A novel isoxazolopyrimidine derivative, pharmaceutical composition and use thereof

Abstract

This invention provides a novel isoxazolopyrimidine derivative, a pharmaceutical composition, and their applications, belonging to the field of pharmaceutical synthesis technology. The novel isoxazolopyrimidine derivative of this invention can be used in drugs that inhibit the activity of human immunodeficiency virus (HIV). This targeted drug achieves highly effective antiviral effects by inhibiting the interaction between viral capsid proteins and the host factor CPSF6, thereby preventing nuclear importation and transcriptional activation region localization processes during HIV-1 infection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and in particular to a novel isoxazolopyrimidine derivative, a pharmaceutical composition, and its application. Background Technology

[0002] HIV-1 (Human Immunodeficiency Virus 1) is the main pathogen causing AIDS (Acquired Immunodeficiency Syndrome). HIV-1 infected individuals can control their viral load to a low level through antiretroviral therapy. However, with the increasing drug resistance rate in recent years, there is a shortage of clinically available HIV-1 drugs. Developing novel small-molecule compounds against HIV-1 meets the needs of clinical medication. In drug screening, traditional cell screening cannot meet the needs of target confirmation and therefore cannot provide explanations at the molecular level. Target confirmation experiments such as SPR (surface plasmon resonance) and BLI (membrane interferometry) require target fixation, necessitating large amounts of protein and sample, thus increasing experimental costs. Developing inhibitor screening technologies based on novel target-based compound libraries can realize a full-chain R&D model from molecular target to compound screening, providing new ideas and technological platform support for the development of innovative HIV-1 drugs.

[0003] The HIV-1 capsid protein (CA) is a crucial component of the viral structure, exhibiting relatively conserved sequences across different HIV-1 subtypes, thus reducing the occurrence of drug resistance mutations. CA plays a vital role in infecting host cells by binding to various host factors, with its binding to cleavage and polyadenylation factor 6 (CPSF6) proven to guide viral particles into the nucleus and influence nuclear localization. Therefore, inhibiting CA / CPSF6 binding is significant for controlling HIV-1 infection. Pfizer, through high-throughput screening, identified PF74, the first small molecule to inhibit capsid assembly. PF74 inhibits the assembly of HIV capsid protein pentamers while promoting hexamer assembly, resulting in a large number of incomplete HIV capsids within the cell, which cannot subsequently infect the virus. Subsequent studies revealed that PF74 can also inhibit the interaction between CA and CPSF6. This dual antiviral mechanism significantly enhances the drug development potential of PF74.

[0004] Gilead Sciences' LEN (Lenacapavir), derived from the PF74 structural framework, is the first FDA-approved compound with long-acting activity and the ability to prevent HIV-1 infection. It has demonstrated long-lasting pharmacokinetic properties and an extremely low half-maximal effective concentration (EC50) in in vitro, in vivo, and clinical trials. 50 However, because LEN inhibits the assembly of CA pentamers, a large number of damaged capsids remain in the cytoplasm, activating the cGAS-STING pathway, triggering innate immunity, and subsequently inducing the bystander effect, ultimately leading to a large amount of CD4+ in the body.+ T-cell death significantly reduces patients' immune levels. In summary, the development of drugs targeting this target, from the lead compound PF74 to the marketed LEN, still faces challenges: the former suffers from inherent defects such as poor metabolic stability; the latter, as an innovative therapy, also warrants attention for its potential immune side effects. Therefore, continued development of novel inhibitors targeting this target remains of significant research value. Summary of the Invention

[0005] The purpose of this invention is to provide a novel isoxazolopyrimidine derivative, a pharmaceutical composition and its application, in order to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a novel isoxazolopyrimidine derivative having the following general structural formula:

[0007] R 1 Selected from substituted or unsubstituted alkyl, cycloalkyl or heterocycloalkyl, substituted or unsubstituted aryl or heteroaryl; R 2 Selected from substituted or unsubstituted alkyl, cycloalkyl or heterocycloalkyl, substituted or unsubstituted aryl or heteroaryl; R 3 Selected from substituted or unsubstituted morpholino, substituted or unsubstituted thiomorpholino, substituted or unsubstituted piperidinyl, substituted or unsubstituted piperazine, substituted or unsubstituted pyridazine, substituted or unsubstituted pyrrole, substituted or unsubstituted imidazolyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted amino; R 1 and R 2 The alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, R 3 The morpholino, thiomorpholino, piperidino, piperazino, pyridazino, pyrrolo, imidazolyl, pyrazolo, and amino groups are optionally substituted with 1, 2, 3, 4, or 5 R groups; R is selected from hydrogen, deuterium, olefin, alkynyl, halogen, hydroxyl, hydroxymethyl, amino, trifluoromethyl, cyano, ester, carbonyl, acyl, aminoacyl, amide, sulfonyl, aminosulfonyl, sulfonamide, alkyl, cycloalkyl, heterocycloalkyl, morpholine ethyl, alkoxy, urea, thiourea, aryl, pyridinyl, pyrimidinyl, pyridazinyl, aminopyrimidinyl, quinolinyl, benzofuranyl, indolyl, inzolyl, benzothiophenyl, furanyl, thiophenyl, pyridinyl, oxazolyl, and pyrazolyl.

[0008] The present invention also provides a pharmaceutical composition comprising the novel isoxazolopyrimidine derivative thereof or a pharmaceutically acceptable salt, solvent compound or deuterated derivative thereof, and a pharmaceutically acceptable excipient.

[0009] Furthermore, the pharmaceutical composition comprises at least one other therapeutic agent selected from the following: combination drugs for HIV, other drugs for treating HIV, HIV protease inhibitors, non-nucleoside or non-nucleotide inhibitors of HIV reverse transcriptase, nucleoside or nucleotide inhibitors of HIV reverse transcriptase, HIV integrase inhibitors, HIV non-catalytic site integrase inhibitors, HIV allosteric integrase inhibitors, HIV entry inhibitors, HIV maturation inhibitors, latency reversal agents, compounds targeting the HIV capsid, phosphatidylinositol 3-kinase inhibitors, HIV antibodies, bispecific antibodies and "antibody-like" therapeutic proteins, HIV p17 matrix protein inhibitors, IL-13 antagonists, peptidyl-prolyl cis-trans isomerase A modulators, protein disulfide isomerase inhibitors, complement C5a receptor antagonists, DNA methyltransferase inhibitors, HIV vif gene modulators, Vif dimerization antagonists, HIV-1 viral infection factor inhibitors, TAT protein inhibitors, HIV-1 Nef modulators, Hck tyrosine kinase modulators, mixed lineage kinase-3 inhibitors, HIV-1 splicing inhibitors, Rev protein inhibitors, integrin antagonists, nucleoprotein inhibitors, splicing factor modulators, COMM domain-containing protein 1 modulators, HIV ribonuclease H inhibitors, retrocyclin modulators, CDK-9 inhibitors, dendritic ICAM-3 capture non-integrin 1 inhibitors, HIV GAG protein inhibitors, HIV POL protein inhibitors, complement factor H modulators, ubiquitin ligase inhibitors, deoxycytidine kinase inhibitors, cyclin-dependent kinase inhibitors, proprotein convertase PC9 stimulators, ATP-dependent RNA helicase DDX3X inhibitors, reverse transcriptase initiation complex inhibitors, G6PD and NADH-oxidase inhibitors, pharmacokinetic enhancers, HIV vaccines.

[0010] Furthermore, the pharmaceutical composition may be in the form of an injection or a solid dosage form.

[0011] The present invention also provides the application of the above-mentioned novel isoxazolopyrimidine derivative in drugs that inhibit the activity of human immunodeficiency virus.

[0012] The present invention also provides the use of the above-described pharmaceutical composition in a medicament for inhibiting the activity of human immunodeficiency virus.

[0013] The beneficial effects of this invention are: The 4c ​​small molecule compound provided by this invention significantly inhibits HIV-1 replication at the cellular level and in a humanized mouse infection model. Its antiviral activity is stable and reproducible, and its activity intensity is superior to existing small molecule compounds of the same class. Experimental results show that 4c effectively blocks key viral steps, exhibits a clear mechanism of action, does not interfere with the main physiological processes of host cells, and has good selectivity and safety. The sustained antiviral effect demonstrated by the compound in in vitro models allows it to exert significant inhibitory effects in both high-risk exposure-related prophylaxis and post-infection treatment, meeting the clinical need for protective effects with a single dose. Compared with existing technologies, the 4c ​​small molecule has significant advantages in inhibitory intensity, duration of action, mechanism specificity, and dual-indication availability, making it suitable for further development into a novel oral small molecule drug for the prevention and treatment of HIV-1 infection, demonstrating outstanding technological advancement and broad application prospects. Attached Figure Description

[0014] Figure 1 The diagrams show the preparation process of two Raman probes and the setup of the screening platform (a and b), as well as the screening process of inhibitors from the combined compound library (c).

[0015] Figure 2 Characterization images of silver nanoparticles. a) UV-Vis spectrum of silver nanoparticles (AgNPs) (diluted three times); b) Transmission electron microscopy (TEM) image of AgNPs.

[0016] Figure 3 The images show TEM characterization of the silver magnetic probe complex. a) TEM image of the silver magnetic probe without interaction. b) TEM image of the silver magnetic probe with interaction.

[0017] Figure 4 This is a SERS screening diagram of a small molecule compound library.

[0018] Figure 5 This graph shows the changes in viral load in HIV-1-infected humanized mice after treatment with LEN and 4c compounds. The red background represents two consecutive weeks of LEN and 4c administration. The blue background represents administration at two-week intervals, with administration occurring once in week four and week six.

[0019] Figure 6 This is a flowchart illustrating the synthesis method of the small molecule combinatorial compound library of the present invention. Detailed Implementation

[0020] This invention provides a novel isoxazolopyrimidine derivative having the following general structural formula:

[0021] R1 Selected from substituted or unsubstituted alkyl, cycloalkyl or heterocycloalkyl, substituted or unsubstituted aryl or heteroaryl; R 2 Selected from substituted or unsubstituted alkyl, cycloalkyl or heterocycloalkyl, substituted or unsubstituted aryl or heteroaryl; R 3 Selected from substituted or unsubstituted morpholino, substituted or unsubstituted thiomorpholino, substituted or unsubstituted piperidinyl, substituted or unsubstituted piperazine, substituted or unsubstituted pyridazine, substituted or unsubstituted pyrrole, substituted or unsubstituted imidazolyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted amino; R 1 and R 2 The alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, R 3 The morpholino, thiomorpholino, piperidino, piperazino, pyridazino, pyrrolo, imidazolyl, pyrazolo, and amino groups are optionally substituted with 1, 2, 3, 4, or 5 R groups; R is selected from hydrogen, deuterium, olefin, alkynyl, halogen, hydroxyl, hydroxymethyl, amino, trifluoromethyl, cyano, ester, carbonyl, acyl, aminoacyl, amide, sulfonyl, aminosulfonyl, sulfonamide, alkyl, cycloalkyl, heterocycloalkyl, morpholine ethyl, alkoxy, urea, thiourea, aryl, pyridinyl, pyrimidinyl, pyridazinyl, aminopyrimidinyl, quinolinyl, benzofuranyl, indolyl, inzolyl, benzothiophenyl, furanyl, thiophenyl, pyridinyl, oxazolyl, and pyrazolyl.

