tetrahydrofuran-2-yl-6H-pyrazolo[4,3-d]pyrimidine-7-one (20.7 mg, 49.82 μmol, yield 21.71%, purity 94.90%) was obtained as a yellow solid. 1 1H 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 m / z; Measured mass: 394.0 m / z [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]pyrimidine-7(6H)-one

[0102] Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-3-(2-ethoxyvinyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-7-one (0.5 g, 1.11 mmol, 1 equivalent), 2-[(E)-2-ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (880.18 mg, 4.44 mmol, 4 equivalents), K2CO3 (460.62 mg, 3.33 mmol, 3 equivalents), and Pd(dppf)Cl2 (121.94 mg, 166.64 μmol, 0.15 equivalents) in dioxane (12 mL) and H2O (3 mL) was stirred at 100°C under N2 for 10 hours. LC-MS and HPLC showed that 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, 50 mL / min, eluate with a 0-100% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure. Compound 5-[(3,4-dichlorophenyl)methylamino]-3-[(E)-2-ethoxyvinyl]-1-methyl-6H-pyrazolo-[4,3-d]pyrimidine-7-one (0.34 g, 862.39 μmol, yield 77.63%) was obtained as a grayish-white solid. 1 1H 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]pyrimidine-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]pyrimidine-7-one (0.15 g, 380.46 μmol, 1 equivalent) and Rh(PPh3)3Cl (35.20 mg, 38.05 μmol, 0.1 equivalent) in EtOH (50 mL) and THF (20 mL) was stirred at 30°C under H2 (20 psi) for 5 hours. HPLC and LC-MS showed that 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 freeze-drying. Compound 5-[(3,4-dichlorophenyl)methylamino]-3-(2-ethoxyethyl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (71.7 mg, 180.94 μmol, yield 47.56%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 396.1 m / z [M+H] + .

[0105] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-1-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-7-one (0.15 g, 380.46 μmol, 1 equivalent) in 10 mL of DCM, 1 mL of TFA was added dropwise at 0°C. The mixture was then stirred at 20°C for 5 hours. LC-MS indicated that the reaction was complete. The mixture was poured into 10 mL of water, and the organic layer was separated. The aqueous solution was extracted with 5 x 20 mL of DCM. The combined organic layers were washed with saturated NaHCO3 to pH=7, dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was used in the next step without further purification. Compound 2-[5-[(3,4-dichlorophenyl)methylamino]-1-methyl-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-3-yl]acetaldehyde (0.1 g, 273.07 μmol, yield 71.77%) was obtained as a yellow solid. 1 1H 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]pyrimidine-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]pyrimidine-3-yl]acetaldehyde (0.1 g, 273.07 μmol, 1 equivalent) in MeOH (5 mL), NaBH4 (30.99 mg, 819.22 μmol, 3 equivalents) was gradually added at 0°C. The mixture was then stirred at 20°C for 1 hour. LC-MS showed that the reaction was complete. The mixture was slowly quenched with ice water (5 mL) and extracted with siRNA (10 mL x 4). The combined organic layers were washed with brine (5 mL x 2), dehydrated with Na2SO4, 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%~40%, 10 min). The eluate was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-3-(2-hydroxyethyl)-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (83.8 mg, 227.58 μmol, yield 83.34%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 368.1 m / z [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]pyrimidine-7(6H)-one

[0110] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 1 in Scheme B-6) [ka] 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.1 g, 251.10 μmol, 1 equivalent) and NIS (73.44 mg, 326.43 μmol, 1.3 equivalents) were dissolved in MeCN (2 mL), to which HBF4 (55.12 mg, 627.76 μmol, 39.10 μL, 2.5 equivalents) was added dropwise. The mixture was stirred at 100 °C for 10 hours. TLC showed that the reaction was complete. H2O (5 mL) was added to the reaction mixture. The reaction mixture was extracted with HCl (10 mL × 3). The combined organic layers were washed with brine (5 mL × 2), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-iodo-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (0.12 g, 228.95 μmol, yield 91.18%) was obtained as a brown solid. 1 1H 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]pyrimidine-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]pyrimidine-7-one (55 mg, 104.93 μmol, 1 equivalent), tributyl(oxazole-2-yl) stannan (375.77 mg, 1.05 mmol, 10 equivalents), and Pd(dppf)Cl2 (15.36 mg, 20.99 μmol, 0.2 equivalents) in dioxane (2 mL) was degassed, purged three times with N2, and then stirred at 100°C for 12 hours under an N2 atmosphere. LC-MS and HPLC showed that 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%~50%, 10 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-3-(oxazol-2-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (14.0 mg, 28.83 μmol, yield 27.48%, purity 95.832%) was obtained as a pale yellow solid. 11H 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 m / z; Measured mass: 465.0 m / z [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]pyrimidine-7(6H)-one (Step 1 in Scheme B-7) [ka] 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (230 mg, 577.54 μmol, 1 equivalent) and NBS (113.07 mg, 635.29 μmol, 1.1 equivalents) were mixed in MeCN (3 mL) and HBF4 (126.79 mg, 1.44 mmol, 89.92 μL, 2.5 equivalents) was added. The mixture was stirred at 100°C for 30 minutes. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (5 mL) and extracted with SiO (8 mL × 3). The combined organic layer was washed with saturated NaHCO3 (10 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 4 g, 30 mL / min, eluate with 0-100% ethyl acetate / methanol). The eluate was removed under reduced pressure. Compound 3-bromo-5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (190 mg, crude) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 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]pyrimidine-7-one (40 mg, 83.83 μmol, 1 equivalent), Zn(CN)2 (29.53 mg, 251.50 μmol, 3 equivalents), dppf (7.44 mg, 13.41 μmol, 0.16 equivalents), and Pd2(dba)3 (6.14 mg, 6.71 μmol, 0.08 equivalents) in DMA (0.5 mL) was degassed, purged three times with N2, and then stirred at 170°C for 40 minutes under an N2 atmosphere. LC-MS showed that the reaction was complete. After filtration, the filtrate was separated and purified by preparative HPLC (column: Phenomenex Gemini-NX 150 30 mm × 5 μm; mobile phase: [water (10 mM NH4HCO3)-MeCN]; B%: 20%~50%, 8 min). The solvent was removed under freeze-drying conditions. 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 1H 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 m / z; Measured mass: 423.0 m / z [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]pyrimidine-7(6H)-one

[0118] Preparation of 5-chloro-3-fluoro-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 1 in Scheme B-8) [ka] A mixture of 5-chloro-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.5 g, 2.71 mmol, 1 equivalent) and Select F (2.88 g, 8.13 mmol, 3 equivalents) in MeCN (15 mL) was stirred at 100 °C for 40 hours. LC-MS and HPLC showed that approximately 20% of 5-chloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidine-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 minutes. After filtration, the solid was collected. The residue was used in the next step without further purification. Compound 5-chloro-3-fluoro-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.4 g, 1.97 mmol, yield 72.90%) was obtained as a yellow solid. 1 1H 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]pyrimidine-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]pyrimidine-7-one (0.1 g, 493.65 μmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (173.81 mg, 987.30 μmol, 131.67 μL, 2 equivalents) in t-BuOH (3 mL) was stirred at 100 °C under N2 for 10 hours. LC-MS and HPLC showed that 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%~60%, 10 min). The eluate was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1-methyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (15.6 mg, 45.59 μmol, yield 9.24%) was obtained as a white solid. 1 1H 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 m / z, measured value: 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]pyrimidine-7(6H)-one and 3-chloro-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one

[0122] Preparation of 5-chloro-3-fluoro-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one and 3,5-dichloro-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 1 in Scheme B-9) [ka] A mixture of 5-chloro-3-fluoro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (100 mg, 493.65 μmol, 1 equivalent) and 3,5-dichloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (493.65 μmol, 1 equivalent) in MeCN (1 mL) was mixed with NaI (295.97 mg, 1.97 mmol, 4 equivalents) and TMSCl (214.52 mg, 1.97 mmol, 250.61 μL, 4 equivalents) at 0°C. The mixture was then stirred at 25°C for 5 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (1 mL) and concentrated under reduced pressure. The aqueous solution was extracted with SiO (1 mL × 3). The combined organic layers were washed with brine (1 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. A mixture of compound 5-chloro-3-fluoro-1,6-dihydropyrazolo[4,3-d]pyrimidine-7-one (100 mg, crude) and 3,5-dichloro-1,6-dihydropyrazolo[4,3-d]pyrimidine-7-one was obtained as a brown solid. MS (ESI): Calculated mass of C5H2ClFN4O: 187.99 m / z; Measured mass: 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]pyrimidine-7(6H)-one and 3-chloro-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 2 in Scheme B-9) [ka] (3,4-dichlorophenyl)methaneamine (240.19 mg, 1.36 mmol, 181.96 μL, 2 equivalents) was added to a mixture of 5-chloro-3-fluoro-1,3a,6,7a-tetrahydropyrazolo[4,3-d]pyrimidine-7-one (130 mg, 682.19 μmol, 1 equivalent) and 3,5-dichloro-1,3a,6,7a-tetrahydropyrazolo[4,3-d]pyrimidine-7-one (682.19 μmol, 1 equivalent) in t-BuOH (4 mL). The mixture was stirred at 100 °C for 12 hours. HPLC showed that 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%~50%, 12 min). The solvent was removed by lyophilization. 5-[(3,4-dichlorophenyl)methylamino]-3-fluoro-1,6-dihydropyrazolo[4,3-d]pyrimidine-7-one (compound 54) (6.6 mg, 18.94 μmol, yield 2.78%, purity 94.16%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 328.0 m / z [M+H] + 3-Chloro-5-[(3,4-dichlorophenyl)methylamino]-1,6-dihydropyrazolo[4,3-d]pyrimidine-7-one (compound 55) (8.2 mg, 23.34 μmol, yield 4.45%, purity 98.1%) was obtained as a grayish-white solid. 1 1H 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 m / z; Measured mass: 344.0 [M+H] + .

[0124] Scheme C-1 [ka]

[0125] General procedure for preparing compounds in Scheme C-1 Preparation of 1-allyl-4-nitropyrazole (Step 1 in Scheme C-1) [ka] To a solution of 4-nitro-1H-pyrazole (200 g, 1.77 mol, 1 equivalent) in DMF (1 L), 3-bromopropa-1-ene (235.29 g, 1.94 mol, 1.1 equivalents) and K2CO3 (488.74 g, 3.54 mol, 2 equivalents) were added under N2 conditions at 25°C. The mixture was then stirred at 80°C for 1 hour. TLC showed that the reaction was complete. The reaction mixture was quenched with ice water (500 mL) at 0°C and then extracted with MTBE (500 mL x 3). The combined organic layers were washed with brine (300 mL x 6), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 1-allyl-4-nitropyrazole (270 g, 1.76 mol, yield 99.71%) as a brownish oil. 1 1H 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-nitropyrazole-3-carboxylic acid (Step 2 in Scheme C-1) [ka] To a solution of 1-allyl-4-nitropyrazole (50 g, 326.50 mmol, 1 equivalent) in THF (500 mL), LiHMDS (1 M, 489.75 mL, 1.5 equivalents) was added dropwise under N2 at -78°C. The mixture was then stirred at -78°C for 1 hour. Dry CO2 was then blown into the mixture for 30 minutes. TLC showed that approximately 30% of the starting material remained, and new spots with high polarity had been formed. The reaction mixture (5 batches) was quenched with water (2.5 L) at 0°C, and then the pH was adjusted to 8-9 with saturated Na2CO3. The mixture was extracted with MTBE (1 L x 3) to recover 1-allyl-4-nitropyrazole. The aqueous layer was then adjusted to pH 1 with 2N HCl, and then extracted with ELISA (2 L x 3). The combined organic layers were washed with brine (1 L), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 2-allyl-4-nitropyrazole-3-carboxylic acid (200 g, 1.01 mol, yield 62.14%) as a brownish oil. 1 1H 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-nitropyrazole-3-carboxamide (Step 3 in Scheme C-1) [ka] A solution of 2-allyl-4-nitropyrazole-3-carboxylic acid (65 g, 329.70 mmol, 1 equivalent) in SOCl2 (200 mL) and DMF (5 mL) was stirred at 80°C for 2 hours. TLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain 1-allyl-5-(chlorosylmethyl)-4-nitropyrazole (80 g, crude) as a brownish oil. A solution of 2-allyl-4-nitropyrazole-3-carbonyl chloride (80 g, 371.07 mmol, 1 equivalent) in DCM (50 mL) was added dropwise to NH3·H2O (200 mL) at 0°C, and the mixture was then stirred at 25°C for 1 hour. TLC showed that the reaction was complete. Some solid was produced. After filtration, the solid was collected, and the filtrate was extracted with siRNA (200 mL × 3). The combined organic layers were washed with brine (100 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The combined residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 330 g, eluate with a 0-50% ethyl acetate / petroleum ether concentration gradient at 200 mL / min) to obtain 2-allyl-4-nitropyrazole-3-carboxamide (22 g, 112.15 mmol, yield 30.22%) 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-aminopyrazole-3-carboxamide (Step 4 in Scheme C-1) [ka] To a solution of 2-allyl-4-nitropyrazole-3-carboxamide (22 g, 112.15 mmol, 1 equivalent) in MeOH (150 mL) and H2O (50 mL), NH4Cl (12.00 g, 224.30 mmol, 2 equivalents) and Fe (18.79 g, 336.45 mmol, 3 equivalents) were added at 25 °C. The mixture was stirred at 85 °C for 2 hours. TLC showed that 2-allyl-4-nitropyrazole-3-carboxamide was completely consumed, and one major novel 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 (volume:volume = 3:1, 100 mL x 3). The combined organic layers were washed with brine (50 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 2-allyl-4-aminopyrazole-3-carboxamide (17.5 g, 105.31 mmol, yield 93.90%) as a brownish oil. 1 1H 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-aminopyrazole-3-carboxamide (17.5 g, 105.31 mmol, 1 equivalent) in MeCN (250 mL), CDI (22.20 g, 136.90 mmol, 1.3 equivalents) was gradually added over 1 hour at 100°C under N2. The mixture was then heated at 100°C for 12 hours. LC-MS showed that the reaction was complete. The reaction mixture was filtered at 100°C, and the cake was washed with MeCN (100 mL x 3) to obtain 1-allyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione (15.2 g, 79.09 mmol, yield 75.11%) as a grayish-white solid. 1 1H 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-dichloropyrazolo[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 equivalent) in POCl3 (119.68 g, 780.54 mmol, 72.53 mL, 10 equivalents), DBU (71.30 g, 468.32 mmol, 70.59 mL, 6 equivalents) was added dropwise at 50°C under N2. The mixture was then stirred at 85°C for 12 hours. TLC showed that 1-allyl-4H-pyrazolo[4,3-d]pyrimidine-5,7-dione was completely consumed, and a novel 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, and then extracted with siRNA (200 mL x 3). The combined organic layers were washed with saturated Na2CO3 to a pH of 7-8, then washed with brine (100 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 1-allyl-5,7-dichloropyrazolo[4,3-d]pyrimidine (17 g, 74.21 mmol, yield 95.08%) as a brownish oil. 1 1H 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]pyrimidine-7(6H)-one (Step 7 in Scheme C-1) [ka] To a solution of 1-allyl-5,7-dichloropyrazolo[4,3-d]pyrimidine (6.8 g, 29.69 mmol, 1 equivalent) in dioxane (60 mL), NaOH (1 M, 29.69 mL, 1 equivalent) was added at 25 °C. The mixture was stirred at 100 °C for 10 hours. TLC and LC-MS showed that the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was diluted to pH=5 with 2N HCl. Some solid was formed. After filtration, the solid was collected 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]pyrimidine-7-one (5.5 g, 26.11 mmol, yield 87.97%) 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]pyrimidine-7(6H)-one (Step 8 in Scheme C-1) [ka] A solution of 1-allyl-5-chloro-6H-pyrazolo[4,3-d]pyrimidine-7-one (4 g, 18.99 mmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (10.03 g, 56.97 mmol, 7.60 mL, 3 equivalents) in t-BuOH (60 mL) was heated at 100 °C for 10 hours. LC-MS showed that the reaction was complete. The solvent was removed under reduced pressure. The residue was dissolved in HCl (200 mL) and washed with 0.005 N HCl (50 mL × 10⁻¹). The organic layer was dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was ground with HCl (30 mL). After filtration, the solid was collected. Compound 1-allyl-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one (3 g, 8.49 mmol, yield 44.70%, purity 99.09%) was obtained as a white solid, and 59.6 mg of it was used next. 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 m / z, measured value: 350.0 [M+H] + .