[0022] Furthermore, the R 1 Selected from substituted or unsubstituted cycloalkyl or heterocycloalkyl groups; R 2 Selected from substituted or unsubstituted aryl or heteroaryl groups; R 3 Selected from substituted or unsubstituted morpholino or substituted or unsubstituted piperidino.

[0023] In this invention, the novel isoxazolopyrimidine derivative is selected from one of the following structural formulas: 1a~1e: , , , , ; 3a~3ab: , , , , , , , , , , , , , , , , , , , , , , , , , , , ; 4a~4v: , , , , , , , , , , , , , , , , , , , , , ; 5a~5j: , , , , , , , , , ; 6a~6k: , , , , , , , , , , ; The present invention also provides a pharmaceutical composition comprising the novel isoxazolopyrimidine derivative thereof or a pharmaceutically acceptable salt, solvent compound or deuterated derivative thereof, and a pharmaceutically acceptable excipient.

[0024] In this invention, at least one other therapeutic agent is included, said other therapeutic agent being selected from the following: combination drugs for HIV, other drugs for treating HIV, HIV protease inhibitors, non-nucleoside or non-nucleotide inhibitors of HIV reverse transcriptase, nucleoside or nucleotide inhibitors of HIV reverse transcriptase, HIV integrase inhibitors, HIV non-catalytic site integrase inhibitors, HIV allosteric integrase inhibitors, HIV entry inhibitors, HIV maturation inhibitors, latency reversal agents, compounds targeting the HIV capsid, phosphatidylinositol 3-kinase inhibitors, HIV antibodies, bispecific antibodies and "antibody-like" therapeutic proteins, HIV p17 matrix protein inhibitors, IL-13 antagonists, peptidyl-prolyl cis-trans isomerase A modulators, protein disulfide isomerase inhibitors, complement C5a receptor antagonists, DNA methyltransferase inhibitors, HIV vif gene modulators, Vif dimerization antagonists, HIV-1 viral infection factor inhibitors, TAT protein inhibitors, HIV-1 Nef modulators, Hck tyrosine kinase modulators, mixed lineage kinase-3 inhibitors, HIV-1 splicing inhibitors, Rev protein inhibitors, integrin antagonists, nucleoprotein inhibitors, splicing factor modulators, COMM domain-containing protein 1 modulators, HIV ribonuclease H inhibitors, retrocyclin modulators, CDK-9 inhibitors, dendritic ICAM-3 capture non-integrin 1 inhibitors, HIV GAG protein inhibitors, HIV POL protein inhibitors, complement factor H modulators, ubiquitin ligase inhibitors, deoxycytidine kinase inhibitors, cyclin-dependent kinase inhibitors, proprotein convertase PC9 stimulators, ATP-dependent RNA helicase DDX3X inhibitors, reverse transcriptase initiation complex inhibitors, G6PD and NADH-oxidase inhibitors, pharmacokinetic enhancers, HIV vaccines.

[0025] In this invention, the pharmaceutical composition is formulated in the form of an injection or a solid dosage form.

[0026] The present invention also provides the application of the above-mentioned novel isoxazolopyrimidine derivatives in drugs that inhibit the activity of human immunodeficiency virus.

[0027] The present invention also provides the use of the above-described pharmaceutical composition in a medicament for inhibiting the activity of human immunodeficiency virus.

[0028] This invention builds upon the previously established SERScreen screening platform based on surface-enhanced Raman spectroscopy (SERS) to simulate the protein-protein interaction (PPI) of HIV-1 CA / CPSF6 in vitro. Through the ultrasensitive sensing of SERS, small molecules capable of inhibiting the binding of these two proteins are identified. In previous work, the SERScreen screening platform has enabled the construction of an in vitro PPI platform and inhibitor screening for multiple protein-protein interaction targets, including PD1 / PD-L1 and KRas / PDEδ. The small molecules identified through screening were validated for activity in both in vitro and in vivo experiments, and their targets and sites of action were verified at the molecular dynamics level, achieving the screening of small molecule inhibitors targeting HIV-1 CA / CPSF6.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] The synthetic route for ethyl 4-nitroisoxazole-5-carboxylate S4 is as follows: .

[0032] Under a nitrogen atmosphere, S1 (30 mmol) and CDI (9.72 g, 60 mmol) were dissolved in anhydrous THF (200 mL). The reaction mixture was stirred at room temperature for 1–2 h. Nitromethane (4 mL, 90 mmol) and DBU (20 mL, 135 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 36 h. After dilution with ethyl acetate (100 mL), 2 N The pH was adjusted to 1-2 with 150 mL of HCl aqueous solution, and then extracted with ethyl acetate (200 mL × 3). The combined organic layers were extracted with water (200 mL × 3) and brine (200 mL × 3) respectively, dried over anhydrous Na₂SO₄, and compound S2 was given in 95-98% yield. It could be used for the next step without further purification.

[0033] Compound S2 (29 mmol), hydroxylamine hydrochloride (2.0 g, 29 mmol), and NaHCO3 (2.4 g, 29 mmol) were dissolved in ethanol (150 mL), and the mixture was stirred at 50 °C for 3 hours. After the starting materials had reacted completely, the mixture was concentrated under vacuum, diluted with water (150 mL), and extracted with ethyl acetate (100 mL × 3). The combined organic layers were then washed with H2O (150 mL × 3) and brine (150 mL × 3), dried with anhydrous Na2SO4, and concentrated under vacuum to give compound S3 in 95-99% yield, which could be used for the next step without further purification.

[0034] Under a nitrogen atmosphere, oxaloyl chloride monoethyl ester (4.5 mL, 40 mmol) was added dropwise to a solution of compound S3 (27 mmol) and triethylamine (5.45 g, 54 mmol) in anhydrous diethyl ether (100 mL) at 0 °C, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated under vacuum, diluted with water (100 mL), and extracted with ethyl acetate (100 mL × 3). The combined organic layers were then washed with H2O (100 mL × 3) and brine (100 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound S4 in 38–41% yield.

[0035] Synthetic route of 4-aminoisoxazole-5-formamide 1: (A) Synthetic routes for alkyl-substituted 4-aminoisoxazole-5-carboxamides 1a-1c, 1e:

[0036] Compound S4 (11 mmol) was dissolved in a methanolic solution of ammonia (50 mL), and the mixture was stirred at room temperature for 3 hours. After removing the solvent under reduced pressure, compound S5 was given in 95-97% yield and could be used in the next step without further purification.

[0037] Compound S5 (10 mmol) was dissolved in a solvent (50 mL, ethanol / water = 2 / 1) at 0 °C, and NH4Cl (13.25 g, 250 mmol) and zinc powder (6.5 g, 100 mmol) were added. The mixture was stirred at room temperature for 4 hours, then filtered to remove insoluble matter, and the filter cake was washed with ethanol. The filtrate was concentrated under vacuum and purified on a silica gel column to give compounds 4-aminoisoxazole-5-carboxamide 1a-1c,1e in 76-80% yield.

[0038]

[0039] The above formula is 4-amino-3-cyclopentyloxazol-5-carboxamide 1a: white solid; yield 78%; mp: 148-150℃; 1 HNMR(400MHz, CDCl3)δ6.13(s,1H),5.63(s,1H),4.18(s,2H),3.14–3.03(m,1H),2.11(d, J =8.0Hz,2H),1.96–1.82(m,4H),1.79–1.70(m,2H). 13 CNMR (100MHz, DMSO-) d 6)δ160.5,160.2,140.2,132.3,35.0,30.1,24.9.

[0040] The above formula is 4-amino-3-cyclohexyloxazol-5-carboxamide 1b: white solid; yield 80%; mp: 168-170℃; 1 HNMR(400MHz, CDCl3)δ6.92–5.73(m,2H),4.27(s,2H),2.61(t, J =11.6Hz, 1H), 2.00(d, J =12.6Hz,2H),1.90(d, J =7.2Hz,2H),1.76(d, J =10.4Hz, 1H), 1.62(d, J =12.3Hz,1H),1.41–1.33(m,2H),0.94(t, J =7.4Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ160.7,160.5,140.0,131.9,33.8,32.4,30.1,25.7.

[0041] The above formula is 4-amino-3-(tetrahydro-2-) H 1c: white solid; yield 79%; mp: 187-189℃; 1 HNMR (300MHz, DMSO-) d 6 )δ7.69(s,1H),7.45(s,1H),5.19(s,2H),4.02–3.81(m,2H),3.42(td, J =11.5,2.1Hz,2H),3.02(tt, J =11.3, 3.7 Hz, 1H), 1.85 (dd, J =12.9,1.7Hz,2H),1.74–1.55(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ160.4,159.6,140.2,131.9,66.9,31.1,29.9.

[0042] The above formula is 4-amino-3-isopropylisoxazole-5-carboxamide 1e: white solid; yield 79%; mp: 144-146℃; 1 HNMR (400MHz, DMSO-) d 6 )δ7.73(s,1H),7.48(s,1H),5.16(s,2H),3.07(dt, J =13.5, 6.7 Hz, 1H), 1.25 (d, J =6.8Hz, 6H). 13 CNMR (100MHz, DMSO-) d 6 )δ161.7,160.9,140.7,132.2,25.1,20.5.

[0043] (B) Synthetic route of 3-(1-acetylpiperidin-4-yl)-4-aminoisoxazole-5-carboxamide 1d:

[0044] Trifluoroacetic acid (5 mL) was added to a 50 mL solution of compound S6 (3.69 g, 10 mmol) in dichloromethane, and the mixture was stirred at room temperature for 2 hours. After the starting material had reacted completely, the mixture was concentrated under vacuum to obtain compound S7 in the form of trifluoroacetate, with a yield of 100%, which could be used in the next step without further purification.

[0045] To compound S7 (3.83 g, 10 mmol) N , N Et3N (1.2 g, 12 mmol) was added to a dimethylformamide solution (50 mL), and acetyl chloride solution (936 mg, 12 mmol) was slowly added to the mixture at 0 °C. The reaction mixture was then stirred at room temperature for 6 hours. After the starting materials had reacted completely, the reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were then washed with H2O (150 mL × 3) and brine (150 mL × 3), dried over anhydrous Na2SO4, and the crude product was purified by silica gel column chromatography to give a pale yellow solid compound S8 in 65% yield.

[0046] Compound S8 (6.5 mmol) was dissolved in a methanolic solution of ammonia (30 mL), and the mixture was stirred at room temperature for 3 hours. After removing the solvent under reduced pressure, compound S9 was given in 99% yield and could be used in the next step without further purification.

[0047] Compound S9 (6.5 mmol) was dissolved in a solvent (30 mL, ethanol / water = 2 / 1) at 0 °C, and NH4Cl (8.6 g, 162.5 mmol) and zinc powder (4.2 g, 65 mmol) were added. The mixture was stirred at room temperature for 4 hours, then filtered to remove insoluble matter, and the filter cake was washed with ethanol. The filtrate was concentrated under vacuum and purified on a silica gel column to give compound 3-(1-acetylpiperidin-4-yl)-4-aminoisoxazole-5-carboxamide 1d in 78% yield.

[0048]

[0049] The above formula is 3-(1-acetylpiperidin-4-yl)-4-aminoisoxazole-5-carboxamide. 1d: white solid; yield 78%; mp: 210-212℃; 1 HNMR (400MHz, DMSO-) d 6 )δ7.73(s,1H),7.48(s,1H),5.23(s,2H),4.41(d, J =12.5Hz, 1H), 3.88(d, J =13.4Hz, 1H), 3.13(t, J =12.5Hz, 1H), 3.02(t, J =11.1Hz, 1H), 2.66(t, J =12.2Hz,1H),2.02(s,3H),1.93(t, J =12.4Hz,2H),1.67–1.53(m,1H),1.43(q, J =10.9Hz, 1H). 13 CNMR (100MHz, DMSO-) d 6 )δ168.1,160.4,159.4,140.2,131.9,45.8,40.8,32.0,29.6,29.0,21.4.

[0050] Example 2

[0051] Isoxazolo[4,5- d Pyrimidine-7(6) H Synthesis of 3a-3ab ketones:

[0052] Compound 1 (1.0 mmol), compound 2 (1.2 mmol), I2 (254 mg, 1.0 mmol), and dimethyl sulfoxide (234 mg, 3.0 mmol) were dissolved in CH3CN (5 mL) and reacted overnight at 80 °C. The reaction was monitored by TLC until completion. After the reaction, the mixture was diluted with an aqueous solution of sodium thiosulfate (20 mL) and extracted with ethyl acetate (20 mL × 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography to obtain isoxazolo[4,5- d Pyrimidine-7(6) H )-keto3.

[0053]

[0054] The above formula is 3-cyclopentyl-5-phenylisoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3a: White solid; yield 93%; mp: 209-211℃; 1 HNMR(400MHz, CDCl3): δ11.60(s,1H),8.18(d, J =4.2Hz, 2H), 7.64(s, 3H), 3.58(p, J =8.4Hz, 1H), 2.27(d, J =7.3Hz,2H),2.17–2.08(m,2H),1.97(d, J =5.3Hz,2H), 1.87–1.78(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.3,155.2,152.4,149.4,140.5,132.5,131.9,129.1,128.4,36.0,31.2,25.6.

[0055] The above formula is 3-cyclopentyl-5-(4-methoxyphenyl)isoxazo[4,5- d Pyrimidine-7(6) H )-Ketone 3b: Pale yellow solid; yield 97%; mp: 202-204℃; 1 HNMR(400MHz, CDCl3)δ11.66(s,1H),8.17(d, J =8.7Hz,2H),7.12(d, J =8.7Hz, 2H), 3.95(s, 3H), 3.57(p,J =8.2Hz,1H),2.31–2.23(m,2H),2.12(dd, J =12.0,7.1Hz,2H),1.99(t, J =10.0Hz,2H),1.83(dd, J =14.3, 9.4 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.3,162.4,154.9,152.5,149.0,140.6,130.1,124.6,114.5,56.0,36.0,31.2,25.7.

[0056] The above formula is 3-cyclopentyl-5-(p-tolyl)isoxazo[4,5- d Pyrimidine-7(6) H )-Ketone 3c: White solid; yield 94%; mp: 204-206℃; 1 HNMR(400MHz,CDCl3)δ11.91(s,1H),8.09(d, J =7.9Hz,2H),7.42(d, J =7.8Hz,2H),3.57(p, J =8.0Hz, 1H), 2.49(s, 3H), 2.26(d, J =6.6Hz,2H),2.17–2.07(m,2H),1.98(m,2H),1.88–1.77(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.3,155.2,152.4,149.3,142.0,140.5,129.7,128.3,36.0,31.2,25.6,21.5.

[0057] The above formula is 3-cyclopentyl-5-(4-fluorophenyl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3d: White solid; yield 84%; mp: 209-211℃; 1 HNMR(400MHz, CDCl3)δ12.23(s,1H),8.27–8.21(m,2H),7.30(d, J=8.3Hz,2H),3.54(p, J =8.4Hz, 1H), 2.23(d, J =6.0Hz,2H),2.13–2.04(m,2H),1.96(d, J =14.0Hz,2H),1.80(dd, J =13.8, 7.9 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ165.8,163.3,154.2,152.3,149.3,140.4,131.0,131.0,129.0,116.3,116.1,36.0,31.1,25.6.

[0058] The above formula is 3-cyclopentyl-5-(4-(trifluoromethyl)phenyl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3e: Light brown solid; yield 61%; mp: 203-205℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.40(s,1H),8.25(d, J =8.1Hz,2H),7.93(d, J =8.2Hz,2H),3.47(p, J =8.1Hz, 1H), 2.14(td, J =11.6,7.5Hz,2H),2.02–1.91(m,2H),1.89–1.77(m,2H),1.72(dd, J =14.3, 7.4 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.4,154.0,152.3,149.7,140.3,136.3,132.2,131.8,131.5,131 .2,129.4,128.4,126.1,126.0,125.7,123.0,120.3,36.0,31.1,25.6.

[0059] The above formula is 4-(3-cyclopentyl-7-oxo-6,7-dihydroisoxazolo[4,5- d5-pyrimidinylbenzonitrile 3f: pale yellow solid; yield 91%; mp: 224-226℃; 1 HNMR(400MHz, CDCl3)δ12.78(s,1H),8.42(d, J =8.2Hz,2H),7.94(d, J =8.3Hz,2H),3.59(p, J =8.2Hz, 1H), 2.28(d, J =7.1Hz,2H),2.12(dd, J =16.7, 9.6 Hz, 2H), 2.00 (d, J =14.2Hz,2H),1.85(dd, J =14.1, 8.4 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.4,153.8,152.4,149.8,140.3,136.7,133.1,129.2,118.7,114.2,36.0,31.2,25.6.

[0060] The above formula is 4-(3-cyclopentyl-7-oxo-6,7-dihydroisoxazole[4,5- d Methyl pyrimidin-5-yl)benzoate 3g: pale yellow solid; yield 94%; mp: 214-216℃; 1 HNMR(400MHz,CDCl3)δ12.19(s,1H),8.31(d, J =8.7Hz, 4H), 4.02(s, 3H), 3.59(p, J =8.3Hz, 1H), 2.28(d, J =7.1Hz,2H),2.18–2.08(m,2H),1.97(d, J =6.2Hz,2H), 1.87–1.78(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ166.1,163.5,154.4,152.4,149.7,140.4,136.7,132.3,129.8,128.9,53.0,36.0,31.2,25.6.

[0061] The above formula is 3-cyclopentyl-5-(4-nitrophenyl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3h: Yellow solid; yield 96%; mp: 216-218℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.52(s,1H),8.41(d, J =8.6Hz,2H),8.32(d, J =8.7Hz,2H),3.51(p, J =8.0Hz,1H),2.21–2.13(m,2H),1.98(dd, J =12.5,7.0Hz,2H),1.89–1.80(m,2H),1.79–1.70(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.4,153.5,152.3,149.8,149.4,140.2,138.2,129.9,124.2,36.0,31.2,25.6.

[0062] The above formula is 3-cyclopentyl-5-(2-hydroxyphenyl)isoxazo[4,5- d Pyrimidine-7(6) H )-Ketone 3i: White solid; yield 94%; mp: 213-215℃; 1 HNMR (400MHz, DMSO-) d 6 )δ8.04(d, J =7.9Hz,1H),7.46(t, J =7.6Hz, 1H), 7.02(dd, J =19.3,8.0Hz,2H),3.51(dd, J =16.5,8.3Hz,1H),2.16(d, J =7.4Hz,2H),2.01–1.92(m,2H),1.82(d, J =5.8Hz,2H), 1.78–1.69(m,2H). 13 CNMR (100MHz, DMSO-) d 6)δ162.8,157.9,155.2,151.6,149.2,139.1,133.8,129.5,119.9,117.8,116.5,35.9,31.0,25.7.

[0063] The above formula is 3-cyclopentyl-5-(3-hydroxyphenyl)isoxazo[4,5- d Pyrimidine-7(6) H )-Ketone 3j: White solid; yield 97%; mp: 219-221℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.08(s,1H),9.84(s,1H),7.51(d, J =7.5Hz,2H),7.35(t, J =7.9Hz, 1H), 6.99(d, J =8.7Hz, 1H), 3.48(p, J =8.1Hz, 1H), 2.16(d, J =7.5Hz,2H),2.03–1.94(m,2H),1.86(t, J =9.9Hz,2H),1.75(dd, J =13.3, 8.5 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.3,158.0,155.3,152.4,149.4,140.5,133.8,130.2,119.1,115.2,36.0,31.2,25.6.

[0064] The above formula is 3-cyclopentyl-5-(4-hydroxyphenyl)isoxazo[4,5- d Pyrimidine-7(6) H )-Ketone 3k: White solid; yield 92%; mp: 214-216℃; 1 HNMR (400MHz, DMSO-) d 6 )δ12.91(s,1H),10.21(s,1H),7.99(d, J =8.5Hz,2H),6.91(d, J =8.6Hz, 2H), 3.47(p, J=8.1Hz, 1H), 2.15(d, J =7.3Hz,2H),2.01–1.93(m,2H),1.84(s,2H),1.74(dd, J =14.1, 7.7 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.2,161.1,155.2,152.5,148.8,140.7,130.2,123.1,115.9,36.0,31.1,25.6.

[0065] The above formula is 3-cyclopentyl-5-(2-ethoxyphenyl)isoxazo[4,5- d Pyrimidine-7(6) H )-Ketone 3L: Pale yellow solid; yield 90%; mp: 210-212℃; 1 HNMR(400MHz,CDCl3)δ11.69(s,1H),8.52(d, J =7.8Hz,1H),7.55(t, J =7.8Hz,1H),7.20(t, J =7.6Hz, 1H), 7.11(d, J =8.3Hz, 1H), 4.38(q, J =6.8Hz, 2H), 3.56(p, J =8.0Hz, 1H), 2.26(d, J =5.8Hz,2H),2.17–2.09(m,2H),1.97(d, J =6.3Hz,2H),1.86–1.77(m,2H),1.66(t, J =6.9Hz, 3H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.3,156.9,154.7,151.5,149.2,140.6,133.0,131.1,122.3,121.0,113.3,64.6,36.0,31.1,25.5,14.9.

[0066] The above formula is 3-(3-cyclopentyl-7-oxo-6,7-dihydroisoxazole[4,5- dMethyl pyrimidin-5-yl)benzoate 3m: pale yellow solid; yield 89%; mp: 209-211℃; 1 HNMR(400MHz,CDCl3)δ12.19(s,1H),8.31(d, J =8.7Hz, 4H), 4.02(s, 3H), 3.59(p, J =8.3Hz, 1H), 2.28(d, J =7.1Hz,2H),2.18–2.08(m,2H),1.97(d, J =6.2Hz,2H), 1.87–1.78(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ166.1,163.5,154.4,152.4,149.7,140.4,136.7,132.3,129.8,128.9,53.0,35.95,31.2,25.6.

[0067] The above formula is 3-cyclopentyl-5-(pyridin-4-yl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3n: Pale yellow solid; yield 48%; mp: 204-206℃; 1 HNMR(400MHz,CDCl3)δ12.63(s,1H),8.95(d, J =3.8Hz, 2H), 8.17(d, J =4.2Hz,2H),3.70–3.53(m,1H),2.29(d, J =6.2Hz,2H),2.12(dd, J =12.4,7.4Hz,2H),2.00(s,2H),1.89–1.81(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.4,153.2,152.3,150.8,150.1,140.2,139.6,122.2,35.9,31.2,25.6.