[0134] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-7-one (1 g, 2.86 mmol, 1 equivalent) and K2OsO4·2H2O (105.21 mg, 285.55 μmol, 0.1 equivalent) in THF (20 mL) and H2O (20 mL) was stirred at 25°C for 30 minutes. Then NaIO4 (1.83 g, 8.57 mmol, 3 equivalents) was added, and the mixture was stirred at 25°C for 1.5 hours. TLC showed that the reaction was complete. After filtration, the filtrate was extracted with SiO4 (30 mL × 3). The combined organic layers were washed with saturated Na2SO3 (10 mL) and brine (10 mL × 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was used in the next step without further purification. Compound 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]acetaldehyde (0.8 g, 2.27 mmol, yield 79.55%) was obtained as a grayish-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]pyrimidine-1-yl]acetaldehyde (4.26 mmol, 1 equivalent) and R'NHR'' (5.11-85.2 mmol, 1.2-20 equivalents) in MeOH or DCM (4 mL / mmol), NaBH3CN (5.11-12.78 mmol, 1.2-3 equivalents) was gradually added at 0°C. The mixture was then stirred at 0°C-80°C for a set period (2-10 hours). LC-MS and HPLC showed that the reaction was complete. The reaction mixture was quenched with H2O at 0°C and then extracted with ELISA. The combined organic layer was washed with brine, dehydrated with Na2SO4, 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 min, 10 min, or 12 min; c) [Water (0.04% NH3·H2O + 10mM NH4HCO3)-MeCN]; B%: 40%~60%, 10.5 min. MeCN was removed under reduced pressure. The aqueous solution was dehydrated by freeze-drying.

[0137] compound 57 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholinoethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride (Step 10 in Scheme C-1) [ka] 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]acetaldehyde (1.5 g, 4.26 mmol, 1 equivalent) and morpholine (1.11 g, 12.78 mmol, 1.12 mL, 3 equivalents) were mixed in MeOH (15 mL) and NaBH3CN (401.49 mg, 6.39 mmol, 1.5 equivalents) was gradually added at 0°C, and the mixture was then stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The mixture was quenched in ice water (10 mL) at 0°C, and the organic solvent was removed under reduced pressure. The aqueous solution was extracted with SiO2 (20 mL x 5). The combined organic layers were washed with brine (20 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The filtrate was separated and purified by preparative HPLC (HCl conditions: column: Phenomenex luna C18 250 mm × 50 mm 10 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 5%~35%, 20 min). The solvent was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-(2-morpholinoethyl)-6H-pyrazolo[4,3-d]pyrimidine-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 m / z; Measured mass: 423.1 m / z [M+H] + .

[0138] compound 58 5-((3,4-dichlorobenzyl)amino)-1-(2-(4-methylpiperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The procedure yielded the desired compound (58.7 mg, 134.53 μmol, 31.59% yield) as a grayish-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 m / z; Measured mass: 436.2 m / z [M+H] + .

[0139] compound 59 5-((3,4-dichlorobenzyl)amino)-1-(2-(1,1-dioxidethiomorpholino)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (22.3 mg, 47.31 μmol, yield 11.11%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 471.1 m / z [M+H] + .

[0140] compound 60 5-((3,4-dichlorobenzyl)amino)-1-(2-(4-methyl-3-oxopiperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The procedure yielded the desired compound (69.1 mg, 153.45 μmol, 36.03% yield) as a grayish-white solid. 1 1H 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 The calculated mass of Cl2N7O2 is 449.11 m / z, while the measured mass is 450.2 m / z [M+H]. + .

[0141] compound 61 5-((3,4-dichlorobenzyl)amino)-1-(2-(methylamino)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The procedure yielded the desired compound (30.5 mg, 83.05 μmol, 19.50% yield) as a grayish-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 m / z; Measured mass: 367.1 m / z [M+H] + .

[0142] compound 62 1-[2-(tert-butylamino)ethyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The procedure yielded the desired compound (20.3 mg, 48.83 μmol, 24.46% yield) as a white solid. 1 1H 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 m / z; Measured mass: 409.1 m / z [M+H] + .

[0143] compound 63 1-(2-aminoethyl)-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (34.9 mg, 98.81 μmol, yield 34.80%) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 353.1 m / z [M+H] + .

[0144] compound 64 1-(2-(4-acetylpiperazine-1-yl)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] Following the procedure, the desired compound (0.293 g, 585.05 μmol, yield 20.60%) was obtained as a white solid. 1 1H 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 m / z, measured value: 464.1 m / z [M+H] + .

[0145] compound 65 5-((3,4-dichlorobenzyl)amino)-1-(2-(4-hydroxypiperidine-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (25.4 mg, 51.62 μmol, yield 18.18%, HCl) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 437.1 m / z [M+H] + .

[0146] compound 66 1-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethyl)piperidine-4-carboxamide hydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (24.9 mg, 51.41 μmol, yield 18.10%) was obtained as a white solid according to the procedure. 1 1H 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 m / z, measured value: 464.2 m / z [M+H] + .

[0147] compound 67 5-((3,4-dichlorobenzyl)amino)-1-(2-(4-(dimethylamino)piperidine-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (42.0 mg, 76.61 μmol, yield 26.98%) was obtained as a white solid according to the procedure. 1 1H 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 of Cl4N7O: 463.17 m / z; Measured mass: 464.2 m / z [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]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (26.3 mg, 55.26 μmol, 19.46% yield) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 435.0 m / z [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]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (26.9 mg, 57.25 μmol, yield 28.80%) was obtained as a white solid according to the procedure. 1 1H 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 of Cl4N7O: 447.13 m / z; Measured mass: 448.1 m / z [M+H] + .

[0150] compound 70 5-((3,4-dichlorobenzyl)amino)-1-(2-(3-hydroxyazetidine-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (33.8 mg, 71.91 μmol, yield 25.32%) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 409.1 m / z [M+H] + .

[0151] compound 71 5-((3,4-dichlorobenzyl)amino)-1-(2-(4-(2-hydroxyethyl)piperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The desired compound (28.3 mg, 52.38 μmol, yield 18.45%) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 466.1 m / z [M+H] + .

[0152] compound 72 4-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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] Following the procedure, the desired compound (26 mg, 48.14 μmol, yield 16.95%, purity 98.662%, HCl) was obtained 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 m / z, measured value: 494.1 [M+H] + .

[0153] compound 73 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-((dimethylamino)methyl)morpholino)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as step 10 in scheme C-1. [ka] The procedure yielded the desired compound (33.7 mg, 66.33 μmol, 23.36% yield) as a white solid. 1 1H 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 The calculated mass of Cl4N7O2 is 479.16 m / z, and the measured mass is 480.2 m / z [M+H]. + .

[0154] compound 74 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(piperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-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]pyrimidine-1-yl]acetaldehyde (0.18 g, 511.11 μmol, 1 equivalent), tert-butylpiperazine-1-carboxylate (114.23 mg, 613.33 μmol, 1.2 equivalents), and AcOH (3.07 mg, 51.11 μmol, 2.92 μL, 0.1 equivalent) in MeOH (4 mL) was stirred at 25°C for 2 hours. Then, NaBH3CN (38.54 mg, 613.33 μmol, 1.2 equivalents) was added little by little at 0°C, and the mixture was stirred at 25°C for 2 hours. LC-MS showed that 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 SiO2 (10 mL x 3). The combined organic layer was washed with brine (10 mL x 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was used in the next step without further purification. The compound tert-butyl4-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethyl]piperazine-1-carboxylate (0.2 g, 382.83 μmol, yield 74.90%) was obtained as a pale brown solid. MS (ESI): C 23 H 29 Calculated mass of Cl2N7O3: 521.17 m / z; Measured mass: 522.2 m / z [M+H] + .

[0156] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-(piperazin-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one [ka] To a mixture of tert-butyl 4-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethyl]piperazine-1-carboxylate (0.2 g, 382.83 μmol, 1 equivalent) in SiO2 (5 mL), HCl / SiO2 (4 M, 2.87 mL, 30 equivalents) was added, and the mixture was stirred at 25°C for 2 hours. LC-MS and HPLC showed that the reaction was complete. Some solid was produced. After filtration, the solid was collected. 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%~35%, 10 min). Compound 5-[(3,4-dichlorophenyl)methylamino]-1-(2-piperazine-1-ylethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (60.2 mg, 142.55 μmol, yield 37.24%) was obtained as a grayish-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 m / z; Measured mass: 422.1 m / z [M+H] + .

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

[0158] To a solution of 1-allyl-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.1 g, 285.55 μmol, 1 equivalent) (Step 8 in Scheme C-1) in THF (0.5 mL) and H2O (0.5 mL), K2OsO4·2H2O (10.52 mg, 28.55 μmol, 0.1 equivalent) was added. After 30 minutes, NMO (100.35 mg, 856.65 μmol, 90.41 μL, 3 equivalents) was added. The mixture was stirred at 25°C for 3 hours. LC-MS and HPLC showed that the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was extracted with SiO2 (8 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dehydrated with 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%: 25%~40%, 10 min) to obtain 5-((3,4-dichlorobenzyl)amino)-1-(2,3-dihydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (48.5 mg, 126.23 μmol, yield 44.21%, purity 100%) as a yellow solid. 1 1H 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 m / z; Measured mass: 384.1 m / z [M+H] + .

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

[0160] To a mixture of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]acetaldehyde (0.15 g, 425.92 μmol, 1 equivalent) (Step 9 in Scheme C-1) in MeOH (3 mL), NaBH4 (24.17 mg, 638.89 μmol, 1.5 equivalents) was gradually added at 0 °C. The mixture was then stirred at 25 °C for 1 hour. LC-MS showed that the reaction was complete. The mixture was quenched at 0 °C with saturated NH4Cl (1 mL), and the organic solvent was removed under reduced pressure. The residue was dissolved in ELISA (30 mL) and washed with brine (5 mL × 1). The organic layer was dehydrated with 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%: 25%~40%, 10 min). The eluate was removed by lyophilization. Compound 5-((3,4-dichlorobenzyl)amino)-1-(2-hydroxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-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 m / z; Measured mass: 354.1 m / z [M+H] + .

[0161] compound 77 Preparation of 5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-1-yl]acetaldehyde (0.15 g, 425.92 μmol, 1 equivalent), tert-butylpiperazine-1-carboxylate (95.19 mg, 511.11 μmol, 1.2 equivalents), and AcOH (2.56 mg, 42.59 μmol, 2.44 μL, 0.1 equivalent) in DCM (5 mL) was stirred at 25°C for 1 hour. Then NaBH(OAc)3 (108.32 mg, 511.11 μmol, 1.2 equivalents) was added, and the mixture was stirred at 25°C for 3 hours. LC-MS and HPLC showed no residual 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]acetaldehyde. One novel peak was mainly observed on LC-MS, which was 5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one. The mixture was quenched with ice water (5 mL) and then extracted with SiO2 (10 mL × 3). The combined organic layer was washed with brine (10 mL × 1), dehydrated with 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%~45%, 10 min). After freeze-drying, the resulting mixture was washed with MeOH (2 mL) and SiO2 (1 mL). Compound 5-[(3,4-dichlorophenyl)methylamino]-1,6-dihydropyrazolo[4,3-d]pyrimidine-7-one (24.3 mg, 78.35 μmol, yield 18.40%) was obtained 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 m / z; Measured mass: 310.0 m / z [M+H] + .

[0163] compound 78 Preparation of 1,1'-(azandiylbis(ethane-2,1-diyl))bis(5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-1-yl]acetaldehyde (0.1 g, 283.95 μmol, 1 equivalent) and AcONH4 (218.73 mg, 2.84 mmol, 10 equivalents) in MeOH (3 mL) was stirred at 25°C for 2 hours. Then, NaBH3CN (26.77 mg, 425.92 μmol, 1.5 equivalents) was added gradually at 0°C, and the mixture was stirred at 25°C for 10 hours. LC-MS and HPLC showed that the reaction was complete. The main compound produced was 5-[(3,4-dichlorophenyl)methylamino]-1-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethylamino]ethyl]-6H-pyrazolo[4,3-d]pyrimidine-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 SiO2 (10 mL × 3). The combined organic layers were washed with brine (10 mL × 1), dehydrated with 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). Compound 5-[(3,4-dichlorophenyl)-methylamino]-1-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethylamino]ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (38.1 mg, 55.27 μmol, yield 19.46%) 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 m / z; Measured value: 688.2 m / z [M+H] + .