[0068] The above formula is 5-(6-aminopyridin-3-yl)-3-cyclopentylisoxazolo[4,5- d Pyrimidine-7(6) H)-Ketone 3o: Pale yellow solid; yield 28%; mp: 198-200℃; 1 HNMR (400MHz, DMSO-) d 6 )δ12.85(s,1H),8.67(s,1H),8.05(d, J =8.4Hz, 1H), 6.69(s, 2H), 6.51(d, J =8.9Hz,1H),3.48–3.41(m,1H),2.12(d, J =6.8Hz,2H),1.98–1.92(m,2H),1.82(d, J =2.5Hz,2H),1.70(d, J =4.6Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ165.1,164.2,162.7,158.7,153.2,148.8,135.8,134.7,120.8,109.0,36.0,31.1,25.6.

[0069] The above formula is 5-(6-aminopyridin-3-yl)-3-cyclopentylisoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3p: Yellow solid; yield 59%; mp: 312-314℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.05(s,1H),8.89(s,2H),7.43(s,2H),3.47(p, J =8.1Hz, 1H), 2.15(d, J =7.7Hz,2H),2.01–1.92(m,2H),1.83(d, J =5.0Hz,2H),1.74(dd, J =14.3, 7.7 Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ164.6,163.2,158.4,152.7,152.3,148.9,140.6,115.2,35.9,31.1,25.7.

[0070] The above formula is 3-cyclopentyl-5-(quinolin-3-yl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3q: Brown solid; yield 98%; mp: 201-203℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.51(s,1H),9.49(d, J =1.9Hz, 1H), 9.07(d, J =2.0Hz, 1H), 8.13(dd, J =7.9,4.8Hz,2H),7.94–7.89(m,1H),7.74(t, J =7.5Hz, 1H), 3.53(p, J =8.1Hz, 1H), 2.18(dt, J =11.7,5.8Hz,2H),2.00(dd, J =13.7,6.2Hz,2H),1.90–1.81(m,2H),1.79–1.70(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.5,153.6,152.4,149.7,149.5,148.7,140.4,136.7,132.1,129.7,129.3,128.2,126.8,125.6,36.0,31.3,25.7.

[0071] The above formula is 5-(3-cyclopentyl-7-oxo-6,7-dihydroisoxazole[4,5- d ]pyrimidin-5-yl)-1 H 3r indole-1-carboxylic acid tert-butyl ester: pale yellow solid; yield 37%; mp: 184-186℃; 1 HNMR(400MHz, CDCl3)δ10.85(s,1H),8.32(s,2H),8.01(d, J =8.7Hz,1H),7.71(s,1H),6.74(s,1H),3.56(p, J =8.8Hz, 1H), 2.25(dd, J =7.8,2.3Hz,2H),2.15–2.07(m,2H),1.96(d, J=5.2Hz,2H),1.83–1.77(m,2H),1.71(s,9H). 13 CNMR (100MHz, CDCl3) δ163.5,154.6,153.3,149.4,148.4,142.1,137.2,131 .0,127.5,126.1,123.5,120.7,115.8,107.9,84.5,36.2,31.3,28.2,25.7.

[0072] The above formula is 3-cyclopentyl-5-(1 H -Indazole-4-yl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3s: Pale yellow solid; yield 82%; mp: 229-231℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.44(s,1H),13.26(s,1H),8.57(s,1H),7.83(dd, J =15.4, 7.7 Hz, 2H), 7.52(t, J =7.7Hz,1H),3.63–3.51(m,1H),2.20(d, J =5.6Hz,2H),2.10–2.02(m,2H),1.85(d, J =5.1Hz,2H),1.77(d, J =5.8Hz, 2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.4,154.7,152.4,149.6,140.9,140.4,134.5,126.0,125.1,121.9,120.8,114.4,36.0,31.1,25.8.

[0073] The above formula is 3-cyclopentyl-5-(1-methyl-1-yl) H -pyrazole-4-yl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3t: Pale yellow solid; yield 90%; mp: 205-207℃; 1 HNMR (400MHz, DMSO-) d 6)δ12.96(s,1H),8.48(s,1H),8.19(s,1H),3.93(s,3H),3.43(p, J =8.1Hz, 1H), 2.14(d, J =7.8Hz,2H),2.00–1.90(m,2H),1.85(t, J =10.0Hz,2H),1.77–1.67(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.0,152.3,150.4,148.6,140.8,138.9,132.5,116.3,39.5,36.0,31.1,25.6.

[0074] The above formula is 3-cyclopentyl-5-(furan-2-yl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3u: White solid; yield 60%; mp: 190-192℃; 1 HNMR(400MHz, CDCl3)δ11.17(s,1H),7.68(s,1H),7.47(s,1H),6.66(s,1H),3.51(p, J =8.1Hz, 1H), 2.21(d, J =6.7Hz,2H),2.05(dd, J =13.5,6.4Hz,2H),1.92(s,2H),1.82–1.73(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.3,152.0,149.2,147.2,146.6,145.9,140.3,115.2,113.1,35.9,31.2,25.5.

[0075] The above formula is 5-cyclohexyl-3-cyclopentylisoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3v: White solid; yield 94%; mp: 163-165℃; 1 HNMR(400MHz,CDCl3)δ11.73(s,1H),3.48(dd, J =16.4,8.3Hz,1H),2.75(t,J =11.7Hz, 1H), 2.17(d, J =6.7Hz,2H),2.01(d, J =11.2Hz,4H),1.90(d, J =9.2Hz,4H),1.77(d, J =12.4Hz, 3H), 1.66(d, J =8.7Hz,2H),1.45(dd, J =25.0,12.4Hz,2H),1.38–1.28(m,1H). 13 CNMR (100MHz, DMSO-) d 6 )δ163.5,163.0,152.2,149.2,140.3,42.6,35.9,31.1,30.9,25.8,25.7,25.6.

[0076] The above formula is 3-cyclopentyl-5-(tetrahydro-2-) H -pyran-4-yl)isoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3w: White solid; yield 86%; mp: 170-172℃; 1 HNMR(400MHz, CDCl3)δ12.49(s,1H),4.15(d, J =11.2Hz,2H),3.65(t, J =11.4Hz,2H),3.57–3.46(m,1H),3.08(t, J =10.7Hz,1H),2.20(s,2H),2.05(dd, J =20.3,11.5Hz,6H),1.95(s,2H),1.79(s,2H). 13 CNMR (100MHz, CDCl3) δ163.4,160.6,153.8,148.3,141.9,67.2,40.2,36.1,31.3,30.5,25.7.

[0077] The above formula is 3-cyclopentyl-5-isopropylisoxazolo[4,5- d Pyrimidine-7(6) H )-Ketone 3x: White solid; yield 86%; mp: 157-159℃; 1HNMR(400MHz, CDCl3)δ11.76(s,1H),3.47(p, J =8.1Hz, 1H), 3.08(dt, J =13.8, 6.9 Hz, 1H), 2.17 (dd, J =6.3,4.8Hz,2H),2.05–1.98(m,2H),1.95–1.87(m,2H),1.80–1.72(m,2H),1.41–1.36(m,6H). 13 CNMR (100MHz, DMSO-) d 6 )δ164.4,163.1,152.2,149.2,140.3,35.9,33.3,31.1,25.6,21.0.

[0078] The above formula is 3-(3-cyclohexyl-7-oxo-6,7-dihydroisoxazole[4,5- d Methyl pyrimidin-5-yl)benzoate 3y: pale yellow solid; yield 95%; mp: 228-230℃; 1 HNMR(400MHz, CDCl3)δ12.34(s,1H),8.88(s,1H),8.46(d, J =7.9Hz, 1H), 8.29(d, J =7.8Hz,1H),7.70(t, J =7.8Hz,1H),4.05(s,3H),3.27–3.17(m,1H),2.18(d, J =13.0Hz,2H),1.95–1.81(m,5H),1.66(s,1H),1.51–1.37(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ165.6,163.1,154.1,151.9,149.0,139.8,132.8,132.7,131.8,130.1,129.3,128.8,52.5,34.7,30.1,25.5,25.3.

[0079] The above formula is 3-(7-oxo-3-(tetrahydro-2-) H -pyran-4-yl)-6,7-dihydroisoxazolo[4,5-] dMethyl pyrimidin-5-yl)benzoate 3z: pale yellow solid; yield 90%; mp: 224-226℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.43(s,1H),8.63(s,1H),8.32(d, J =7.6Hz, 1H), 8.16(d, J =7.7Hz,1H),7.72(t, J =7.8Hz, 1H), 3.97(d, J =11.0Hz,2H),3.92(s,3H),3.56(t, J =10.9Hz,2H),3.43(t, J =10.2Hz,1H),2.05–1.94(m,4H). 13 CNMR (100MHz, DMSO-) d 6 )δ166.1,162.5,154.7,152.3,149.7,140.2,133.2,132.3,130.6,129.8,129.3,66.9,53.0,32.5,30.3.

[0080] The above formula is 3-(3-(1-acetylpiperidin-4-yl)-7-oxo-6,7-dihydroisoxazole[4,5- d Methyl pyrimidin-5-yl)benzoate 3aa: pale yellow solid; yield 69%; mp: 189-191℃; 1 HNMR (400MHz, DMSO-) d 6 )δ13.44(s,1H),8.65(s,1H),8.32(d, J =7.3Hz, 1H), 8.16(d, J =7.4Hz, 1H), 7.72(t, J =7.8Hz, 1H), 4.40(d, J =12.7Hz,1H),3.92(s,4H),3.51–3.40(m,1H),3.28(d, J =12.3Hz, 1H), 2.86(t, J =11.9Hz, 1H), 2.13(t, J =14.9Hz,2H),2.05(s,3H),1.95–1.85(m,1H),1.75(t,J =13.7Hz, 1H). 13 CNMR (100MHz, DMSO-) d 6 )δ168.6,166.1,162.4,154.7,152.3,149.7,140.2,133.1,132.3,130.6,129.8,129.3,53.0,45.8,40.9,33.3,30.1,29.5,21.8.

[0081] The above formula is 3-(3-isopropyl-7-oxo-6,7-dihydroisoxazole[4,5- d Methyl pyrimidin-5-yl)benzoate 3ab: white solid; yield 99%; mp: 180-182℃; 1 HNMR(400MHz, CDCl3)δ12.33(s,1H),8.89(s,1H),8.47(d, J =7.5Hz, 1H), 8.29(d, J =7.4Hz, 1H), 7.70(t, J =7.7Hz,1H),4.05(s,3H),3.50(s,1H),1.56(d, J =6.8Hz, 6H). 13 CNMR (100MHz, DMSO-) d 6 )δ166.1,164.4,154.4,152.3,149.6,140.2,133.1,133.0,132.3,130.5,129.7,129.2,52.9,26.3,20.7.

[0082] Example 3

[0083] Synthetic routes of isoxazolopyrimidine compounds 4a-4v with morpholine ring substitution:

[0084] POCl3 (383 mg, 5.0 mmol) was added to a CH3CN (10 mL) solution of compound 3 (0.5 mmol), and the mixture was heated to 80 °C and reacted for 5 hours. After the starting materials had reacted completely, the reaction solution was cooled to room temperature, and the mixture was concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to give compound S10 in 75-90% yield.