[0164] Scheme C-2 [ka]

[0165] General procedure for preparing compounds in Scheme C-2 Preparation of the compound (Step 1 in Scheme C-2) [ka] A mixture of 1-(R'-aminoethyl)-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one (283.12 μmol, 1 equivalent), R''COOH (283.12 μmol to 368.06 μmol, 1 to 1.3 equivalents), EDCI (339.74 μmol, 1.2 equivalents), HOBt (56.62 μmol, 0.2 equivalents), and DIEA (849.36 μmol, 147.94 μL, 3 equivalents) in DMF (3 mL / mmol) was stirred at 20°C to 25°C for 10 to 12 hours. The reaction mixture was stirred at 0°C to 25°C for a set period of time (1 to 3 hours). LC-MS showed that the reaction was complete. The reaction mixture was quenched with H2O and then extracted with SiO. The combined organic layers were washed with brine, dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC. Column: Luna C18 100mm×30mm 5μm or Nano-micro Kromasil C18 100mm×30mm 5μm; Mobile phase: [Water (0.1% TFA)-MeCN]; B%: 20%~45%, 10 min or 20 min). The aqueous solution was lyophilized to obtain 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]pyrimidine-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]pyrimidine-7-one (0.1 g, 283.12 μmol, 1 equivalent), 3,5-dimethylisoxazole-4-carboxylic acid (39.96 mg, 283.12 μmol, 48.14 μL, 1 equivalent), EDCI (65.13 mg, 339.74 μmol, 1.2 equivalents), HOBt (7.65 mg, 56.62 μmol, 0.2 equivalents), and DIEA (109.77 mg, 849.36 μmol, 147.94 μL, 3 equivalents) in DMF (1 mL) was stirred at 25°C for 12 hours. LC-MS showed that the reaction was complete. After filtration, the filtrate was separated and 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). Compound N-[2-[5-[(3,4-dichlorophenyl)-methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethyl]-3,5-dimethyl-isoxazole-4-carboxamide (29.2 mg, 58.90 μmol, yield 20.80%, purity 96.077%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 476.1 m / z [M+H] + .

[0168] compound 80 N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound (29.5 mg, 60.16 μmol, 44.19% yield) as a grayish-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 m / z; Measured mass: 490.2 m / z [M+H] + .

[0169] compound 81 6-Chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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] Following the procedure, the desired compound (34.2 mg, 65.30 μmol, yield 23.98%) was obtained 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 m / z; Measured mass: 523.1 m / z [M+H] + .

[0170] compound 82 N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound (25.1 mg, 54.30 μmol, 24.92% yield) as a grayish-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 m / z; Measured mass: 462.1 m / z [M+H] + .

[0171] compound 83 6-Chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethyl)-3-hydroxypyridazine-4-carboxamide was prepared according to the procedure described herein as step 1 in scheme C-2. [ka] Following the procedure, the desired compound (19 mg, 37.27 μmol, yield 18.81%) was obtained 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 m / z, measured value: 509.1 m / z [M+H] + .

[0172] compound 84 3-Chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound (3.7 mg, 6.08 μmol, yield 2.15%) 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 m / z, measured value: 509.1 m / z [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]pyrimidine-1-yl)ethyl)-N-methylpyridazine-4-carboxamide was prepared according to the procedure described herein as step 1 in scheme C-2. [ka] Following the procedure, the desired compound (35 mg, 64.55 μmol, yield 11.85%) was obtained 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 m / z; Measured mass: 541.1 m / z [M+H] + .

[0174] compound 86 N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethyl)isoxazole-4-carboxamide was prepared according to the procedure described herein as step 1 in scheme C-2. [ka] The desired compound (20.4 mg, 45.24 μmol, yield 10.65%) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 448.1 m / z [M+H] + .

[0175] compound 87 3-Chloro-N-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound (21.4 mg, 40.86 μmol, yield 17.04%) as a white solid. 1 1H 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 m / z; Measured mass: 523.1 m / z [M+H] + .

[0176] Scheme C-3 [ka]

[0177] General procedure for preparing compounds in Scheme C-3 Preparation of the compound (Step 1 in Scheme C-3) [ka] 5-Chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (5.42 mmol, 1 equivalent), R1OH (5.42 mmol to 16.26 mmol, 1 to 3 equivalents), and PPh3 (5.96 mmol to 10.84 mmol, 1.1 to 2 equivalents) were dissolved in THF (3 mL / mmol to 10 mL / mmol), to which DIAD (5.96 mmol to 10.84 mmol, 1.1 to 2 equivalents) was added dropwise at 0°C. The mixture was then stirred at 0°C to 25°C for 2 to 16 hours. TLC showed that the reaction was complete. The mixture was quenched with ice water, and the organic layer was separated. The aqueous solution was extracted with SiO2. The combined organic layers were washed with brine, dehydrated with 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, eluate with a concentration gradient of 0-100% ethyl acetate / petroleum ether or 0-20% MeOH / ethyl acetate ether at 35 mL / min or 80 mL / min). The eluate was removed under reduced pressure to obtain the desired product.

[0178] Preparation of the compound (Step 2 in Scheme C-3) [ka] 5-chloro-7-methoxy-1-R1-1H-pyrazolo[4,3-d]pyrimidine (4.36 mmol, 1 equivalent) was dissolved in MeOH (3 mL / mmol to 5 mL / mmol) or THF (3 mL / mmol to 5 mL / mmol) and H2O (3 mL / mmol to 5 mL / mmol), to which LiOH·H2O (13.08 mmol to 21.80 mmol, 3 to 5 equivalents) was added. The mixture was stirred at 20°C to 25°C for 2 to 16 hours. TLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was diluted to pH 6 with 3N HCl and then extracted with siRNA. The combined organic layers were washed with brine, dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired product.

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

[0180] A mixture of 5-chloro-1-R1-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (3.87 mmol, 1 equivalent), R3NH2 (162.66 mmol to 216.88 mmol, 1.5 to 2 equivalents), and DIEA (11.61 mmol, 3 equivalents; DIEA was added only if R3NH2 was an HCl salt) in t-BuOH, t-AmOH, or NMP (3 mL / mmol to 10 mL / mmol) was stirred at 100°C to 160°C for 3 to 40 hours. LC-MS indicated that 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 30mm×5μm;k) Welch Xtimate C18 150mm×25mm 5μm;l) Xtimate C18 150mm×25mm 5μm;m) Xtimate C18 100mm×30mm 3μm. Mobile phase: a) [Water (0.1% TFA)-MeCN]; B%: 10%~70%, 10 or 20 min; b) [Water (0.05% HCl)-MeCN]; B%: 10%~70%, 10 or 20 min; c) [Water (10mM NH4HCO3)-MeCN]; B%: 10%~85%, 8 or 20 min. The solvent was removed by lyophilization to obtain the desired product.

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

[0182] Preparation of 2-(2-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy)ethanol (Step 1 in Scheme C-3) [ka] 5-Chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (1 g, 5.42 mmol, 1 equivalent), 2-(2-hydroxyethoxy)ethanol (1.15 g, 10.84 mmol, 1.03 mL, 2 equivalents), and PPh3 (2.13 g, 8.13 mmol, 1.5 equivalents) were dissolved in THF (10 mL), to which DIAD (1.64 g, 8.13 mmol, 1.58 mL, 1.5 equivalents) was added dropwise at 0°C. The mixture was stirred at 25°C for 2 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (15 mL) and then concentrated under reduced pressure to remove the organic solvent. The aqueous solution was extracted with SiO2 (20 mL x 3). The combined organic layers were washed with brine (10 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 20 g, 60 mL / min, eluate with a 0-60% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure. Compound 2-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]ethanol (1.19 g, 4.36 mmol, yield 80.55%) was obtained as a yellow oily substance. 1¹H 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). Compound 2-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)ethoxy]ethanol (440 mg, 1.61 mmol, yield 29.78%) 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]pyrimidine-7(6H)-one (Step 2 in Scheme C-3) [ka] A mixture of 2-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]ethanol (1.19 g, 4.36 mmol, 1 equivalent) and LiOH·H2O (732.45 mg, 17.46 mmol, 4 equivalents) in THF (10 mL) and H2O (10 mL) was stirred at 20°C for 5 hours. TLC showed that 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 3N HCl. The aqueous solution was extracted with SiO (25 mL × 4). The combined organic layers were washed with brine (20 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 5-chloro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (1.3 g, crude) as a yellow oily substance. 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]pyrimidine-7(6H)-one (Step 3 in Scheme C-3) [ka] A mixture of (3,4-dichlorophenyl)methaneamine (1.36 g, 7.73 mmol, 1.03 mL, 2 equivalents) and 5-chloro-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (1 g, 3.87 mmol, 1 equivalent) in t-BuOH (6 mL) was stirred at 100 °C for 16 hours. LC-MS showed that 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%~40%, 20 min). The aqueous solution was freeze-dried to obtain 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.99 g, 2.45 mmol, yield 63.34%, purity 98.50%) as a white solid. 30.1 mg was then used. 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 m / z, measured value: 398.0 [M+H] + .

[0185] compound 89 5-((3,4-dichlorobenzyl)amino)-1-(4-hydroxybutyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (378.3 mg, 986.58 μmol, yield 46.04%) as a white solid. 1 1H 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 m / z; Measured mass: 382.2 m / z [M+H] + .

[0186] compound 90 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)butyl acetate was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (0.03 g, 70.71 μmol, yield 77.48%) was obtained as a white solid. 1 1H 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 m / z, measured value: 424.0 [M+H] + .

[0187] compound 91 5-((3,4-dichlorobenzyl)amino)-1-(thiazole-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (71.4 mg, 175.31 μmol, yield 42.66%) was obtained 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 The calculated mass of Cl2N6OS is 406.02 m / z, and the measured mass is 406.9 m / z [M+H]. + .

[0188] compound 92 5-((3,4-dichlorobenzyl)amino)-1-(oxazol-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (290.5 mg, 741.49 μmol, 15.42% yield) as a grayish-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 m / z, measured value: 391.0 [M+H] + .

[0189] compound 93 5-((3,4-dichlorobenzyl)amino)-1-((5-oxopyrrolidine-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (63.7 mg, 156.41 μmol, 46.52% yield) as a white solid. 1 1H 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 m / z; Measured mass: 407.1 m / z [M+H] + .

[0190] compound 94 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-oxopyrrolidine-1-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (90.4 mg, 210.81 μmol, 59.38% yield) as a grayish-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 m / z; Measured mass: 421.1 m / z [M+H] + .

[0191] compound 95 1-(2-(2-(2-butoxyethoxy)ethoxy)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (77.5 mg, 155.50 μmol, yield 29.37%) was obtained as a white solid according to the procedure. 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 of Cl2N5O4: 497.16 m / z; Measured mass: 498.2 m / z [M+H] + .

[0192] compound 96 5-((3,4-dichlorobenzyl)amino)-1-(((2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (20 mg, 41.73 μmol, yield 6.69%) 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 The calculated mass of Cl2N6O4 is 454.09 m / z, and the measured mass is 455.09 m / z [M+H]+.

[0193] compound 97 5-((3,4-dichlorobenzyl)amino)-1-((1-methyl-1H-imidazole-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (21 mg, 51.95 μmol, yield 13.04%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 404.1 m / z [M+H] + .

[0194] compound 98 tert-butyl3-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)pyrrolidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (26.4 mg, 54.15 μmol, 36.80% yield) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 479.2 m / z [M+H] + . compound 99 Preparation of 1-(2-(2-aminoethoxy)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-1-yl)ethoxy)ethyl)carbamate was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 497.2 m / z [M+H] + .

[0196] Preparation of 1-(2-(2-aminoethoxy)ethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-1-yl]ethoxy]ethyl]carbamate (compound 100) (0.1 g, 201.06 μmol, 1 equivalent) in HCl / SiO2 (20 mL) and SiO2 (5 mL) was stirred at 20°C for 5 hours. LC-MS indicated that 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]pyrimidine-7-one (70 mg, 159.76 μmol, yield 79.46%, purity 98.989%, 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 m / z; Measured mass: 397.1 m / z [M+H] + .

[0197] Compound 101 1-((1H-1,2,4-triazol-1-yl)methyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (34.7 mg, 77.16 μmol, 21.34% yield, HCl) as a white solid. 1 1H 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 m / z, measured value: 391.0 [M+H] + .

[0198] Compound 102 5-((3,4-dichlorobenzyl)amino)-1-((tetrahydrofuran-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (48.8 mg, 123.40 μmol, 52.38% yield) as a white solid. 1 1H 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 m / z; Measured mass: 394.1 m / z [M+H] + .

[0199] compound 103 2-((1H-1,2,4-triazol-1-yl)methyl)-5-((3,4-dichlorobenzyl)amino)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (21.1 mg, 45.65 μmol, 12.62% yield, HCl) was obtained as a white solid according to the procedure. 1 1H 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 m / z, measured value: 391.0 [M+H] + .

[0200] Compound 104 5-((3,4-dichlorobenzyl)amino)-1-(2-((2-hydroxyethyl)thio)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (184.5 mg, 436.26 μmol, 52.11% yield) as a white solid. 1 1H 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 m / z; Measured mass: 414.0 m / z [M+H] + .

[0201] Compound 105 1-((1,4-dioxan-2-yl)methyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (89.6 mg, 210.89 μmol, yield 57.08%) was obtained as a white solid. 1 1H 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 m / z; Measured value: 410.0 m / z [M+H] + .

[0202] compound 106 5-((3,4-dichlorobenzyl)amino)-1-(1,3-dimethoxypropan-2-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (68.5 mg, 158.02 μmol, yield 62.01%) was obtained 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 m / z; Measured mass: 412.1 m / z [M+H] + .

[0203] Compound 107 5-((3,4-dichlorobenzyl)amino)-1-(1,3-dihydroxypropan-2-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (17.5 mg, 43.55 μmol, yield 10.65%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 384.1 m / z [M+H] + .

[0204] compound 108 5-((3,4-dichlorobenzyl)amino)-1-(2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one) was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 438.1 m / z [M+H] + .

[0205] Compound 109 5-((3,4-dichlorobenzyl)amino)-1-(pyridine-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (55.4 mg, 117.21 μmol, 29.49% yield) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 401.0 m / z [M+H] + .

[0206] compound 110 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyridine-3-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z, measured value: 415.0 m / z [M+H] + .

[0207] Compound 111 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyridine-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (66.3 mg, 157.39 μmol, 45.68% yield) as a white solid. 1 1H 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 of Cl3N6O: 414.08 m / z; Measured mass: 415.1 [M+H] + .

[0208] compound 112 5-((3,4-dichlorobenzyl)amino)-1-(pyrazine-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z, measured value: 402.0 [M+H] + .

[0209] compound 113 5-[(3,4-dichlorophenyl)methylamino]-1-(3-pyridylmethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (87.3 mg, 196.51 μmol, yield 57.13%) was obtained 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 m / z; Measured mass: 401.0 m / z [M+H] + .

[0210] compound 114 5-((3,4-dichlorobenzyl)amino)-1-(pyrimidine-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 402.0 m / z [M+H] + .

[0211] compound 115 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 442.1 m / z [M+H] + .

[0212] compound 116 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 486.1 m / z [M+H] + .

[0213] Compound 117 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyrimidine-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (9.8 mg, 21.84 μmol, yield 20.14%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 416.0 m / z [M+H] + .