[0085] Morpholine (47 mg, 0.54 mmol) and Et3N (68 mg, 0.68 mmol) were added to a stirred solution of compound S10 (0.45 mmol) in dichloromethane (10 mL), and the mixture was reacted at room temperature for 1 hour. After the starting materials had reacted completely, the reaction mixture was diluted with water (10 mL), extracted with dichloromethane (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v) to give compound 4 in 67-87% yield.

[0086]

[0087] The above formula is 3-cyclopentyl-7-morpholine-5-phenylisoxazo[4,5- d Pyrimidine 4a: white solid; yield 83%; mp: 224-226℃; 1 HNMR (400MHz, CDCl3) δ 8.42 (d, J =5.1Hz,2H),7.45(s,3H),4.16(s,4H),3.89(d, J =4.0Hz, 4H), 3.58(p, J =8.0Hz, 1H), 2.23(d, J =6.9Hz,2H),2.13(td, J =14.7,7.9Hz,2H),2.02–1.90(m,2H),1.85–1.72(m,2H). 13 CNMR (100MHz, CDCl3) δ163.0,160.2,148.2,145.9,143.7,138.0,130.0,128.3,128.2,66.9,45.6,36.2,31.2,25.8.

[0088] The above formula is 3-cyclopentyl-5-(4-methoxyphenyl)-7-morpholinoisoxazo[4,5- d Pyrimidine 4b: white solid; yield 85%; mp: 121-123℃; 1 HNMR (400MHz, CDCl3) δ 8.40 (d, J =8.8Hz,2H),7.01(d, J =8.8Hz,2H),4.20–4.16(m,4H),3.93–3.90(m,7H),3.60(p, J=8.2Hz,1H),2.31–2.22(m,2H),2.20–2.12(m,2H),2.00(dt, J =9.0,5.7Hz,2H),1.88–1.78(m,2H). 13 CNMR (100MHz, CDCl3) δ162.9,161.3,160.1,148.1,145.8,143.4,130.8,129.7,113.6,66.9,55.4,45.7,36.2,31.2,25.8.

[0089] The above formula is 3-cyclopentyl-5-(4-methoxyphenyl)-7-morpholinoisoxazo[4,5- d Pyrimidine 4c: white solid; yield 86%; mp: 131-133℃; 1 HNMR (400MHz, CDCl3) δ 8.30 (d, J =8.2Hz,2H),7.24(s,2H),4.17–4.13(m,4H),3.90–3.86(m,4H),3.57(p, J =8.2Hz,1H),2.41(s,3H),2.27–2.19(m,2H),2.17–2.09(m,2H),2.00–1.91(m,2H),1.79(tt, J =7.9, 3.8 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ162.9,160.3,148.1,145.9,143.6,140.1,135.4,129.1,128.1,66.9,45.6,36.2,31.2,25.8,21.5.

[0090] The above formula is 3-cyclopentyl-5-(4-fluorophenyl)-7-morpholinoisoxazo[4,5- d Pyrimidine 4d: white solid; yield 63%; mp: 125-127℃; 1 HNMR(400MHz, CDCl3)δ8.44–8.39(m,2H),7.13(t, J =8.7Hz,2H),4.17–4.13(m,4H),3.91–3.87(m,4H),3.57(p, J =8.2Hz,1H),2.22(dt, J=11.7,5.9Hz,2H),2.17–2.07(m,2H),2.01–1.92(m,2H),1.80(ddd, J =10.5, 8.0, 3.2 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ165.5,163.0,162.9,159.3,148.2,145.8,143.6,134.2,130.2,130.1,115.3,115.1,66.8,45.5,36.2,31.2,25.8.

[0091] The above formula is 3-cyclopentyl-7-morpholine-5-(4-(trifluoromethyl)phenyl)isoxazo[4,5- d Pyrimidine 4e: white solid; yield 67%; mp: 151-153℃; 1 HNMR (400MHz, CDCl3) δ 8.53 (d, J =8.1Hz,2H),7.71(d, J =8.3Hz,2H),4.19–4.15(m,4H),3.92–3.89(m,4H),3.59(p, J =8.2Hz,1H),2.31–2.21(m,2H),2.18–2.09(m,2H),2.01–1.93(m,2H),1.82(qt, J =10.2, 5.2 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ163.0,158.8,148.2,145.8,143.8,141.3,132.1,131.7,131 .4,131.1,128.4,125.6,125.3,125.2,122.9,120.2,66.8,45.7,36.2,31.2,25.8.

[0092] The above formula is 4-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d 5-pyrimidinylbenzonitrile 4f: white solid; yield 82%; mp: 209-211℃; 1 HNMR (400MHz, CDCl3) δ 8.53 (d, J =8.3Hz,2H),7.75(d, J=8.4Hz,2H),4.19–4.15(m,4H),3.91–3.88(m,4H),3.57(p, J =8.3Hz, 1H), 2.23(dt, J =11.7,6.0Hz,2H),2.16–2.07(m,2H),2.00–1.92(m,2H),1.81(tt, J =8.1, 4.0 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ163.0,158.3,148.2,145.7,143.9,142.1,132.2,128.7,119.0,113.2,66.8,45.9,36.18,31.2,25.8.

[0093] The above formula is 4-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Methyl pyrimidin-5-yl)benzoate 4g: white solid; yield 81%; mp: 219-221℃; 1 HNMR (400MHz, CDCl3) δ 8.48 (d, J =8.5Hz,2H),8.12(d, J =8.5Hz,2H),4.19–4.14(m,4H),3.96(s,3H),3.92–3.88(m,4H),3.59(p, J =8.2Hz,1H),2.29–2.21(m,2H),2.18–2.09(m,2H),2.01–1.92(m,2H),1.86–1.75(m,2H). 13 CNMR (100MHz, CDCl3) δ167.1,163.0,159.2,148.2,145.8,143.8,142.2,131.2,129.6,128.1,66.9,52.3,45.6,36.2,31.2,25.8.

[0094] The above formula is 2-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenol 4h: white solid; yield 82%; mp: 179-181℃; 1 HNMR(400MHz,CDCl3)δ13.38(s,1H),8.36(d, J =7.8Hz, 1H), 7.36(t,J =7.5Hz, 1H), 7.02(d, J =8.0Hz,1H),6.93(t, J =7.7Hz,1H),4.19(s,4H),3.90(s,4H),3.59–3.48(m,1H),2.24(dd, J =14.6, 6.8 Hz, 2H), 2.07 (dd, J =12.4,7.3Hz,2H),1.93(s,2H),1.81(dd, J =12.1, 6.5 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ161.7,161.0,160.2,147.9,143.5,142.2,132.4,129.4,119.0,118.8,117.7,66.8,45.4,36.2,30.9,25.8.

[0095] The above formula is 3-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenol 4i: white solid; yield 81%; mp: 221-223℃; 1 HNMR (400MHz, CDCl3) δ 8.00 (d, J =7.5Hz, 1H), 7.91(s, 1H), 7.33(t, J =7.8Hz, 1H), 6.93(d, J =7.8Hz,1H),5.12(s,1H),4.15(s,4H),3.88(s,4H),3.57(t, J =8.1Hz,1H),2.22(s,2H),2.12(s,2H),1.95(s,2H),1.78(s,2H). 13 CNMR (100MHz, CDCl3) δ163.0,160.2,158.4,148.7,145.8,144.3,139.8,130.3,119.7,118.2,115.6,66.9,46.2,36.5,31.6,26.2.

[0096] The above formula is 4-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenol 4j: white solid; yield 82%; mp: 220–222℃; 1HNMR (400MHz, CDCl3) δ 7.99 (d, J =7.6Hz, 1H), 7.91(s, 1H), 7.33(t, J =7.8Hz, 1H), 6.92(d, J =7.5Hz,1H),5.35(s,1H),4.15(s,4H),3.88(s,4H),3.62–3.52(m,1H),2.22(d, J =6.5Hz,2H),2.16-2.08(m,2H),1.94(s,2H),1.77(s,2H). 13 CNMR (100MHz, CDCl3) δ162.7,160.0,158.1,148.5,145.5,144.0,139.5,130.0,119.5,117.9,115.4,66.7,45.9,36.3,31.3,26.0.

[0097] The above formula is 3-cyclopentyl-5-(2-ethoxybenzene)-7-morpholinoisoxazo[4,5- d Pyrimidine 4K: Yellow solid; yield 85%; mp: 80-82℃; 1 HNMR (400MHz, CDCl3) δ 7.76 (dd, J =7.6,1.7Hz,1H),7.39-7.33(m,1H),7.02(dd, J =13.0,7.9Hz,2H),4.15-4.11(m,4H),4.10-4.06(m,2H),3.86-3.83(m,4H),3.57(p, J =8.1Hz,1H),2.24-2.17(m,2H),2.15-2.07(m,2H),1.93-1.84(m,2H),1.75(tt, J =8.0, 3.8Hz, 2H), 1.35(d, J =7.0Hz, 3H). 13 CNMR (100MHz, CDCl3) δ162.8,161.2,157.3,147.9,145.6,143.1,131.9,130.4,129.1,120.5,113.4,66.9,64.3,45.7,36.3,31.2,25.5,15.0.

[0098] The above formula is 3-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Methyl pyrimidin-5-yl)benzoate 4L: white solid; yield 84%; mp: 138-140℃; 1 HNMR(400MHz,CDCl3)δ9.09(s,1H),8.66(d, J =7.6Hz, 1H), 8.15(d, J =7.6Hz, 1H), 7.57(t, J =7.7Hz,1H),4.21(s,4H),4.01(s,3H),3.93(s,4H),3.63(p, J =8.1Hz, 1H), 2.28(d, J =6.8Hz,2H),2.20–2.12(m,2H),2.00(s,2H),1.85(dd, J =14.2, 8.5 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ167.2,163.0,159.3,148.2,145.8,143.8,138.5,132.6,130.9,130.4,129.4,128.4,66.9,52.3,45.7,36.1,31.25,25.8.

[0099] The above formula is 3-cyclopentyl-7-morpholino-5-(pyridin-4-yl)isoxazolo[4,5- d Pyrimidine 4m: white solid; yield 82%; mp: 135-137℃; 1 HNMR (400MHz, CDCl3) δ 8.73 (d, J =5.1Hz, 2H), 8.26(d, J =5.8Hz,2H),4.20–4.15(m,4H),3.92–3.88(m,4H),3.58(t, J =8.2Hz, 1H), 2.24(dd, J =12.5,4.6Hz,2H),2.17–2.07(m,2H),1.97(dd, J =15.4,7.0Hz,2H),1.85–1.77(m,2H). 13CNMR (100MHz, CDCl3) δ163.0,158.0,150.2,148.2,145.7,145.2,144.1,122.1,66.8,45.6,36.2,31.2,25.8.

[0100] The above formula is 5-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)pyrimidin-2-amine 4n: white solid; yield 82%; mp: 227-229℃; 1 HNMR(400MHz, CDCl3)δ9.24(s,2H),5.33(s,2H),4.13(s,4H),3.88(s,4H),3.60–3.49(m,1H),2.25–2.17(m,2H),2.11(dd, J =13.4,5.7Hz,2H),1.94(s,2H),1.82–1.74(m,2H). 13 CNMR (100MHz, CDCl3) δ163.4,162.8,158.6,157.2,148.2,145.4,143.6,122.1,66.8,45.2,36.1,31.2,25.8.