[0214] compound 118 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (84.1 mg, 207.55 μmol, yield 24.97%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 380.0 m / z [M+H] + .

[0215] Compound 119 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 380.1 m / z [M+H] + .

[0216] compound 120 5-((3,4-dichlorobenzyl)amino)-1-((tetrahydrofuran-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (72.6 mg, 181.68 μmol, yield 21.03%) 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 m / z; Measured mass: 394.0 m / z [M+H] + .

[0217] compound 121 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 394.0 m / z [M+H] + .

[0218] compound 122 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (8.0 mg, 19.89 μmol, yield 11.97%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 380.0 m / z [M+H] + .

[0219] compound 123 5-((3,4-dichlorobenzyl)amino)-1-(3-hydroxypropyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (27.8 mg, 75.50 μmol, 19.18% yield) as a grayish-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 m / z, measured value: 368.0 [M+H] + .

[0220] compound 124 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyrazine-2-yl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (24.1 mg, 49.93 μmol, yield 19.19%) was obtained as a white solid according to the procedure. 1 1H 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 m / z; Measured mass: 416.0 m / z [M+H] + .

[0221] compound 125 5-((3,4-difluorobenzyl)amino)-1-(oxetan-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (18.3 mg, 52.58 μmol, yield 50.62%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 348.2 m / z [M+H] + .

[0222] compound 126 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-methoxyethyl)-1H-pyrazolo[4,3-d]pyrimidine-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] A mixture of 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (1 g, 5.42 mmol, 1 equivalent) and 1-bromo-2-methoxyethane (978.90 mg, 7.04 mmol, 661.42 μL, 1.3 equivalents) in DMF (10 mL) was mixed with Cs₂CO₃ (3.53 g, 10.84 mmol, 2 equivalents) at 20°C. The mixture was then stirred at 20°C for 3 hours. TLC showed that the reaction was complete. The mixture was slowly poured into ice water (20 mL) and then extracted with siRNA (20 mL x 3). The combined organic layers were washed with brine (10 mL x 4), dehydrated with Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 12 g, 40 mL / min, eluate with a 0-35% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure. Compound 5-chloro-7-methoxy-2-(2-methoxyethyl)pyrazolo[4,3-d]pyrimidine (0.6 g, 2.47 mmol, yield 45.64%) was obtained as a white solid. 1 1H 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). Compound 5-chloro-7-methoxy-1-(2-methoxyethyl)pyrazolo[4,3-d]pyrimidine (0.5 g, 2.06 mmol, yield 38.033%) was obtained as a white solid. 1 1H 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]pyrimidine-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 equivalent) and LiOH·H2O (311.25 mg, 7.42 mmol, 3 equivalents) in MeOH (5 mL) and H2O (5 mL) was stirred at 20°C for 3 hours. TLC showed that the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was slowly adjusted to pH 6-7 with 2N HCl, and some solid was produced. The solid was collected after filtration. The aqueous solution was then extracted with SiO2 (20 mL x 2). The combined organic layers were washed with brine (5 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 5-chloro-1-(2-methoxyethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.5 g, 2.19 mmol, yield 88.45%) was obtained as a white solid. 1 1H 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]pyrimidine-7(6H)-one (Step 3 in Scheme C-3) [ka] A solution of 5-chloro-1-(2-methoxyethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (500.00 mg, 2.19 mmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (769.97 mg, 4.37 mmol, 583.31 μL, 2 equivalents) in t-BuOH (5 mL) was heated at 100 °C for 30 hours. TLC showed that 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%~55%, 10 min). Compound 5-[(3,4-dichlorophenyl)methylamino]-1-(2-methoxyethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (510.7 mg, 1.38 mmol, yield 63.00%, purity 99.34%) was obtained as a white solid. 1 1H 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 m / z, measured value: 368.0 [M+H] + .

[0226] compound 127 1-benzyl-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (119.5 mg, 294.94 μmol, 38.44% yield) as a pale 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 m / z; Measured mass: 400.0 m / z [M+H] + .

[0227] compound 128 5-((3,4-dichlorobenzyl)amino)-2-(2-methoxyethyl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 368.1 m / z [M+H] + .

[0228] compound 129 2-benzyl-5-((3,4-dichlorobenzyl)amino)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (49.5 mg, 123.67 μmol, yield 21.49%) 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 m / z; Measured mass: 400.1 m / z [M+H] + .

[0229] compound 130 5-((3,4-dichlorobenzyl)amino)-1-isopropyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (77.2 mg, 219.18 μmol, yield 51.27%) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 352.1 m / z [M+H] + .

[0230] compound 131 5-((3,4-dichlorobenzyl)amino)-1-(5-methoxypentyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (73.6 mg, 175.20 μmol, yield 18.97%) 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 m / z; Measured mass: 410.0 m / z [M+H] + .

[0231] compound 132 5-((3,4-dichlorobenzyl)amino)-2-isopropyl-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 352.1 m / z [M+H] + .

[0232] compound 133 5-((3,4-dichlorobenzyl)amino)-2-(5-methoxypentyl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (68.3 mg, 166.46 μmol, yield 18.03%) 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 m / z; Measured mass: 410.0 m / z [M+H] + .

[0233] compound 134 1-(cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (150 mg, 396.56 μmol, 52.58% yield) was obtained as a white solid according to the procedure. 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 m / z; Measured mass: 378.1 m / z [M+H] + .

[0234] compound 135 2-(cyclobutylmethyl)-5-((3,4-dichlorobenzyl)amino)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 378.1 m / z [M+H] + .

[0235] compound 136 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-methoxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 412.1 m / z [M+H] + .

[0236] compound 137 5-((3,4-dichlorobenzyl)amino)-2-(2-(2-methoxyethoxy)ethyl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 412.1 m / z [M+H] + .

[0237] compound 138 5-((3,4-dichlorobenzyl)amino)-1-((2-methoxyethoxy)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z; Measured mass: 398.1 m / z [M+H] + .

[0238] compound 139 5-((3,4-dichlorobenzyl)amino)-1-(pyrimidine-2-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z, measured value: 388.1 [M+H] + .

[0239] compound 140 5-((3,4-dichlorobenzyl)amino)-2-(pyrimidine-2-yl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (8 mg, 19.56 μmol, yield 12.16%) was obtained 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 m / z, measured value: 388.0 [M+H] + .

[0240] compound 141 2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-2H-pyrazolo[4,3-d]pyrimidine-2-yl)ethoxy)acetic acid was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (6.1 mg, 14.08 μmol, yield 5.48%) was obtained as a white solid according to the procedure. 1 1H 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 m / z, measured value: 412.0 [M+H] + .

[0241] compound 142 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The desired compound (19.6 mg, 50.10 μmol, 45.42% yield) was obtained as a white solid according to the procedure. 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 m / z, measured value: 366.0 [M+H] + .

[0242] compound 143 5-((3,4-dichlorobenzyl)amino)-2-(oxetan-3-yl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] Following the procedure, the desired compound (16 mg, 41.96 μmol, 36.57% yield) was obtained 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 m / z, measured value: 366.0 [M+H] + .

[0243] compound 144 5-((3,4-dichlorobenzyl)amino)-1-(oxazol-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 m / z, measured value: 391.0 [M+H] + .

[0244] compound 145 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)butanoic acid was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded 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 then used. 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 m / z, measured value: 396.2 [M+H] + .

[0245] compound 146 5-((3,4-dichlorobenzyl)amino)-1-(4-(dimethylphosphoryl)butyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound (4.4 mg, 9.55 μmol, yield 9.64%, purity 96.037%) 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-methylmorpholine-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-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]pyrimidine-1-yl)methyl]morpholine-4-carboxylate (1 g, 1.08 mmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (380.84 mg, 2.16 mmol, 288.51 μL, 2 equivalents) in t-BuOH (10 mL) was stirred at 100 °C for 12 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove t-BuOH. The residue was diluted with SiO2 (10 mL) and washed with aqueous HCl (2 N, 10 mL x 6). The combined organic layer was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The compound tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]morpholine-4-carboxylate (1 g, crude product) was obtained as a white solid. The residue (200 mg) was then purified by preparative HPLC (neutral conditions column: Welch Xtimate C18 150 mm × 25 mm 5 μm; mobile phase: [water (10 mM NH4HCO3)-MeCN]; B%: 50%~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]pyrimidine-1-yl]methyl]morpholine-4-carboxylate (23.4 mg, 45.88 μmol, yield 4.24%, purity 99.87%) was obtained as a white solid and used thereafter. 1 1H 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 of Cl2N6O4: 508.14 m / z; Measured mass: 509.2 m / z [M+H] + .

[0248] compound 149 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(morpholine-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] A solution of tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]morpholine-4-carboxylate (200 mg, 392.63 μmol, 1 equivalent) in HCl / siRNA (2 mL) and siRNA (1 mL) was stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove siRNA. The residue was purified by preparative HPLC (HCl conditions column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15%~50%, 8 min). The solvent was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-(morpholine-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (35.4 mg, 79.42 μmol, yield 20.23%, purity 100%, HCl) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 409.1 m / z [M+H] + .

[0249] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-((4-methylmorpholine-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-(morpholine-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (400 mg, 897.40 μmol, 1 equivalent, HCl) in MeOH (5 mL), HCHO (218.48 mg, 2.69 mmol, 200.44 μL, purity 37%, 3 equivalents) was added 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 equivalent) and NaBH3CN (451.16 mg, 7.18 mmol, 8 equivalents) were added at 0°C. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl conditions: column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15%~45%, 10 min). The solvent was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[(4-methylmorpholine-2-yl)methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (49.1 mg, 106.69 μmol, yield 11.89%, purity 99.9%, HCl) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 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]pyrimidine-1-yl)-1-isonicotinoylpyrrolidine-2-carboxylate hydrochloride

[0251] Preparation of 1-(tert-butoxycarbonyl)-4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrzolo[4,3-d]pyrimidine-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]pyrimidine-1-yl)pyrrolidine-2-carboxylic acid (520 mg, 1.35 mmol, 1 equivalent) and (3,4-dichlorophenyl)methanamine (477.05 mg, 2.71 mmol, 361.40 μL, 2 equivalents) in 2-methylbutan-2-ol (3 mL) was stirred at 130 °C for 10 hours. LC-MS and HPLC showed that 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%~50%, 12 min). The mixture was dried under freeze-drying conditions to obtain 1-(tert-butoxycarbonyl)-4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrzolo[4,3-d]pyrimidine-1-yl)pyrrolidine-2-carboxylic acid (140 mg, 267.50 μmol, yield 19.74%) as a white solid. 1 1H 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]pyrimidine-1-yl)pyrrolidine-2-carboxylate hydrochloride (Step 1 in Scheme C-3) [ka] 1-tert-butoxycarbonyl-4-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]pyrrolidine-2-carboxylic acid (140 mg, 267.50 μmol, 1 equivalent) was dissolved in HCl (3 mL) at 0°C and HCl / HCl (4 M, 3 mL) was added. The mixture was stirred at 25°C for 4 hours. LC-MS and HPLC showed that 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%~50%, 12 min). The mixture was dried under freeze-drying conditions to obtain 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)pyrrolidine-2-carboxylate hydrochloride (120 mg, 249.49 μmol, yield 93.27%, purity 95.577%, HCl) as a white solid. 1 1H 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 m / z; Measured mass: 423.1 m / z [M+H] + .

[0254] Preparation of 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)-1-isonicotinoylpyrrolidine-2-carboxylate 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]pyrimidine-1-yl]pyrrolidine-2-carboxylic acid (70 mg, 152.27 μmol, 1 equivalent, HCl) in DCM (3 mL), TEA (61.63 mg, 609.07 μmol, 84.78 μL, 4 equivalents) was added. Then, pyridine-4-carbonyl chloride (70.48 mg, 395.90 μmol, 2.6 equivalents, HCl) was gradually added to the mixture at 0°C. The mixture was stirred at 25°C for 3 hours. LC-MS showed that 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%~40%, 12 min). The mixture was dried under freeze-drying conditions to obtain 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)-1-isonicotinoylpyrrolidine-2-carboxylic acid hydrochloride (25.0 mg, 43.68 μmol, yield 28.69%, purity 98.683%, HCl) as a white solid. 1 1H 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 of Cl3N7O4: 527.09 m / z; Measured mass: 528.1 m / z [M+H] + .

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

[0256] compound 153 tert-butyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)piperidine-1-carboxylate was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound tert-butyl4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)piperidine-1-carboxylate (33.7 mg, 65.68 μmol, yield 46.48%, purity 96.163%) as a white solid. 1 1H 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 m / z; Measured mass: 493.2 m / z [M+H] + .

[0257] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one [ka] Compound 5-((3,4-dichlorobenzyl)amino)-1-(piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (1 g, 2.33 mmol, yield 39.64%, HCl) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 393.1 m / z [M+H] + .

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

[0259] tert-butyl3-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound, tert-butyl 3-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl)piperidine-1-carboxylate (430 mg, crude product), as a white solid. 1 1H 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-(piperidine-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 1 in scheme C-3. [ka] Following the procedure, the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(piperidine-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (500 mg, crude) was obtained as a white solid. 80 mg (crude) was separated and purified by HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15%~45%, 10 min) to obtain 20.9 mg of pure product, which was then used. 1 1H 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 m / z, measured mass: 407.11 [M+H] + .

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

[0261] tert-butyl 3-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound tert-butyl 3-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]pyrrolidine-1-carboxylate (220 mg, 445.90 μmol, yield 43.82%) as a white solid. 1 1H 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-(pyrrolidine-3-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] To a solution of tert-butyl 3-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]pyrrolidine-1-carboxylate (compound 165) (220 mg, 445.90 μmol, 1 equivalent) in SiO2 (2 mL), HCl / SiO2 (4 M, 2 mL, 17.94 equivalents) was added at 0°C. The mixture was stirred at 25°C for 4 hours. LC-MS showed that 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%~35%, 12 min). The mixture was freeze-dried to obtain 5-[(3,4-dichlorophenyl)methylamino]-1-(pyrrolidine-3-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (180 mg, 418.87 μmol, yield 93.94%, HCl) as a white solid. 1 1H 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 m / z, measured value: 393.1 m / z [M+H] + .

[0263] compound 156 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-ylsulfonyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] 5-[(3,4-dichlorophenyl)methylamino]-1-(4-piperidyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (80 mg, 186.16 μmol, 1 equivalent, HCl) and TEA (75.35 mg, 744.65 μmol, 103.65 μL, 4 equivalents) were mixed in 3 mL of DCM, to which pyridine-3-sulfonyl chloride (33.06 mg, 186.16 μmol, 8.53 μL, 1 equivalent) was added dropwise at 0°C. The mixture was then stirred at 20°C for 1 hour. LC-MS showed that 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 x 4). The combined organic layers were washed with brine (2 mL x 1), dehydrated with Na2SO4, filtered, and 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%: 45%~70%, 10 min). The eluate was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(3-pyridylsulfonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (26.7 mg, 46.07 μmol, yield 24.75%, purity 98.51%, HCl) was obtained as a white solid. 1 1H 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 The calculated mass of Cl3N7O3S is 533.08 m / z, while the measured mass is 354.2 [M+H]. + .