[0101] The above formula is 3-cyclopentyl-7-morpholino-5-(quinolino-3-yl)isoxazolo[4,5- d Pyrimidine 4O: White solid; yield 81%; mp: 170-172℃; 1 HNMR(400MHz, CDCl3)δ9.94(s,1H),9.10(s,1H),8.16(d, J =8.4Hz, 1H), 7.98(d, J =7.8Hz,1H),7.77(t, J =7.2Hz, 1H), 7.60(t, J =7.1Hz,1H),4.22(s,4H),3.92(s,4H),3.68–3.56(m,1H),2.26(d, J =2.6Hz,2H),2.18(d, J =7.2Hz,2H),2.00(s,2H),1.82(s,2H). 13CNMR (100MHz, CDCl3) δ162.9,158.2,150.7,148.6,148.1,145.6,143.7,135 .2,130.5,130.1,129.2,128.7,127.5,126.7,66.8,45.9,36.1,31.2,25.8.

[0102] The above formula is 3-cyclopentyl-5-(1 H -Indazole-5-yl)-7-morpholinoisoxazo[4,5- d Pyrimidine 4p: white solid; yield 82%; mp: 238-240℃; 1 HNMR(400MHz, CDCl3)δ10.26(s,1H),9.05(s,1H),8.30(d, J =7.2Hz, 1H), 7.65(d, J =8.0Hz,1H),7.54(t, J =7.7Hz,1H),4.24(s,4H),3.95(s,4H),3.72–3.61(m,1H),2.28(dd, J =19.6,9.4Hz,4H),2.02(s,2H),1.88(d, J =18.4Hz, 2H). 13 CNMR (100MHz, CDCl3) δ162.9,160.3,148.2,145.7,143.6,141.0,136.8,131.5,126.6,122.5,121.6,111.7,66.9,45.7,36.4,31.1,25.7.

[0103] The above formula is 3-cyclopentyl-5-(1-methyl-1-yl) H -pyrazol-4-yl)-7-morpholinoisoxazo[4,5- d Pyrimidine 4q: white solid; yield 83%; mp: 134-136℃; 1 HNMR(400MHz, CDCl3)δ8.12(s,1H),8.04(s,1H),4.13(s,4H),3.99(s,3H),3.89(s,4H),3.56(p, J =7.9Hz, 1H), 2.23(d, J=6.9Hz,2H),2.14–2.06(m,2H),1.95(s,2H),1.84–1.76(m,2H). 13 CNMR (100MHz, CDCl3) δ162.6,156.8,148.2,145.5,143.1,139.6,130.8,123.6,66.8,45.5,39.2,36.1,31.2,25.7.

[0104] The above formula is 3-cyclopentyl-5-(furan-2-yl)-7-morpholinoisoxazo[4,5- d Pyrimidine 4r: white solid; yield 80%; mp: 93-95℃; 1 HNMR(400MHz, CDCl3)δ7.60(s,1H),7.19(s,1H),6.53(s,1H),4.13(s,4H),3.87(s,4H),3.60(p, J =8.1Hz, 1H), 2.24(d, J =7.4Hz,2H),2.04(dd, J =12.2,6.9Hz,2H),1.91(s,2H),1.78(dd, J =13.8, 8.0 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ163.0,153.7,152.5,148.2,145.4,144.4,143.2,112.4,111.8,66.8,45.4,35.9,31.4,25.5.

[0105] The above formula is 5-cyclohexyl-3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidine 4s: white solid; yield 87%; mp: 80-82℃; 1 HNMR(400MHz, CDCl3)δ4.06(s,4H),3.84(s,4H),3.51(m,1H),2.77(t, J =11.5Hz,1H),2.17(s,2H),2.06–1.95(m,4H),1.92–1.80(m,4H),1.74(s,3H),1.65–1.56(m,2H),1.45–1.35(m,2H),1.33–1.26(m,1H). 13CNMR (100MHz, CDCl3) δ170.2,162.6,148.1,145.3,143.3,66.8,47.1,45.2,36.1,32.3,31.2,26.3,26.2,25.5.

[0106] The above formula is 3-cyclopentyl-7-morpholine-5-(tetrahydro-2-) H -pyran-4-yl)isoxazolo[4,5- d Pyrimidine 4t: white solid; yield 90%; mp: 80-82℃; 1 HNMR(400MHz, CDCl3)δ4.07(s,6H),3.84(s,4H),3.59–3.47(m,3H),3.01(t, J =11.1Hz,1H),2.16(s,2H),2.09–1.97(m,4H),1.91(d, J =11.5Hz,4H),1.73(d, J =14.9Hz, 2H). 13 CNMR (100MHz, CDCl3) δ168.1,162.5,148.2,145.3,143.4,67.9,66.8,45.4,43.8,36.1,31.8,31.1,25.5.

[0107] 51-(4-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Synthetic route of pyrimidin-5-yl)phenyl)-3-methylurea 4v:

[0108] POCl3 (383 mg, 5.0 mmol) was added to a CH3CN (10 mL) solution of compound 3h (163 mg, 0.5 mmol), and the mixture was heated to 80 °C and reacted for 5 hours. After the starting materials had reacted completely, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v). Then, morpholine (47 mg, 0.54 mmol) and Et3N (68 mg, 0.68 mmol) were added to a dichloromethane (10 mL) solution of the purified product, and the mixture was reacted at room temperature for 1 hour. After the starting materials had reacted completely, the reaction mixture was diluted with water (10 mL), extracted with dichloromethane (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v) to give compound S11 in 87% yield.

[0109] Compound S11 (198 mg, 0.5 mmol) was dissolved in a solvent (10 mL, ethanol / water = 2 / 1) at 0 °C, and NH4Cl (663 mg, 12.5 mmol) and zinc powder (325 mg, 5 mmol) were added. The mixture was stirred at room temperature for 4 hours, then filtered to remove insoluble matter, and the filter cake was washed with ethanol. The filtrate was concentrated under vacuum and purified on a silica gel column to give compound 4u in 78% yield.

[0110] BTC (29.6 mg, 0.1 mmol) and Et3N (30.3 mg, 0.3 mmol) were added to a 2 mL solution of compound 4v (37 mg, 0.1 mmol) in dichloromethane at 0 °C, and the mixture was reacted for 15 min to give compound S12. Then, methylamine hydrochloride (34 mg, 0.5 mmol) was added to the reaction solution at room temperature, and the mixture was reacted overnight. The reaction mixture was diluted with water (10 mL), extracted with EA (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (ethyl acetate) to give compound 4v as a white solid in 93% yield.

[0111]

[0112] The above formula is 4-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)aniline 4u: pale yellow solid; yield 78%; mp: 190-192℃; 1 HNMR (400MHz, CDCl3) δ 8.28 (d, J =8.3Hz,2H),6.77(d, J=8.3Hz,2H),4.17(s,4H),3.91(s,4H),3.69–3.54(m,1H),2.26(d, J =6.3Hz,2H),2.22–2.12(m,2H),1.99(s,2H),1.88–1.78(m,2H). 13 CNMR (100MHz, DMSO-) d 6 )δ162.4,160.6,151.4,148.2,145.4,143.1,129.7,125.2,113.6,66.4,45.6,36.0,31.1,25.7.

[0113] The above formula is 1-(4-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)-3-methylurea 4v: white solid; yield 93%; mp: 266-268℃; 1 HNMR(400MHz, CDCl3)δ8.77(s,1H),8.23(d, J =8.7Hz,2H),7.51(d, J =8.7Hz,2H),6.08(d, J =4.6Hz,1H),4.07–4.02(m,4H),3.82–3.77(m,4H),3.51(p, J =8.1Hz, 1H), 2.66(d, J =4.6Hz, 3H), 2.15(dt, J =11.8, 6.0 Hz, 2H), 2.01(dd, J =12.5,7.3Hz,2H),1.91–1.83(m,2H),1.79–1.69(m,2H). 13 CNMR (100MHz, CDCl3) δ162.9,160.3,156.6,148.7,145.8,144.0,143.6,130.9,129.5,129.4,117.9,66.9,46.3,36.5,31.5,27.2,26.2.

[0114] Example 4

[0115] (3-(isoxazolo[4,5-) d Synthetic routes of pyrimidin-5-yl)phenyl)methanol 5a-5j:

[0116] POCl3 (383 mg, 5.0 mmol) was added to a CH3CN (10 mL) solution of compounds 3n, 3aa-3ab (0.5 mmol), and the mixture was heated to 80 °C and reacted for 5 hours. After the starting materials reacted completely, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v). Morpholine (47 mg, 0.54 mmol) and Et3N (68 mg, 0.68 mmol) were added to a dichloromethane (10 mL) solution of the purified product (0.45 mmol), and the mixture was reacted at room temperature for 1 hour. After the starting materials reacted completely, the reaction mixture was diluted with water (10 mL), extracted with dichloromethane (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v) to give compound S13 in 85% yield.

[0117] LiAlH4 (0.2 mL, 0.2 mmol, 1.0 mol / L THF solution) was added to an anhydrous tetrahydrofuran solution (5 mL) of S13 (0.2 mmol), and the mixture was reacted at 0 °C for 30 min. Subsequently, the mixture was reacted at room temperature for 1 h. The reaction mixture was quenched with an aqueous NaOH solution (3 mL), and filtered to remove insoluble matter. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a white solid compound in 64–78% yield.

[0118]

[0119] The above formula is (3-(3-cyclopentyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 5a: pale yellow solid; yield 77%; mp: 134-136℃; 1 HNMR(400MHz, CDCl3)δ8.39(s,1H),8.35(s,1H),7.46(d, J =3.6Hz,2H),4.79(s,2H),4.15(s,4H),3.88(s,4H),3.58(p, J =7.8Hz, 1H), 2.24(d, J =7.1Hz,2H),2.16–2.07(m,2H),1.97(d, J =13.6Hz,2H),1.83–1.77(m,2H). 13CNMR(100MHz, CDCl3)δ163.0,160.0,148.1,145.8,143.6,141.0,138.3,128.8,128.6,127.6,126.8,66.9,65.5,45.6,36.2,31.2,25.7.

[0120] The above formula is (3-(3-cyclopentyl-7-((2) S 6 R )-2,6-dimethylmorpholino)isoxazolo[4,5- d 5b pyrimidin-5-yl)phenylmethanol: white solid; yield 74%; mp: 130-132℃; 1 HNMR(400MHz, CDCl3)δ8.38(s,1H),8.34(t, J =4.5Hz, 1H), 7.46(t, J =6.2Hz,2H),4.88(d, J =10.4Hz,2H),4.80(s,2H),3.77(ddd, J =10.4, 6.3, 2.4 Hz, 2H), 3.59 (p, J =8.1Hz,1H),2.92(s,2H),2.31–2.20(m,2H),2.16–2.07(m,2H),1.99–1.91(m,2H),1.82–1.77(m,2H),1.33(d, J =6.2Hz, 6H). 13 CNMR (100MHz, CDCl3) δ163.0,160.1,147.9,145.7,143.6,141.0,138.4,128.8,128.7,127.7,126.8,71.9,65.5,50.3,36.2,31.2,25.7,18.9.