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

[0265] 5-[(3,4-dichlorophenyl)methylamino]-1-(4-piperidyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one hydrochloride (60 mg, 139.62 μmol, 1 equivalent, HCl) and 1-bromo-2-methoxyethane (58.22 mg, 418.87 μmol, 39.34 μL, 3 equivalents) were dissolved in CH3CN (1 mL), to which K2CO3 (57.89 mg, 418.87 μmol, 3 equivalents) was added. The mixture was stirred at 80°C for 16 hours. LC-MS and HPLC showed that the reaction was complete. The mixture was filtered. The filtrate was separated and purified by preparative HPLC (column: Phenomenex Luna C18 100 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 20%~40%, 10 min). The mixture was freeze-dried to obtain 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(2-methoxyethyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (18.6 mg, 40.51 μmol, yield 29.01%, purity 98.295%) as a white solid. 1 1H 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 m / z; Measured mass: 451.2 m / z [M+H] + .

[0266] compound 158 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholino-2-oxoethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-morpholino-2-oxoethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (16.8 mg, 37.65 μmol, yield 17.24%, purity 98.005%) as a white solid. 1 1H 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 m / z; Measured mass: 437.1 m / z [M+H] + .

[0267] compound 159 5-((3,4-dichlorobenzyl)amino)-1-((1-methyl-5-oxopyrrolidine-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-((1-methyl-5-oxopyrrolidine-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (56.2 mg, 132.45 μmol, yield 30.33%, purity 99.285%) as a white solid. 1 1H 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 m / z; Measured mass: 421.1 m / z [M+H] + .

[0268] compound 160 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-morpholinoethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-morpholinoethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (54.9 mg, 105.69 μmol, yield 17.32%, purity 96.991%, HCl) as a white solid. 1 1H 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 m / z, measured value: 467.1 m / z [M+H] + .

[0269] compound 161

[0270] 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-2-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-2-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride (40.9 mg, 85.66 μmol, yield 14.17%, purity 98.510%) as a white solid. 1 1H 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 m / z; Measured mass: 470.1 m / z [M+H] + .

[0271] compound 162 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-4-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(4-pyridyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (17.6 mg, 37.42 μmol, yield 10.31%, purity 95.01%, HCl) as a pale yellow solid. 1 1H 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 m / z; Measured mass: 470.1 m / z [M+H] + .

[0272] compound 163 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride (70.0 mg, 144.42 μmol, yield 25.01%, purity 97.038%) as a white solid. 1 1H 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 m / z; Measured mass: 470.1 m / z [M+H] + .

[0273] compound 164 Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(2-((2-hydroxyethyl)sulfonyl)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one [ka] A mixture of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethylsulfanyl)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.1 g, 241.37 μmol, 1 equivalent) (steps 1-3 in scheme C-3), sodium periodate (206.50 mg, 965.46 μmol, 53.50 μL, 4 equivalents), and trichlororuthenium (5.01 mg, 24.14 μmol, 1.61 μL, 0.1 equivalent) in THF (2.5 mL) and H2O (2.5 mL) was stirred at 50°C for 3 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was extracted with ELISA (10 mL x 3). The combined organic layers were washed with brine (10 mL), dehydrated with Na2SO4, filtered, and 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%: 20%~55%, 12 min). The aqueous solution was lyophilized to obtain 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethylsulfonyl)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (41 mg, 91.03 μmol, yield 37.72%, purity 99.093%) as a white solid. 1 1H 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 m / z; Measured mass: 446.0 m / z [M+H] + .

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

[0275] Preparation of 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)acetic acid [ka] To a solution of 2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)acetic acid (0.32 g, 1.40 mmol, 1 equivalent) in t-BuOH (3 mL), (3,4-dichlorophenyl)methaneamine (369.66 mg, 2.10 mmol, 280.04 μL, 1.5 equivalents) was added. The mixture was stirred at 100 °C for 24 hours. LC-MS showed that 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%~45%, 12 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by freeze-drying. Compound 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]acetic acid (50 mg, 135.81 μmol, yield 9.70%) was obtained as a white solid. 1 1H 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]pyrimidine-1-yl)-N-(methylsulfonyl)acetamide [ka]

[0277] To a solution of 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]acetic acid (40 mg, 108.64 μmol, 1 equivalent) (steps 1-3 in scheme C-3) in DCM (1 mL), methanesulfonamide (20.67 mg, 217.29 μmol, 2 equivalents), DMAP (6.64 mg, 54.32 μmol, 0.5 equivalents) and DCC (22.42 mg, 108.64 μmol, 21.98 μL, 1 equivalent) were added. The mixture was stirred at 40°C under N2 for 10 hours. LC-MS indicated that 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%~45%, 12 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound 2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)-N-(methylsulfonyl)acetamide (21.1 mg, 46.89 μmol, yield 43.16%, purity 98.960%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 445.0 m / z [M+H] + .

[0278] compound 166 tert-butyl4-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound, tert-butyl 4-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]ethyl]piperazine-1-carboxylate (20 mg, 34.80 μmol, yield 9.29%, purity 98.58%), as a white solid, which was then used. 1 1H 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 of Cl2N7O4: 565.20 m / z; Measured mass: 566.2 m / z [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]pyrimidine-7(6H)-one dihydrochloride [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-piperazine-1-ylethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (43.3 mg, 86.11 μmol, yield 24.39%, HCl) as a white solid. 1 1H 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 m / z; Measured mass: 466.1 m / z [M+H] + .

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

[0281] 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-tetrahydropyran-2-yloxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-tetrahydropyran-2-yloxypropoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (180 mg, crude product) as a white solid. 1 1H 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]pyrimidine-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 eq) in MeOH (1 mL) and HCl / MeOH (5 mL, 4 M) was stirred at 25 °C for 1 h. HPLC indicated that 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% - 50%, 10.5 min). The solvent was removed under lyophilization. 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, yield 32.03%, purity 98.74%) 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 = 5.6 Hz, 2H), 4.31 (t, J = 5.2 Hz, 1H), 3.73 (t, J = 5.6 Hz, 2H), 3.39 (t, J = 6.4 Hz, 2H), 3.36~3.34 (m, 2H), 1.57~1.51 (m, 2H). HPLC: 98.74% (220 nm), 98.25% (215 nm), 100.00% (254 nm). MS (ESI): C 17 H 19 The calculated mass of Cl2N5O3 is 411.09, m / z found 412.0 [M+H] + .

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

[0284] 1-[3-(2-benzyloxyethoxy)propyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 1-[3-(2-benzyloxyethoxy)propyl]-5-[(3,4-dichlorophenyl)methylamino]-6H-pyrazolo[4,3-d]pyrimidine-7-one (180 mg, 358.29 μmol, yield 44.82%) as a white solid. 1 1H 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]pyrimidine-7-one [ka] 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 eq) in EtOAc (10 mL) was added with Pd / C (10%, 5 mg) under a N2 atmosphere. The suspension was degassed and purged three times with H2. The mixture was stirred at 25 °C for 12 h under H2 (15 psi). LC-MS indicated that the reaction was complete. The reaction mixture was filtered to remove insolubles 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% - 50%, 10.5 min). The solvent was removed under lyophilization. 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%) was obtained 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 alculated mass for C18H18Cl2N5O3 411.09, m / z found 412.2 [M+H] + .

[0286] Compound 170 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyridine-3-yloxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-pyridyloxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (202 mg, 431.32 μmol, yield 69.90%, purity 99.873%, HCl) as a white solid. 59.0 mg was then used. 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 m / z; Measured mass: 431.1 m / z [M+H] + .

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

[0288] Preparation of methyl 4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl)oxazole-2-carboxylate and methyl 4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidine-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 equivalent) in THF (5 mL), PPh3 (852.59 mg, 3.25 mmol, 1.5 equivalents) and methyl 4-(hydroxymethyl)oxazole-2-carboxylate (408.59 mg, 2.60 mmol, 1.2 equivalents) were added. DIAD (657.30 mg, 3.25 mmol, 632.02 μL, 1.5 equivalents) was then added dropwise at 0°C. The mixture was stirred at 25°C for 12 hours. TLC (PE:Â=1:1) indicated that the reaction was complete. The reaction mixture was quenched with H2O (5 mL), and a small amount of white solid was produced. After filtration, the solid was collected. The solid was washed with Â(2 mL) and concentrated under reduced pressure. A mixture of the compound methyl 4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl)oxazole-2-carboxylate (0.45 g, 1.39 mmol, yield 64.15%) and methyl 4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)oxazole-2-carboxylate was obtained as a white solid. 1 1H 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 1H 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]pyrimidine-1-yl)methyl)oxazol-2-yl)methanol and (4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)oxazol-2-yl)methanol [ka] A mixture of methyl 4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)methyl]oxazole-2-carboxylate (0.36 g, 1.11 mmol, 1 equivalent) and methyl 4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)methyl]oxazole-2-carboxylate (1.11 mmol, 1 equivalent) in MeOH (2 mL) was gradually added at 0°C with NaBH4 (167.97 mg, 4.44 mmol, 4 equivalents). The mixture was stirred at 25°C for 13 hours. Then, NaBH4 (167.97 mg, 4.44 mmol, 4 equivalents) was gradually added again at 0°C. The mixture was stirred at 25°C for 12 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (10 mL) and then extracted with ELISA (15 mL x 3). The combined organic layer was washed with brine (10 mL x 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. A mixture of the compound (4-((5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl)oxazole-2-yl)methanol (0.27 g, 913.15 μmol, yield 82.27%) and (4-((5-chloro-7-methoxy-2H-pyrazolo[4,3-d]pyrimidine-2-yl)methyl)oxazole-2-yl)methanol was obtained as a pale yellow oil. 1 1H 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 1H 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]pyrimidine-7(6H)-one and 5-chloro-2-((2-(hydroxymethyl)oxazol-4-yl)methyl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 2 in Scheme C-3) [ka]

[0291] [4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)methyl]oxazole-2-yl]methanol (0.27 g, 913.15 μmol, 1 equivalent) and [4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)methyl]oxazole-2-yl]methanol (913.15 μmol, 1 equivalent)] were mixed in H2O (2 mL) and MeOH (4 mL) to which LiOH·H2O (114.95 mg, 2.74 mmol, 3 equivalents) was added. The mixture was stirred at 25°C for 10 hours. TLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was adjusted to pH=6 with HCl (3N). The mixture was then extracted with HCl (10 mL × 3). The combined organic layers were washed with brine (6 mL x 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. A mixture of compound 5-chloro-1-((2-(hydroxymethyl)oxazole-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (0.25 g, crude) and 5-chloro-2-((2-(hydroxymethyl)oxazole-4-yl)methyl)-2H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was obtained as a pale yellow oil. 1 1H 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 1H 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]pyrimidine-7(6H)-one) (Step 3 in Scheme C-3) [ka] A mixture of 5-chloro-2-[[2-(hydroxymethyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (781.10 μmol, 1 equivalent) and 5-chloro-1-[[2-(hydroxymethyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.22 g, 781.10 μmol, 1 equivalent) in 2-methylbutan-2-ol (5 mL) was mixed with (3,4-dichlorophenyl)methaneamine (275.01 mg, 1.56 mmol, 208.34 μL, 2 equivalents). The mixture was stirred at 130 °C for 10 hours. LC-MS and HPLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions column: Phenomenex Luna C18 150×30mm 5μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%~35%, 12 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound 5-[(3,4-dichlorophenyl)methylamino]-2-[[2-(hydroxymethyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (33.1 mg, 78.58 μmol, yield 10.06%) was obtained as a white solid. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(hydroxymethyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (30.5 mg, 68.96 μmol, yield 8.83%, purity 95.236%) was obtained as a pale yellow solid, and 20.1 mg was used next. 1 1H 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 m / z; Measured mass: 421.0 m / z [M+H] + .

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

[0294] tert-butyl3-[4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound tert-butyl3-[4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]oxazole-2-yl]piperidine-1-carboxylate (220 mg, 382.97 μmol, yield 97.97%) as a white solid. 1 1H 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-(piperidine-3-yl)oxazol-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] A mixture of tert-butyl 3-[4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]oxazole-2-yl]piperidine-1-carboxylate (40 mg, 69.63 μmol, 1 equivalent) in HCl (1 mL) and HCl / HCl (4 M, 3 mL) was stirred at 25°C for 2 hours. HPLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The filtrate was purified by preparative HPLC (HCl conditions column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 10%~40%, 12 min). The solvent was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(3-piperidyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (17 mg, 30.86 μmol, yield 44.33%, purity 92.74%, HCl) was obtained as a white solid. 1 1H 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 m / z, measured value: 474.1 m / z [M+H] + .

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

[0297] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(3-piperidyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (130 mg, 254.50 μmol, 1 equivalent, HCl) in MeOH (2 mL), formaldehyde (61.97 mg, 763.50 μmol, 56.85 μL, purity 37%, 3 equivalents) was added at 0°C. The mixture was stirred at 0°C for 10 minutes. Then, AcOH (1.53 mg, 25.45 μmol, 1.46 μL, 0.1 equivalent) and NaBH3CN (79.96 mg, 1.27 mmol, 5 equivalents) were added at 0°C. The mixture was stirred at 25°C for 10 hours. LC-MS showed that the reaction was complete. The reaction mixture was quenched with H2O (2 mL) at 0°C and then concentrated under reduced pressure. The aqueous solution was extracted with RINKAN (2 mL x 3). The combined organic layer was washed with brine (2 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl conditions column: Phenomenex Luna C18 150 mm x 30 mm 5 μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 10%~40%, 12 min). The solvent was removed by lyophilization. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[2-(1-methyl-3-piperidyl)oxazole-4-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (50.6 mg, 93.65 μmol, yield 36.80%, purity 97.14%, HCl) was obtained as a white solid. 1 1H 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 m / z, measured value: 488.1 m / z [M+H] + .