[0121] The above formula is ( R )–(3-(3-cyclopentyl-7-(3-methylmorpholino)isoxazolo[4,5- d Pyrimidin-5-yl)phenyl)methanol 5c: white solid; yield 73%; mp: 128-130℃; 1 HNMR(400MHz, CDCl3)δ8.39(s,1H),8.35(s,1H),7.46(s,2H),5.02(d, J=4.4Hz, 1H), 4.79(s, 2H), 4.70(d, J =12.8Hz, 1H), 4.10(d, J =10.6Hz,1H),3.92–3.81(m,2H),3.69(t, J =11.8Hz, 1H), 3.59(dd, J =16.5,8.9Hz,2H),2.23(s,2H),2.12(s,2H),1.95(s,2H),1.84(s,1H),1.79(s,2H),1.48(d, J =6.6Hz, 3H). 13 CNMR (100MHz, CDCl3) δ163.0,160.1,148.1,145.7,143.7,141.0,138.4,128.7, 128.6,127.6,126.8,71.1,67.1,65.5,48.6,40.0,36.2,31.2,31.2,25.7,15.0.

[0122] The above formula is (3-(3-cyclopentyl-7-thiomorpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 5d: white solid; yield 75%; mp: 159-161℃; 1 HNMR(400MHz, CDCl3)δ8.38(s,1H),8.36-8.31(m,1H),7.46(dd, J =4.2,1.1Hz,2H),4.79(s,2H),4.46-4.42(m,4H),3.58(p, J =8.2Hz,1H),2.82-2.78(m,4H),2.23(dt, J =11.5,5.9Hz,2H),2.16-2.08(m,2H),1.99-1.91(m,2H),1.84-1.78(m,2H). 13 CNMR (100MHz, CDCl3) δ163.0,160.1,147.7,145.9,143.6,141.0,138.3,128.7,128.6,127.6,126.7,65.4,48.4,36.2,31.2,27.4,25.7.

[0123] The above formula is (3-(3-cyclopentyl-7-((2-morpholinoethyl)amino)isoxazole[4,5- d 5-pyrimidinyl)phenyl)methanol 5e: colorless oil; yield 64%; 1 HNMR(400MHz, CDCl3)δ8.43(s,1H),8.38(s,1H),7.46(s,2H),6.09(s,1H),4.79(s,2H),3.86(s,2H),3.76(s,4H),3.58(dd, J =16.1,8.0Hz,1H),2.72(s,2H),2.55(s,4H),2.23(s,2H),2.16–2.09(m,2H),1.96(s,3H),1.79(s,2H). 13 CNMR (100MHz, CDCl3) δ163.2,160.5,147.9,143.5,141.1,138.4,128.7,128.6,127.6,126.8,66.9,65.4,56.9,53.4,36.3,31.3,25.7.

[0124] The above formula is (3-(3-cyclopentyl-7-(4-methylpiperazin-1-yl)isoxazole[4,5- d Pyrimidin-5-yl)phenyl)methanol 5f: white solid; yield 78%; mp: 170-172℃; 1 HNMR(400MHz, CDCl3)δ8.40(s,1H),8.35(dd, J =6.3, 2.7 Hz, 1H), 7.46 (d, J =5.1Hz,2H),4.79(s,2H),4.18–4.13(m,4H),3.57(p, J =8.2Hz,1H),2.60–2.56(m,4H),2.36(s,3H),2.23(dd, J =12.6,4.7Hz,2H),2.11(dd, J =12.7,7.6Hz,2H),1.98–1.90(m,2H),1.83–1.74(m,2H). 13 CNMR (100MHz, CDCl3) δ162.9,160.0,148.0,145.6,143.7,141.1,138.5,128.7,128.6,127.6,126.8,65.5,54.9,46.1,44.7,36.2,31.2,25.7.

[0125] The above formula is (3-(3-cyclopentyl-7-(piperidin-1-yl)isoxazolo[4,5- d 5 g of pyrimidin-5-yl)phenyl)methanol: white solid; yield 73%; mp: 155-157℃; 1 HNMR(400MHz, CDCl3)δ8.40(s,1H),8.38–8.35(m,1H),7.45(d, J =5.1Hz,2H),4.79(s,2H),4.11(d, J =5.5Hz,4H),3.59(dd, J =16.4,8.2Hz,1H),2.24(dd, J =12.7,4.8Hz,2H),2.12(dd, J =12.7, 7.6 Hz, 2H), 1.95 (dd, J =8.8, 5.9 Hz, 2H), 1.77(t, J =7.4Hz, 8H). 13 CNMR (100MHz, CDCl3) δ162.9,160.1,148.0,145.3,143.9,141.0,138.6,128.5,128.5,127.5,126.7,65.4,46.4,36.2,31.2,26.1,25.7,24.8.

[0126] The above formula is (3-(3-cyclohexyl-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 5h: pale yellow solid; yield 74%; mp: 140-142℃; 1 HNMR(400MHz, CDCl3)δ8.39(s,1H),8.37–8.33(m,1H),7.47(d, J =4.8Hz,2H),4.80(s,2H),4.18–4.14(m,4H),3.92–3.87(m,4H),3.24(ddd, J =11.6,7.6,3.3Hz,1H),2.19(d, J =11.8Hz,2H),1.94–1.79(m,6H),1.52–1.42(m,2H). 13CNMR (100MHz, CDCl3) δ163.3,160.2,148.3,145.8,143.7,141.1,138.5,128.9,128.8,127.8,126.9,67.0,65.7,45.8,35.6,30.7,26.2,26.1.

[0127] The above formula is (3-(7-morpholine-3-(tetrahydro-2-)) H -pyran-4-yl)isoxazole[4,5- d Pyrimidin-5-yl)phenyl)methanol 5i: white solid; yield 72%; mp: 152-154℃; 1 HNMR(400MHz, CDCl3)δ8.39(s,1H),8.35(s,1H),7.48(s,2H),4.81(d, J =5.2Hz,2H),4.16(s,4H),4.12(s,2H),3.89(s,4H),3.67(t, J =11.3Hz,2H),3.49(s,1H),2.27–2.13(m,4H),1.85(t, J =5.7Hz, 1H). 13 CNMR (100MHz, CDCl3) δ161.6,160.3,148.1,145.4,143.8,141.1,138.1,128.9,128.7,127.7,126.8,67.7,66.8,65.4,45.7,32.8,30.1.

[0128] The above formula is (3-(3-isopropyl-7-morpholinoisoxazole[4,5- d Pyrimidin-5-yl)phenyl)methanol 5j: white solid; yield 72%; mp: 143-145℃; 1 HNMR(400MHz, CDCl3)δ8.40(s,1H),8.36(s,1H),7.47(s,2H),4.80(d, J =4.2Hz,2H),4.16(s,4H),3.89(s,4H),3.52(dt, J =13.4,6.6Hz,1H),1.80(s,1H),1.57(d, J =6.8Hz, 6H). 13CNMR (100MHz, CDCl3) δ164.0,160.1,148.2,145.6,143.6,141.0,138.3,128.8,128.6,127.6,126.8,66.9,65.5,45.6,26.5,20.5.

[0129] Example 5

[0130] Acetyl chloride, ethyl and hydrogen-substituted piperidinyl isoxazolo[4,5- d Synthetic routes for pyrimidin-5-ylphenylmethanol 6a-6c:

[0131] POCl3 (383 mg, 5.0 mmol) was added to a CH3CN (10 mL) solution of compound 3aa (198 mg, 0.5 mmol), and the mixture was heated to 80 °C and reacted for 5 hours. After the starting materials reacted completely, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v). Morpholine (47 mg, 0.54 mmol) and Et3N (68 mg, 0.68 mmol) were then added to a dichloromethane (10 mL) solution of the purified product (186 mg, 0.45 mmol), and the mixture was reacted at room temperature for 1 hour. After the starting materials reacted completely, the reaction mixture was diluted with water (10 mL), extracted with dichloromethane (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v) to give compound S14 in 83% yield.

[0132] LiAlH4 (5 mL, 5 mmol, 1.0 mol / L THF solution) was added to an anhydrous tetrahydrofuran (5 mL) stirred solution of S14 (2.33 g, 5 mmol) at 0 °C, and the mixture was reacted for 6 hours. The reaction mixture was then quenched with an aqueous NaOH solution (10 mL) and filtered to remove insoluble matter. The combined organic layers were dried over anhydrous Na2SO4, the mixture was concentrated under reduced pressure, and purified by rapid column chromatography (dichloromethane / methanol = 10 / 1, v / v) to give white solid compound 6a in 15% yield, white solid compound 6b in 30% yield, and white solid compound 6c in 50% yield.

[0133]

[0134] The above formula is 1-(4-(5-(3-(hydroxymethyl)phenyl)-7-morpholinoisoxazo[4,5- dPyrimidin-3-yl)piperidin-1-yl)ethyl-1-one 6a: white solid; yield 15%; mp: 194-196℃; 1 HNMR(400MHz, CDCl3)δ8.39(s,1H),8.32(s,1H),7.47(s,2H),4.80(s,2H),4.65(d, J =13.1Hz,1H),4.16(s,4H),3.96(d, J =13.3Hz,1H),3.89(s,4H),3.48(s,1H),3.34(t, J =12.4Hz, 1H), 2.94(t, J =12.0Hz,1H),2.25(t, J =12.0Hz,2H),2.16(s,3H),2.06(dd, J =20.7, 12.8 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ169.2,161.3,160.4,148.1,145.3,143.8,141.2,138.1,129.0, 128.7,127.6,126.8,66.8,65.4,60.5,46.3,41.4,33.6,29.8,29.2,21.6,21.1,14.3.

[0135] The above formula is (3-(3-(1-ethylpiperidin-4-yl)-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 6b: white solid; yield 30%; mp: 196-198℃; 1 HNMR (400MHz, DMSO-) d 6 )δ10.92(s,1H),8.35(d, J =42.1Hz,2H),7.44(s,2H),5.37(s,1H),4.59(s,2H),4.08(s,4H),3.82(s,4H),3.60(d, J =7.4Hz,3H),3.13(s,4H),2.39(s,4H),2.02(s,1H),1.27(d, J =31.3Hz, 7H). 13CNMR(100MHz,CDCl3)δ162.0,160.4,148.2,145.6,143.7,141.5,138.2,1 28.8,128.6,127.5,126.8,66.9,65.2,53.0,52.7,45.6,33.8,29.4,12.0.

[0136] The above formula is (3-(7-morpholino-3-(piperidin-4-yl)isoxazolo[4,5- d Pyrimidin-5-yl)phenyl)methanol 6c: white solid; yield 50%; mp: 225-227℃; 1 HNMR (400MHz, DMSO-) d 6 )δ9.28(s,1H),8.38(s,1H),8.33–8.28(m,1H),7.45(d, J =4.5Hz,2H),5.37(t, J =5.7Hz, 1H), 4.60(d, J =5.3Hz,2H), 4.08(s,4H), 3.82(d, J =3.6Hz, 4H), 3.57(dtd, J =11.8,7.9,4.1Hz,2H),3.15(t, J =10.2Hz,2H),2.36–2.24(m,4H). 13 CNMR (100MHz, DMSO-) d 6 )δ160.5,159.6,147.9,144.5,143.6,142.8,137.1,128.6,128.2,126.5,126.2,66.0,62.9,45.2,42.5,30.7,25.8.