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

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

[0300] 5-Chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (400 mg, 2.17 mmol, 1 equivalent), methyl 5-(2-hydroxyethoxy)pyridine-2-carboxylate (555.51 mg, 2.82 mmol, 1.3 equivalents), and PPh3 (852.59 mg, 3.25 mmol, 1.5 equivalents) were dissolved in THF (5 mL), to which DIAD (657.30 mg, 3.25 mmol, 632.02 μL, 1.5 equivalents) was added dropwise at 0°C. The mixture was stirred at 25°C for 5 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (2 mL) and extracted with ₹ (2 mL × 3). The combined organic layers were washed with brine (2 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 12 g, 45 mL / min, eluate with 0-100% ethyl acetate / methanol). The eluate was removed under reduced pressure. Compound methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]pyridine-2-carboxylate (580 mg, 1.59 mmol, yield 73.58%) was obtained as a white solid. Compound methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)ethoxy]pyridine-2-carboxylate (280 mg, 769.75 μmol, yield 35.52%) was obtained as a white solid. 1 1H 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]pyrimidine-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]pyrimidine-1-yl)ethoxy]pyridine-2-carboxylate (100 mg, 274.91 μmol, 1 equivalent) in MeOH (1 mL) and H2O (2 mL), LiOH·H2O (34.61 mg, 824.73 μmol, 3 equivalents) was added. The mixture was stirred at 25°C for 2 hours. TLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was diluted to pH=6 with 3N HCl, and a small amount of white solid was produced. After filtration, the solid was collected and concentrated under reduced pressure. Compound 5-[2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]pyridine-2-carboxylic acid (95 mg, crude) was obtained as a white solid. 1 1H 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]pyrimidine-1-yl)ethoxy)picolinate (Step 3 in Scheme C-3) [ka] A mixture of 5-[2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]pyridine-2-carboxylic acid (90 mg, 268.09 μmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (94.39 mg, 536.19 μmol, 71.51 μL, 2 equivalents) in 2-methyl-2-butanol (2 mL) was stirred at 140 °C for 4 hours. LC-MS showed that 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%~45%, 12 min). The solvent was removed by lyophilization. Compound 5-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]pyridine-2-carboxylic acid (45.1 mg, 86.74 μmol, yield 32.35%, purity 98.42%, HCl) was obtained as a white solid. 1 1H 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 m / z, measured value: 475.0 [M+H] + .

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

[0304] To a solution of methyl 5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]pyridine-2-carboxylate (150 mg, 412.37 μmol, 1 equivalent) in THF (1 mL), DIBAL-H (1 M, 2.06 mL, 5 equivalents) was added dropwise at 0°C. The mixture was then stirred at 25°C for 6 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (2 mL) and extracted with ELISA (2 mL × 3). The combined organic layers were washed with brine (2 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The compound [5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]-2-pyridyl]methanol (130 mg, 387.20 μmol, yield 93.90%) was obtained as a white solid. 1 1H 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]pyrimidine-7-one (Step 2 in Scheme C-3) [ka] [5-[2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy]-2-pyridyl]methanol (130 mg, 387.20 μmol, 1 equivalent) was dissolved in MeOH (1 mL) and H2O (1 mL), to which LiOH·H2O (48.74 mg, 1.16 mmol, 3 equivalents) was added. The mixture was stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was diluted to pH=6 with 3N HCl and extracted with ELISA (3 mL × 3). The combined organic layers were washed with brine (3 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 5-chloro-1-[2-[[6-(hydroxymethyl)-3-pyridyl]oxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (100 mg, 310.83 μmol, yield 80.28%) was obtained as a white solid. 1 1H 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)pyridine-3-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as step 3 in scheme C-3. [ka]

[0306] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-[[6-(hydroxymethyl)-3-pyridyl]oxy]ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (82.6 mg, 162.01 μmol, yield 52.12%, purity 97.63%, HCl) as a white solid. 11H 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 m / z; Measured mass: 461.1 m / z [M+H] + .

[0307] compound 176 5-((3,4-dichlorobenzyl)amino)-1-((1-(2-methoxyethyl)-5-oxopyrrolidine-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-((1-(2-methoxyethyl)-5-oxopyrrolidine-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (306 mg, 642.75 μmol, yield 52.34%, purity 97.743%) as a white solid. 1 1H 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 m / z; Measured mass: 465.1 m / z [M+H] + .

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

[0309] 5-[(3,4-dichlorophenyl)methylamino]-1-[[1-(2-methoxyethyl)-5-oxo-pyrrolidine-3-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (50 mg, 107.45 μmol, 1 equivalent) was dissolved in 2 mL of DCM, to which BBr3 (80.76 mg, 322.35 μmol, 31.06 μL, 3 equivalents) was added dropwise at 0°C. The mixture was stirred at 0°C for 1 hour. LC-MS and HPLC showed that the reaction was complete. 2 mL of ice water was added to the mixture. The mixture was extracted with HCl (5 mL x 3). The combined organic layers were dehydrated with Na2SO4, filtered, and 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%: 20%~50%, 12 min). The mixture was dried under lyophilization to obtain 5-((3,4-dichlorobenzyl)amino)-1-((1-(2-hydroxyethyl)-5-oxopyrrolidine-3-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (13.2 mg, 29.25 μmol, yield 27.22%) as a white solid. 1 1H 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; Measured mass: 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 to 3 in Scheme C-3. [Chemical formula] By the procedure, 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, yield 52.10%, purity 94.451%) was obtained 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, m / z found 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 to 3 in Scheme C-3. [Chemical formula] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[3-(3-hydroxycyclobutoxy)propyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (88.5 mg, 200.74 μmol, yield 37.48%, purity 99.42%) as a white solid. 1 1H 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 m / z; Measured mass: 438.1 m / z [M+H] + .

[0312] compound 180 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-4-yl)pyrrolidine-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-4-yl)pyrrolidine-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride (12.5 mg, 27.30 μmol, yield 18.91%, purity 99.647%) as a white solid. 1 1H 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 of Cl4N7O: 455.10 m / z; Measured mass: 456.1 [M+H] + .

[0313] compound 181 5-((3,4-dichlorobenzyl)amino)-1-(2-(pyridine-3-ylmethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(3-pyridylmethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (183.7 mg, 378.49 μmol, yield 55.10%, purity 99.26%, HCl) as a white solid. 1 1H 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 m / z; Measured mass: 445.1 m / z [M+H] + .

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

[0315] tert-butyl 2-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]pyrrolidine-1-carboxylate (0.13 g, 263.49 μmol, yield 23.31%) as a white solid. 1 1H 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-(pyrrolidine-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] To a solution of tert-butyl 2-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrrolidine-2-ylmethyl]methyl]pyrrolidine-1-carboxylate (0.13 g, 263.49 μmol, 1 equivalent) in HCl (1 mL), HCl / HCl (2 mL, 4 N) was added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-(pyrrolidine-2-ylmethyl)-6H-pyrrolidine-7-one (0.1 g, crude) was obtained as a white solid. 1 1H 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-(pyridine-2-yl)pyrrolidine-2-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-(pyrrolidine-2-ylmethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (0.1 g, 232.70 μmol, 1 equivalent, HCl) in DMF (1 mL), 2-fluoropyridine (45.19 mg, 465.41 μmol, 39.99 μL, 2 equivalents) and K2CO3 (96.49 mg, 698.11 μmol, 3 equivalents) were added. The mixture was stirred at 100 °C for 12 hours. LC-MS and HPLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (HCl conditions: column: Phenomenex Luna C18 150×30mm 5μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 15%~40%, 12 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[1-(2-pyridyl)pyrrolidine-2-yl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (37.0 mg, 72.55 μmol, yield 31.18%, purity 99.374%, HCl) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 470.1 m / z [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]pyrimidine-7(6H)-one hydrochloride

[0319] tert-butyl(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound tert-butyl(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy)-3-methoxypropyl)(methyl)carbamate (0.13 g, 234.04 μmol, yield 54.07%) as a white solid. 1 1H 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-(pyrrolidine-2-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-1-yl]ethoxy]-3-methoxypropyl]-N-methyl-carbamate (0.13 g, 234.04 μmol, 1 equivalent) in HCl / SiO2 (2 mL) was stirred at 25°C for 10 hours. LC-MS indicated that the reaction was complete. The reaction mixture was filtered under reduced pressure. A slightly pale yellow solid was produced. The solid was collected after filtration. Compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-methoxy-3-(methylamino)propan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (28.5 mg, 55.75 μmol, yield 23.82%, purity 96.203%, HCl) was obtained as a pale yellow solid and used next. Compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-methoxy-3-(methylamino)propan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (80 mg, HCl) was obtained as a pale yellow solid. 1 1H 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 m / z, measured value: 455.1 m / z [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]pyrimidine-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]pyrimidine-7-one (80 mg, 175.69 μmol, 1 equivalent) in MeOH (1 mL), formaldehyde (42.77 mg, 527.08 μmol, 39.24 μL, 3 equivalents) and AcOH (1.06 mg, 17.57 μmol, 1.00 μL, 0.1 equivalent) were added at 0°C. Then, NaBH3CN (33.12 mg, 527.08 μmol, 3 equivalents) was added little by little at 0°C. The mixture was stirred at 25°C for 3 hours. LC-MS and HPLC showed that the reaction was complete. H2O (1 mL) was added to the reaction mixture. The mixture was filtered under reduced pressure. The filtrate was purified by preparative HPLC (HCl conditions: column: Phenomenex Luna C18 150×30 mm 5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 15%~45%, 12 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((1-(dimethylamino)-3-methoxypropan-2-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (40.1 mg, 79.28 μmol, yield 45.12%, HCl) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 469.1 m / z [M+H] + .

[0323] compound 185 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-methoxy-1-(pyridine-3-yl)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-(2-methoxy-1-(pyridine-3-yl)ethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (98.1 mg, 185.02 μmol, yield 38.07%, purity 99.172%, HCl) as a white solid. 1 1H 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 m / z, measured value: 489.1 m / z [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]pyrimidine-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]pyrimidine-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]pyrimidine-7-one (0.6 g, 1.36 mmol, 1 equivalent) (steps 1-3 in scheme C-3) in 30 mL of DCM, DMP (2.30 g, 5.43 mmol, 1.68 mL, 4 equivalents) was gradually added at 0°C. The mixture was stirred at 25°C for 32 hours. TLC (ethyl acetate:methanol = 10:1, Rf = 0.58) indicated that the reaction was complete. The reaction mixture was quenched with H2O (10 mL) at 0°C. The organic layer was separated, and the aqueous solution was extracted with 3 x 10 mL of DCM. The combined organic layers were washed with brine (10 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]ethoxy]acetaldehyde (230 mg, crude product) was obtained as a yellow oily substance.

[0326] compound 187 Preparation of 2-(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)ethoxy)ethoxy)acetic acid) [ka] Sodium chlorite (51.97 mg, 574.63 μmol, 1.1 equivalents) was gradually added at 0°C to a solution of 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]ethoxy]acetaldehyde (230 mg, 522.40 μmol, 1 equivalent) and 2-methylbuta-2-ene (732.74 mg, 10.45 mmol, 1.11 mL, 20 equivalents) in H2O (1 mL), THF (1 mL), and t-BuOH (1 mL). The mixture was stirred at 20°C for 3 hours. LC-MS showed that the reaction was nearly complete. The reaction mixture was quenched with H2O (5 mL) at 20°C and then extracted with ELISA (10 mL × 3). The combined organic layers were washed with brine (5 mL), dehydrated with 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%~40%, 10 min). The aqueous solution was lyophilized. Compound 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]ethoxy]acetic acid (31 mg, 65.58 μmol, yield 12.55%, purity 96.527%) was obtained as a brown solid. 10.8 mg was used next. The desired compound (31 mg, 65.58 μmol, yield 12.55%) 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 m / z; Measured mass: 456.1 m / z [M+H] + .

[0327] Preparation of methyl 2-(2-(2-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-1-yl]ethoxy]ethoxy]acetic acid (20 mg, 43.83 μmol, 1 equivalent) in MeOH (10 mL), SOCl2 (26.07 mg, 219.16 μmol, 15.90 μL, 5 equivalents) was added dropwise at 0°C. The mixture was stirred at 25°C for 5 hours. LC-MS showed that 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%~50%, 10 min). The aqueous solution was freeze-dried to obtain methyl 2-[2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]ethoxy]acetate (3.6 mg, 7.37 μmol, yield 16.81%, purity 96.244%) 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 m / z; Measured mass: 470.1 m / z [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]pyrimidine-7(6H)-one

[0330] 5-((3,4-dichlorobenzyl)amino)-1-(2-((tetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-((3,4-dichlorobenzyl)amino)-1-(2-((tetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (72 mg, 156.23 μmol, yield 27.32%, purity 98.591%) 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; Measured mass: 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]pyrimidine-7(6H)-one [ka] 5-[(3,4-dichlorophenyl)methylamino]-1-(2-tetrahydrothiopyran-4-yloxyethyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (30 mg, 66.03 μmol, 1 equivalent) was added to a mixture of THF (3 mL) and H2O (3 mL) with RuCl3 (1.37 mg, 6.60 μmol, 0.44 μL, 0.1 equivalent), and then NaIO4 (56.49 mg, 264.10 μmol, 14.63 μL, 4 equivalents) was added to the mixture at 0°C. The mixture was stirred at 50°C for 16 hours. LC-MS and HPLC showed that the reaction was complete. The mixture was poured into H2O (5 mL) and then extracted with SiO4 (5 mL x 3). The combined organic layer was dehydrated with Na2SO4, 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%~50%, 10 min). The mixture was concentrated under reduced pressure to obtain 5-((3,4-dichlorobenzyl)amino)-1-(2-((1,1-dioxidetetrahydro-2H-thiopyran-4-yl)oxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-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; Measured mass: 486.0 [M+H] + .

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

[0333] compound 190 5-((3,4-dichlorobenzyl)amino)-1-(3,4-dimethoxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one was prepared according to the procedure described herein as steps 1-3 in scheme C-3. [ka] The procedure yielded the desired compound 5-[(3,4-dichlorophenyl)methylamino]-1-[(3,4-dimethoxyphenyl)methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (40 mg, 86.90 μmol, yield 36.44%, purity 100.00%) as a white solid. 5.8 mg was then used. 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 m / z; Measured mass: 460.0 m / z [M+H] + .

[0334] Preparation of 5-((3,4-dichlorobenzyl)amino)-1-(3,4-dihydroxybenzyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[(3,4-dimethoxyphenyl)methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (30 mg, 65.17 μmol, 1 equivalent) in DCM (1 mL), BBr3 (65.31 mg, 260.69 μmol, 25.12 μL, 4 equivalents) was added dropwise at 0°C. The mixture was stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete. The reaction mixture was quenched with H2O (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%~45%, 10 min). The solution was freeze-dried to obtain 5-[(3,4-dichlorophenyl)methylamino]-1-[(3,4-dihydroxyphenyl)methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (7.2 mg, 15.48 μmol, yield 23.75%, purity 92.908%) as a gray solid. The desired compound (7.2 mg, 15.48 μmol, yield 23.75%) 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 m / z, measured value: 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]pyrimidine-1-yl)ethoxy)acetaldehyde oxime

[0336] Preparation of 2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]acetaldehyde [ka] To a solution of 5-[(3,4-dichlorophenyl)methylamino]-1-[2-(2-hydroxyethoxy)ethyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (70 mg, 175.77 μmol, 1 equivalent) in DCM (10 mL), DMP (134.19 mg, 316.39 μmol, 97.95 μL, 1.8 equivalents) was gradually added at 0°C. The mixture was stirred at 25°C for 15 hours. LC-MS showed that 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 SiO (5 mL × 5). The combined organic layers were washed with saturated NaHCO3 (4 mL × 1) and brine (4 mL × 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]acetaldehyde (60 mg, 151.43 μmol, yield 86.15%) 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]pyrimidine-1-yl)ethoxy)acetaldehyde oxime [ka] To a solution of 2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]acetaldehyde (50 mg, 126.19 μmol, 1 equivalent) in EtOH (2 mL), NH2OH·HCl (17.54 mg, 252.38 μmol, 2 equivalents) and TEA (31.92 mg, 315.48 μmol, 43.91 μL, 2.5 equivalents) were added at 0°C. The mixture was stirred at 25°C for 10 hours. LC-MS indicated that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (neutral conditions: column: Phenomenex Gemini-NX C18 75mm × 30mm 3μm; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 25%~50%, 10.5 min). MeCN was removed under reduced pressure at 30°C. The residue was dehydrated by freeze-drying. Compound (1E)-2-[2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]ethoxy]acetaldehyde oxime (15.0 mg, 35.10 μmol, yield 27.81%, purity 96.223%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 411.0 m / z [M+H] + .

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

[0339] Preparation of (E)-1-(4-bromobuta-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-dibromobuta-2-ene (2.90 g, 13.54 mmol, 5 equivalents) in DMF (5 mL), Cs2CO3 (1.77 g, 5.42 mmol, 2 equivalents) was added. Then, 5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.5 g, 2.71 mmol, 1 equivalent) was added dropwise to DMF at 0°C. The mixture was stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete. H2O (10 mL) was added. The reaction mixture was extracted with RINKAN (15 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 12 g, 50 mL / min, eluate with a 0-50% ethyl acetate / petroleum ether concentration gradient). Compound (E)-1-(4-bromobuta-2-en-1-yl)-5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (0.4 g, 1.26 mmol, yield 46.50%) was obtained as a pale yellow solid. 1 1H 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). Compound 2-[(E)-4-bromobuta-2-enyl]-5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine (0.28 g, 881.70 μmol, yield 32.55%) was obtained as a white solid.

[0340] Preparation of (E)-5-chloro-7-methoxy-1-(4-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine [ka] 1-[(E)-4-bromobuta-2-enyl]-5-chloro-7-methoxypyrazolo[4,3-d]pyrimidine (0.12 g, 377.87 μmol, 1 equivalent) was dissolved in MeOH (1.5 mL) and NaOMe (30.62 mg, 566.81 μmol, 1.5 equivalents) was added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. Compound (E)-5-chloro-7-methoxy-1-(4-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine (0.12 g, crude) was obtained as a white solid. 1 1H 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-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 2 in Scheme C-3) [ka] 5-Chloro-7-methoxy-1-[(E)-4-methoxybuta-2-enyl]pyrazolo[4,3-d]pyrimidine (0.12 g, 446.60 μmol, 1 equivalent) was dissolved in H2O (1 mL) and THF (1 mL), to which LiOH·H2O (56.22 mg, 1.34 mmol, 3 equivalents) was added. The mixture was stirred at 25°C for 10 hours. LC-MS showed that 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 (2N) and extracted with ELISA (10 mL × 5). The combined organic layers were washed with brine (5 mL × 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound (E)-5-chloro-1-(4-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (70 mg, crude product) was obtained as a white solid. 1 1H 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-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 3 in Scheme C-3) [ka] To a solution of 5-chloro-1-[(E)-4-methoxybuta-2-enyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (70 mg, 274.86 μmol, 1 equivalent) in t-BuOH (2 mL), (3,4-dichlorophenyl)methaneamine (96.78 mg, 549.73 μmol, 73.31 μL, 2 equivalents) was added. The mixture was stirred at 100 °C for 12 hours. LC-MS and HPLC showed that 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%~45%, 10 min). Compound (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-methoxybuta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (54.8 mg, 133.12 μmol, yield 48.43%, purity 95.77%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 394.1 m / z [M+H] + .

[0343] compound 193 Preparation of (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-(2-methoxyethoxy)buta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-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), 2-methoxyethanol (1 g, 13.14 mmol, 1.04 mL, 1 equivalent) was added dropwise at 0°C. The mixture was stirred at 0°C for 2 hours. TLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. Compound sodium 2-methoxyethanolate (0.4 g, 4.08 mmol, yield 31.03%) was obtained as a white solid.

[0345] Preparation of (E)-5-chloro-7-(2-methoxyethoxy)-1-(4-(2-methoxyethoxy)buta-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 equivalents) in 2-methoxyethanol (2 mL), 1-[(E)-4-bromobuta-2-enyl]-5-chloro-7-methoxypyrazolo[4,3-d]pyrimidine (0.16 g, 503.83 μmol, 1 equivalent) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete. H2O (5 mL) was added. The reaction mixture was extracted with ELISA (10 mL x 3). The combined organic layers were washed with brine (5 mL x 2), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column 4 g, eluate with a 0-60% ethyl acetate / petroleum ether concentration gradient at 36 mL / min). The eluate was then concentrated under reduced pressure. Compound (E)-5-chloro-7-(2-methoxyethoxy)-1-(4-(2-methoxyethoxy)buta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine (70 mg, 196.19 μmol, yield 38.94%) was obtained as a yellow oily substance. 1 1H 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)buta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 2 in Scheme C-3) [ka]

[0347] 5-Chloro-7-(2-methoxyethoxy)-1-[(E)-4-(2-methoxyethoxy)buta-2-enyl]pyrazolo[4,3-d]pyrimidine (70 mg, 196.19 μmol, 1 equivalent) was dissolved in H2O (1 mL) and THF (1 mL), to which LiOH·H2O (24.70 mg, 588.56 μmol, 3 equivalents) was added. The mixture was stirred at 25°C for 10 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove THF. The mixture was adjusted to pH=5 with HCl (2N) and then extracted with ₹ (10 mL × 5). The combined organic layers were washed with brine (5 mL × 1), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound (E)-5-chloro-1-(4-(2-methoxyethoxy)buta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (60 mg, crude product) was obtained as a pale yellow oily substance. 1 1H 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)buta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (Step 3 in Scheme C-3) [ka] To a solution of 5-chloro-1-[(E)-4-(2-methoxyethoxy)buta-2-enyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (60 mg, 200.85 μmol, 1 equivalent) in t-BuOH (2 mL), (3,4-dichlorophenyl)methaneamine (70.72 mg, 401.71 μmol, 53.57 μL, 2 equivalents) was added. The mixture was stirred at 100 °C for 12 hours. LC-MS and HPLC showed that 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%~55%, 10 min). MeCN was removed under reduced pressure at 30 °C. The residue was dehydrated by freeze-drying. Compound (E)-5-((3,4-dichlorobenzyl)amino)-1-(4-(2-methoxyethoxy)buta-2-en-1-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (59.8 mg, 136.43 μmol, yield 67.93%) was obtained as a white solid. 1 1H 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 m / z; Measured mass: 438.1 m / z [M+H] + .

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

[0350] 1-tert-butyl4-ethyl4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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 yielded the desired compound, 1-tert-butyl 4-ethyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-1,4-dicarboxylate (0.22 g, 379.65 μmol, yield 66.80%), as a white solid. 1 1H 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]pyrimidine-1-yl)methyl)-4-(hydroxymethyl)piperidine-1-carboxylate [ka] To a solution of O1-tert-butylO4-ethyl4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-1,4-dicarboxylate (0.1 g, 172.57 μmol, 1 equivalent) in THF (3 mL), LiBH4 (15.04 mg, 690.28 μmol, 4 equivalents) was added at 0 °C. The mixture was then stirred at 55 °C for 6 hours. LC-MS and TLC showed that 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 SiO2 (5 mL x 4). The combined organic layers were washed with brine (2 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain tert-butyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]-4-(hydroxymethyl)piperidine-1-carboxylate (90 mg, 167.46 μmol, yield 97.04%) as a white solid. 1 1H NMR (DMSO-d 6, 400 MHz) 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)piperidine-4-yl)methyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride [ka] To a mixture of tert-butyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]-4-(hydroxymethyl)piperidine-1-carboxylate (0.13 g, 241.89 μmol, 1 equivalent) in SiO (4 mL), HCl / SiO (4 M, 6.05 mL, 100 equivalents) was added, and the mixture was stirred at 20°C for 2 hours. LC-MS showed that the reaction was complete. A small amount of white solid was produced. 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%~35%, 12 min). The eluate was removed under reduced pressure. Compound 5-[(3,4-dichlorophenyl)methylamino]-1-[[4-(hydroxymethyl)-4-piperidyl]methyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (102 mg, 210.83 μmol, yield 87.16%, purity 97.93%, HCl) was obtained as a white solid. 1 1H 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 The calculated mass of Cl3N6O2 is 436.12 m / z, while the measured mass is 437.1 [M+H]. + .

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

[0354] O1-tert-butyl O4-ethyl 4-[(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)methyl]piperidine-1,4-dicarboxylate (0.1 g, 220.30 μmol, 1 equivalent) and LiOH·H2O (46.22 mg, 1.10 mmol, 5 equivalents) were mixed in MeOH (1 mL), THF (1 mL), and H2O (1 mL) and stirred at 20°C for 3 hours. Then NaOH (17.62 mg, 440.61 μmol, 2 equivalents) was added, and the mixture was stirred at 70°C for 5 hours. TLC showed that the reaction was complete. The organic solvent was removed under reduced pressure. The aqueous solution was slowly adjusted to pH 6-7 with 2N HCl, and some solid was produced. The solid was collected after filtration. The aqueous solution was then extracted with SiO (10 mL × 3). The combined organic layers were washed with brine (5 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 1-tert-butoxycarbonyl-4-[(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl]piperidine-4-carboxylic acid (80 mg, 194.25 μmol, yield 88.17%) as a white solid. 1 1H 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]pyrimidine-1-yl)methyl)piperidine-4-carboxylic acid (Step 3 in Scheme C-3) [ka] A solution of (3,4-dichlorophenyl)methaneamine (68.39 mg, 388.50 μmol, 51.81 μL, 2 equivalents) and 1-tert-butoxycarbonyl-4-[(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl]piperidine-4-carboxylic acid (80 mg, 194.25 μmol, 1 equivalent) in t-BuOH (2 mL) was heated at 100 °C for 30 hours. LC-MS showed that the reaction was nearly complete. The solvent was removed under reduced pressure. Compound 1-tert-butoxycarbonyl-4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-4-carboxylic acid (0.13 g, crude product) was obtained as a grayish-white solid. MS (ESI): C 24 H 28 Calculated mass of Cl2N6O5: 550.15 m / z; Measured mass: 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]pyrimidine-1-yl)methyl)piperidine-4-carboxylate hydrochloride [ka] A mixture of 1-tert-butoxycarbonyl-4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-4-carboxylic acid (0.11 g, 199.48 μmol, 1 equivalent) in HCl (5 mL) was mixed with HCl / HCl (4 M, 4.99 mL, 100 equivalents), and the mixture was stirred at 20°C for 2 hours. LC-MS showed that the reaction was complete. Some solid was produced. 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%~40%, 12 min). The eluate was dried under lyophilization. Compound 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-4-carboxylic acid (40.2 mg, 81.00 μmol, yield 40.60%, purity 98.28%, HCl) was obtained as a white solid. 1 1H 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 mass: 451.0 [M+H] + .

[0357] Preparation of ethyl 4-((5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)methyl)piperidine-4-carboxylate hydrochloride [ka] To a mixture of O1-tert-butylO4-ethyl4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-1,4-dicarboxylate (30 mg, 51.77 μmol, 1 equivalent) in HCl (1 mL), HCl / HCl (4 M, 1.29 mL, 100 equivalents) was added, and the mixture was stirred at 20°C for 2 hours. TLC showed that the reaction was complete. A small amount of white solid was produced. After filtration, the solid was collected. The solid was concentrated under reduced pressure to obtain ethyl 4-[[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]methyl]piperidine-4-carboxylate (15.6 mg, 29.46 μmol, yield 56.90%, purity 97.41%, HCl) as a white solid. 1 1H 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 25The calculated mass of Cl3N6O3 is 478.13 m / z, while the measured mass is 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]pyrimidine-1-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)acetamide

[0359] Preparation of 2-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-1-yl)acetamide (Step 1 in Scheme C-3) [ka] 5-Chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine (700 mg, 3.79 mmol, 1 equivalent) and 2-chloroacetamide (709.26 mg, 7.58 mmol, 2 equivalents) were dissolved in DMF (5 mL) and K2CO3 (1.05 g, 7.58 mmol, 2 equivalents) was added. The mixture was stirred at 70°C for 10 hours. TLC showed that the reaction was complete. The mixture was poured into H2O (7 mL) and the pH of the mixture was adjusted to 7 with HCl (2 M). The mixture was extracted with DCM and i-PrOH (3:1, 10 mL x 3). The combined organic layers were dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain a mixture of 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)acetamide (900 mg, 3.72 mmol, yield 98.21%) and 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)acetamide as a white solid. 1 1H 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]pyrimidine-1-yl)-N-((2-(trimethylsilyl)ethoxy)methyl)acetamide [ka] To a mixture of 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)acetamide (392 mg, 1.62 mmol, 1 equivalent) and 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)acetamide (1.00 equivalent) in DMF (5 mL), DIEA (1.05 g, 8.11 mmol, 1.41 mL, 5 equivalents) and SEM-Cl (1.03 g, 6.16 mmol, 1.09 mL, 3.8 equivalents) were added dropwise at 0°C. The mixture was then stirred at 40°C for 10 hours. TLC showed that the reaction was complete. The mixture was poured into H2O (5 mL) and the pH was adjusted to 7 with HCl (3 M) at 0°C. The mixture was extracted with SiO (8 mL × 3). The combined organic layers were dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 4 g, 40 mL / min, eluate with a 0-55% ethyl acetate / petroleum ether concentration gradient). The eluate was concentrated under reduced pressure to obtain 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (30 mg, 80.67 μmol, yield 4.97%) and 2-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-2-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (60 mg, 161.34 μmol, yield 9.94%) as white solids. 1 1H 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]pyrimidine-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]pyrimidine-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (75 mg, 201.67 μmol, 1 equivalent) in MeOH (1 mL) and H2O (1 mL), LiOH·H2O (25.39 mg, 605.01 μmol, 3 equivalents) was added. The mixture was stirred at 25°C for 10 hours. LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the aqueous solution was then adjusted to pH=7 with HCl (2 M). The aqueous solution was extracted with ELISA (3 mL × 3). The combined organic layers were dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain 2-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (72 mg, 201.19 μmol, yield 99.76%) as a white solid. 1 1H 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]pyrimidine-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]pyrimidine-1-yl)-N-(2-trimethylsilylethoxymethyl)acetamide (50 mg, 139.72 μmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (49.19 mg, 279.43 μmol, 37.27 μL, 2 equivalents) in t-BuOH (2 mL) was stirred at 110 °C for 10 hours. LC-MS showed that 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%~60%, 12 min). The mixture was dried under lyophilization. The residue was purified twice by preparative HPLC (column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-MeCN]; B%: 45%~65%, 12 min). The mixture was dried under lyophilization to obtain 2-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-N-(2-trimethylsilylethoxymethyl)acetamide (5.4 mg, 10.20 μmol, yield 7.30%, purity 94.00%) as a white solid. 1 1H 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 m / z; Measured mass: 497.1 m / z [M+H] + .

[0364] Compound 198 Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-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]pyrimidine-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 yielded the desired compound, 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-2,2-dimethylpentanoic acid (93.4 mg, 212.99 μmol, yield 42.42%, purity 99.953%), as a white solid. 26.3 mg was then used. 1 1H 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 m / z; Measured mass: 438.1 m / z [M+H] + .

[0366] Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)-N,2,2-trimethylpentanamide [ka] A mixture of 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-2,2-dimethylpentanoic acid (30 mg, 68.45 μmol, 1 equivalent), methanamine hydrochloride (9.24 mg, 136.89 μmol, 2 equivalents), EDCI (15.75 mg, 82.13 μmol, 1.2 equivalents), HOBt (1.85 mg, 13.69 μmol, 0.2 equivalents), and DIEA (26.54 mg, 205.34 μmol, 35.77 μL, 3 equivalents) in DMF (1 mL) was stirred at 20°C for 16 hours. LC-MS showed that the reaction was complete. After filtration, the filtrate was separated and purified by HPLC (column: Phenomenex Luna C18 150mm × 30mm 5μm; mobile phase: [water (0.05% HCl)-MeCN]; B%: 15%~35%, 12 min). The aqueous solution was freeze-dried to obtain compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-N,2,2-trimethylpentanamide (12.7 mg, 27.80 μmol, yield 40.62%, purity 98.800%) as a grayish-white solid. 1 1H 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 m / z; Measured mass: 451.1 m / z [M+H] + .

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

[0368] Preparation of diethyl 2-(3-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-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 equivalent), diethyl 2-(3-bromopropyl)-2-phenyl-propanediote (870.92 mg, 2.44 mmol, 470.47 μL, 1.5 equivalents), and Cs2CO3 (1.06 g, 3.25 mmol, 2 equivalents) in DMF (3 mL) was stirred at 25°C for 5 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (5 mL) and extracted with RINKAN (5 mL x 3). The combined organic layers were washed with brine (5 mL x 3), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 4 g, 65 mL / min, eluate with a 0-40% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure. The compound diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)propyl]-2-phenyl-propanediote (450 mg, 976.33 μmol, yield 60.07%) was obtained as a colorless oil. 1 1H 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]pyrimidine-2-yl)propyl]-2-phenyl-propanediote (270 mg, 585.80 μmol, yield 36.04%) was obtained as a colorless oil. 1 1H 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]pyrimidine-1-yl)-2-phenylpentanoic acid [ka] A mixture of diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)propyl]-2-phenyl-propanedioate (200 mg, 433.92 μmol, 1 equivalent) and NaOH (104.13 mg, 2.60 mmol, 6 equivalents) in MeOH (1 mL) and H2O (1 mL) was stirred at 80°C for 3 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The aqueous solution was diluted to pH=6 with 3N HCl and extracted with ELISA (6 mL x 3). The combined organic layers were washed with brine (6 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 5-(5-chloro-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl)-2-phenylpentanoic acid (175 mg, crude product) was obtained as a white solid. 1 1H 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]pyrimidine-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]pyrimidine-1-yl)-2-phenylpentanoic acid (175 mg, 504.66 μmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (177.68 mg, 1.01 mmol, 134.61 μL, 2 equivalents) in 2-methyl-2-butanol (3 mL) was stirred at 140 °C for 4 hours. LC-MS showed that 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%~60%, 10 min). The solvent was removed by lyophilization. Compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-2-phenylpentanoic acid (131.2 mg, 264.99 μmol, yield 52.51%, purity 98.23%) was obtained as a white solid. 31.2 mg was then used. 1 1H 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 m / z; Measured mass: 486.1 m / z [M+H] + .

[0372] Preparation of 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)-N-methyl-2-phenylpentanamide [ka] A mixture of 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-2-phenylpentanoic acid (30 mg, 61.68 μmol, 1 equivalent), methanamine hydrochloride (8.33 mg, 123.37 μmol, 2 equivalents), DIEA (23.92 mg, 185.05 μmol, 32.23 μL, 3 equivalents), HOBt (1.67 mg, 12.34 μmol, 0.2 equivalents), and EDCI (14.19 mg, 74.02 μmol, 1.2 equivalents) in DMF (0.5 mL) was stirred at 25°C for 12 hours. LC-MS showed that the reaction was complete. The reaction mixture was filtered to remove insoluble matter. The filtrate was separated and purified by preparative HPLC (HCl conditions: column: Phenomenex Luna C18 150 mm × 30 mm 5 μm; mobile phase: [water (0.04% HCl)-MeCN]; B%: 25%~50%, 12 min). The solvent was removed by lyophilization. Compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-N-methyl-2-phenylpentanamide (13.8 mg, 27.59 μmol, yield 44.73%, purity 99.85%) was obtained as a white solid. 1 1H 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 m / z, measured value: 499.1 m / z [M+H] + .

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

[0374] Diethyl 2-(3-(5-chloro-7-methoxy-1H-pyrazolo[4,3-d]pyrimidine-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 equivalent), diethyl 2-(3-bromopropyl)-2-methyl-propanediote (623.65 mg, 2.11 mmol, 201.22 μL, 1.3 equivalents), and Cs2CO3 (1.06 g, 3.25 mmol, 2 equivalents) in DMF (2 mL) was stirred at 25°C for 4 hours. TLC showed that the reaction was complete. The reaction mixture was quenched with H2O (15 mL) at 20°C and then extracted with RINKAN (15 mL x 3). The combined organic layer was washed with brine (10 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Biotage®; SepaFlash® silica flash column, 12 g, 35 mL / min, eluate with a 0-30% ethyl acetate / petroleum ether concentration gradient). The eluate was removed under reduced pressure. The compound diethyl 2-[3-(5-chloro-7-methoxy-pyrazolo[4,3-d]pyrimidine-1-yl)propyl]-2-methyl-propanediote (440 mg, 1.10 mmol, yield 67.88%) 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]pyrimidine-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]pyrimidine-1-yl)propyl]-2-methyl-propanediote (380 mg, 952.76 μmol, 1 equivalent) and NaOH (266.77 mg, 6.67 mmol, 7 equivalents) in H2O (2 mL) and MeOH (2 mL) was stirred at 100°C for 3 hours. LC-MS showed that 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 siRNA (10 mL x 3). The combined organic layers were washed with brine (5 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Compound 2-[3-(5-chloro-7-hydroxy-pyrazolo[4,3-d]pyrimidine-2-yl)propyl]-2-methyl-propanediic acid (230 mg, 699.71 μmol, yield 73.44%) 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]pyrimidine-1-yl)-2-methylpentanoic acid [ka]

[0377] A mixture of 2-[3-(5-chloro-7-hydroxy-pyrazolo[4,3-d]pyrimidine-2-yl)propyl]-2-methyl-propanedioic acid (170 mg, 517.18 μmol, 1 equivalent) and (3,4-dichlorophenyl)methaneamine (182.09 mg, 1.03 mmol, 137.95 μL, 2 equivalents) in 2-methylbutan-2-ol (3 mL) was stirred at 140 °C for 16 hours. LC-MS showed that 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%~50%, 10 min). The aqueous solution was removed by freeze-drying to obtain compound 5-[5-[(3,4-dichlorophenyl)methylamino]-7-oxo-6H-pyrazolo[4,3-d]pyrimidine-1-yl]-2-methylpentanoic acid (80 mg, 187.63 μmol, yield 36.28%, purity 99.510%) as a white solid. 30.0 mg was then used. 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 m / z; Measured mass: 424.1 m / z [M+H] + .

[0378] compound 203 Preparation of ethyl 5-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)pentanoate [ka] To a solution of 5-[5-[(3,4-dichlorophenyl)m...

Claims

1. Compounds corresponding to the following formula I, 【Chemistry 1】 (In the formula, A and B are N, n is 1, R １ This is selected from the group consisting of oxetanyl, tetrahydrofuranil, tetrahydropyranil, phenyl, pyridyl, 1-(tetrahydropyranil)piperidinyl, 1-(2-methoxyethyl)piperidinyl, 1-(tert-butoxycarbonyl)piperidinyl, 1-(pyridinyl)piperidinyl, 1-(pyridinylmethyl)piperidinyl, 1-(pyridinylsulfonyl)piperidinyl, 1-isonicotinoylpiperidinyl, 1-nicotinoylpiperidinyl, 1-picolinoylpiperidinyl, 1-(methoxypicolinoyl)piperidinyl, 1-(methylpicolinoyl)piperidinyl, 1-(oxazole-carbonyl)piperidinyl, and 1-(thiazole-carbonyl)piperidinyl. R ２ is H, CN, F, or Cl, R ３ is a phenyl group 3- or 4-substituted with F, Cl, Me or ethyl; or a 3,4-dichloro, 3,4-difluoro, 3-Me-4-Cl, 3-Me-4-F, 3-Cl-4-Me, 3-F-4-Me, 3-ethyl-4-Cl, 3-ethyl-4-F, 3-Cl-4-ethyl, or 3-F-4-ethyl substituted phenyl group. R ０ is H, or CH ２ OPO (OH) ２ (It is) or its optical isomers, isotopic isomers, or pharmaceutically acceptable salts; However, the following compounds are excluded: 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (compound 121), 5-((3,4-dichlorobenzyl)amino)-1-(oxetan-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one (compound 142), 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-ylsulfonyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride (compound 156), 5-[(3,4-dichlorophenyl)methylamino]-1-phenyl-6H-pyrazolo[4,3-d]pyrimidine-7-one (compound 212), 5-[(3,4-dichlorophenyl)methylamino]-1-(4-pyridyl)-6H-pyrazolo[4,3-d]pyrimidine-7-one (compound 222), 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-2-ylmethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride (compound 244), 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-ylmethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride (compound 245), 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-4-ylmethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride (compound 246), 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(oxazole-4-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (compound 253), 5-[(3,4-dichlorophenyl)methylamino]-1-[1-(thiazole-2-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (compound 254), 5-((3,4-dichlorobenzyl)amino)-1-(1-(3-methylpicolinoyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride (compound 262), 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methoxypicolinoyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride (compound 263), 5-((3,4-dichlorobenzyl)amino)-1-(1-(4-methylpicolinoyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride (compound 264), and 3-Chloro-5-[(3,4-dichlorophenyl)methylamino]-1-[1-(pyridine-3-carbonyl)-4-piperidyl]-6H-pyrazolo[4,3-d]pyrimidine-7-one (compound 318).

2. A and B are N, n is 1, R １ However, it is selected from the group consisting of oxetanyl, tetrahydrofuranil, tetrahydropyranil, phenyl, pyridyl, 1-(tetrahydropyranil)piperidinyl, 1-(2-methoxyethyl)piperidinyl, 1-(tert-butoxycarbonyl)piperidinyl, 1-(pyridinyl)piperidinyl, 1-(pyridinylmethyl)piperidinyl, 1-(pyridinylsulfonyl)piperidinyl, 1-isonicotinoylpiperidinyl, 1-nicotinoylpiperidinyl, 1-picolinoylpiperidinyl, 1-(methoxypicolinoyl)piperidinyl, 1-(methylpicolinoyl)piperidinyl, 1-(oxazole-carbonyl)piperidinyl, and 1-(thiazole-carbonyl)piperidinyl. R ２ However, it is H, CN, F, or Cl, R ３ is a phenyl group substituted at the 3- or 4-position by F, Cl, Me or ethyl; or a 3,4-dichloro, 3,4-difluoro, 3-Me-4-Cl, 3-Me-4-F, 3-Cl-4-Me, 3-F-4-Me, 3-ethyl-4-Cl, 3-ethyl-4-F, 3-Cl-4-ethyl, or 3-F-4-ethyl substituted phenyl group, R ０ The compound according to claim 1, wherein is H, an optical isomer, an isotopic isomer, or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition comprising the compound described in claim 1 and at least one pharmaceutically acceptable carrier.

4. A surface coating for use in a method for inhibiting the growth of Gram-positive bacteria, wherein the method comprises the step of bringing the surface coating, which contains an effective amount of the compound described in claim 1, into contact with a habitat.

5. The surface coating according to claim 4, wherein the habitat is the surface of a medical device.

6. A pharmaceutical composition for use in a method of treating a subject requiring treatment for a Gram-positive bacterial infection, the method comprising the step of administering to the subject the pharmaceutical composition containing a therapeutically effective amount of the compound described in claim 1.

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

8. 3-bromo-5-((3,4-dichlorobenzyl)amino)-1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, 5-((3,4-dichlorobenzyl)amino)-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one, tert-butyl 4-(5-((3,4-dichlorobenzyl)amino)-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidine-1-yl)piperidine-1-carboxylate, 5-((3,4-dichlorobenzyl)amino)-1-(1-(2-methoxyethyl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-4-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(pyridine-3-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one dihydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-(tetrahydro-2H-pyran-4-yl)piperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-isonicotinoylpiperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, 5-((3,4-dichlorobenzyl)amino)-1-(1-picolinoylpiperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, and Compounds selected from the group consisting of 5-((3,4-dichlorobenzyl)amino)-1-(1-nicotinoylpiperidine-4-yl)-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one hydrochloride, or pharmaceutically acceptable salts thereof.

9. A pharmaceutical composition comprising the compound according to claim 8 and at least one pharmaceutically acceptable carrier.

10. A surface coating for use in a method for inhibiting the growth of Gram-positive bacteria, the method comprising the step of bringing the surface coating, which contains an effective amount of the compound according to claim 8, into contact with a habitat.

11. The surface coating according to claim 10, wherein the habitat is the surface of a medical device.

12. A pharmaceutical composition for use in a method of treating a subject requiring treatment for a Gram-positive bacterial infection, wherein the method comprises the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition containing the compound described in claim 8.

13. A surface coating comprising the compound described in claim 8 and a coating agent, wherein the coating agent is capable of adhering the compound to the habitat.

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