[0137] Example 6

[0138] Piperidinylisoxazolo[4,5-] d Synthesis of pyrimidin-5-ylphenylmethanol 6d-6h:

[0139] At room temperature, an acyl chloride or bromide or acid anhydride (0.24 mmol) and Et3N (41 mg, 0.4 mmol) were added to a dichloromethane solution (5 mL) of compound 6c (79 mg, 0.2 mmol), and the mixture was reacted for 3 hours. After the starting materials had reacted completely, the reaction mixture was diluted with water (10 mL), extracted with DCM (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain the compound.

[0140]

[0141] The above formula is cyclopropyl(4-(5-(3-(hydroxymethyl)phenyl)-7-morpholinoisoxazo[4,5- d Pyrimidin-3-yl)piperidin-1-yl)methyl ketone 6d: white solid; yield 95%; mp: 195-197℃; 1 HNMR(400MHz, CDCl3)δ8.38(s,1H),8.36–8.31(m,1H),7.47(d, J =5.2Hz,2H), 4.80(s,2H), 4.66(d, J =12.5Hz, 1H), 4.36(d, J =12.5Hz,1H),4.18–4.14(m,4H),3.91–3.88(m,4H),3.56–3.48(m,1H),3.41(t, J =10.0Hz,1H),2.97(t, J =12.1Hz,1H),2.32–2.22(m,2H),2.11–1.99(m,2H),1.86–1.79(m,1H),1.03(dd, J =7.3, 3.1Hz, 2H), 0.79(dd, J =7.9, 2.8 Hz, 2H). 13 CNMR (100MHz, CDCl3) δ172.1,161.3,160.4,148.1,145.3,143.8,141.3,138.0,1 28.9,128.6,127.5,126.8,66.8,65.3,45.5,42.2,33.8,30.0,29.2,11.18,7.4.

[0142] The above formula is 4-(5-(3-(hydroxymethyl)phenyl)-7-morpholinoisoxazo[4,5- d6e: white solid; yield 95%; mp: 92-94℃; 1 HNMR(400MHz, CDCl3)δ8.37(s,1H),8.33(d, J =6.3Hz, 1H), 7.46(d, J =6.2Hz,2H), 4.79(s,2H), 4.22(d, J =4.9Hz,2H),4.17–4.13(m,4H),3.90–3.87(m,4H),3.38(ddd, J =11.3,7.7,3.8Hz,1H),3.01(t, J =10.3Hz,2H),2.18(d, J =11.4Hz,2H),2.08–2.02(m,2H),1.50(s,9H). 13 CNMR (100MHz, CDCl3) δ161.7,160.4,154.9,148.1,145.4,143.8,141.3,138. 1,128.9,128.7,127.6,126.8,79.8,66.9,65.4,45.8,43.4,33.8,29.4,28.6.

[0143] The above formula is (4-(5-(3-(hydroxymethyl)phenyl)-7-morpholinoisoxazo[4,5- d Pyrimidin-3-yl)piperidin-1-yl)phenyl)methyl ketone 6f: white solid; yield 94%; mp: 106-108℃; 1 HNMR(400MHz, CDCl3)δ8.40(s,1H),8.34(t, J =4.4Hz, 1H), 7.45 (dd, J =15.7,3.8Hz,7H),4.79(s,2H),4.19–4.15(m,4H),3.92–3.88(m,4H),3.58–3.50(m,1H),3.22(d, J =44.8Hz,2H),2.16(t, J =67.9Hz, 6H). 13CNMR (100MHz, CDCl3) δ170.7,161.3,160.5,148.2,145.4,144.0,141.3,138.1,136.2 ,129.8,129.1,128.8,128.7,127.7,127.0,126.9,66.9,65.5,47.7,33.8,30.0,29.5.

[0144] The above formula is (3-(3-(1-(methylsulfonyl)piperidin-4-yl)-7-morpholinoisoxazo[4,5- d 6 g of pyrimidin-5-yl)phenyl)methanol: white solid; yield 95%; mp: 222-224℃; 1 HNMR(400MHz,CDCl3)δ8.39(s,1H),8.33(dd, J =6.3, 2.6 Hz, 1H), 7.47 (d, J =5.1Hz,2H),4.80(s,2H),4.19–4.15(m,4H),3.92–3.88(m,6H),3.44–3.34(m,1H),3.07–2.99(m,2H),2.86(s,3H),2.35–2.26(m,4H). 13 CNMR (100MHz, CDCl3) δ161.0,160.6,148.2,145.4,144.0,141.2,138.1,129.1,128.8,127.7,126.9,66.9,65.5,45.7,35.2,32.9,29.0.

[0145] The above formula is (3-(3-(1-benzylpiperidin-4-yl)-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 6h: white solid; yield 96%; mp: 87-89℃; 1 HNMR(400MHz, CDCl3)δ8.40(s,1H),8.33(t, J =4.4Hz, 1H), 7.45(d, J =5.0Hz,2H),7.36(dd, J =11.1,7.1Hz,4H),7.29(s,1H),4.79(s,2H),4.16–4.12(m,4H),3.90–3.86(m,4H),3.61(s,2H),3.27–3.18(m,1H),3.04(d,J =8.4Hz,2H),2.22(dd, J =14.7, 5.3 Hz, 6H). 13 CNMR (100MHz, CDCl3) δ162.1,160.3,148.1,145.6,143.7,141.3,138.2,137.8,129.5 ,128.8,128.6,128.4,127.6,127.3,126.8,66.9,65.3,63.3,53.3,45.7,33.7,29.5.

[0146] Example 7

[0147] (3-(7-morpholino-3-(1-(pyridin-3-ylmethyl)piperidin-4-yl)isoxazo[4,5-] d Synthesis of pyrimidin-5-yl)phenyl)methanol 6i:

[0148] Compound 6c (79 mg, 0.2 mmol) was subjected to treatment at 60 °C. N , N 4-(bromomethyl)pyridine hydrobromide (61 mg, 0.24 mmol) and Et3N (41 mg, 0.4 mmol) were added to a 5 mL solution of dimethylformamide, and the mixture was reacted overnight. The reaction mixture was diluted with 10 mL of water, extracted with EA (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (dichloromethane / methanol = 30 / 1, v / v) to give a white solid compound 6i in 99% yield.

[0149]

[0150] The above formula is (3-(7-morpholino-3-(1-(pyridin-3-ylmethyl)piperidin-4-yl)isoxazo[4,5-] d Pyrimidin-5-yl)phenyl)methanol 6i: white solid; yield 99%; mp: 158-160℃; 1 HNMR(400MHz, CDCl3)δ8.66(s,1H),8.53–8.50(m,1H),8.46(s,1H),8.33(dd, J =6.2, 2.7 Hz, 1H), 7.72 (d, J =7.7Hz, 1H), 7.46(d, J=5.1Hz,2H),7.31–7.27(m,1H),4.81(s,2H),4.17–4.14(m,4H),3.91–3.87(m,4H),3.61(s,2H),3.25(s,1H),3.01(d, J =5.9Hz,2H),2.30(d, J =8.5Hz, 4H), 2.16(d, J =6.6Hz, 2H). 13 CNMR (100MHz, CDCl3) δ162.1,160.4,150.5,148.5,148.2,145.7,143.8,141.5,138.3,13 6.9,134.1,129.0,128.6,127.6,126.9,123.5,66.9,65.3,60.5,53.4,45.9,33.6,29.6.

[0151] Example 8

[0152] (3-(3-(1-(2-methoxyethyl)piperidin-4-yl)-7-morpholinoisoxazo[4,5-] d Synthesis of pyrimidin-5-yl)phenyl)methanol 6j:

[0153] 1-Bromo-2-methoxyethane (33 mg, 0.24 mmol) and DIPEA (52 mg, 0.4 mmol) were added to an ethanol solution (5 mL) of compound 6c (79 mg, 0.2 mmol) at 80 °C, and the mixture was reacted overnight. The mixture was concentrated under reduced pressure, and the residue was purified by rapid column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to give a pale yellow solid, compound 6j, in 89% yield.

[0154]

[0155] The above formula is (3-(3-(1-(2-methoxyethyl)piperidin-4-yl)-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 6j: pale yellow solid; yield 89%; mp: 200-202℃; 1 HNMR(400MHz,CDCl3)δ8.43(s,1H),8.32(td, J =4.7, 1.6 Hz, 1H), 7.44 (d, J=4.9Hz,2H),4.79(s,2H),4.17–4.13(m,4H),3.90–3.87(m,4H),3.59(t, J =5.6Hz,2H),3.38(s,3H),3.28–3.19(m,1H),3.14(d, J =8.8Hz,2H),2.70(t, J =5.5Hz,2H),2.33(dd, J =16.7,9.4Hz,4H),2.23(s,2H). 13 CNMR (100MHz, CDCl3) δ161.9,160.4,148.2,145.6,143.8,141.5,138.2,128. 8,128.6,127.6,126.8,70.2,66.9,65.3,59.0,58.1,53.8,45.6,33.5,29.2.

[0156] Example 9

[0157] (3-(3-(1-Cyclopentylpiperidin-4-yl)-7-morpholinoisoxazo[4,5-) d Synthesis of pyrimidin-5-yl)phenyl)methanol 6k:

[0158] Compound 6c (79 mg, 0.2 mmol) was subjected to treatment at 80 °C. N,N Bromocyclopentane (36 mg, 0.24 mmol) and Et3N (41 mg, 0.4 mmol) were added to a dimethylformamide solution (5 mL), and the mixture was reacted overnight. The reaction mixture was diluted with water (10 mL), extracted with EA (10 mL × 3), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by rapid column chromatography (dichloromethane / methanol = 20 / 1, v / v) to give a white solid compound 6K in 62% yield.

[0159]

[0160] The above formula is (3-(3-(1-cyclopentylpiperidin-4-yl)-7-morpholinoisoxazo[4,5- d Pyrimidin-5-yl)phenyl)methanol 6k: white solid; yield 62%; mp: 187-189℃; 1 HNMR(400MHz, CDCl3)δ8.44(s,1H),8.30(t, J =3.6Hz, 1H), 7.44(d, J=4.6Hz,2H),4.79(s,2H),4.18–4.14(m,4H),3.90–3.86(m,4H),3.21(s,2H),2.62(dd, J =15.5,7.8Hz,1H),2.27–2.21(m,7H),1.92(d, J =8.5Hz,2H),1.73(s,2H),1.60–1.50(m,4H). 13 CNMR (100MHz, CDCl3) δ161.3,160.5,148.1,145.5,143.8,141.7,138.1,128. 9,128.5,127.4,127.0,68.0,66.9,65.1,52.0,45.7,33.1,29.7,28.4,24.1.

[0161] Example 10

[0162] Screening for CA-CPSF6 interaction inhibitors using surface-enhanced Raman spectroscopy

[0163] 1. Preparation of silver nanoparticles

[0164] Silver nanoparticles (AgNPs): A three-necked flask was immersed in an acid bath overnight, then rinsed with ultrapure water to ensure the walls were clean and free of impurities. 33.4 mg of silver nitrate powder was accurately weighed and poured into the three-necked flask, along with 150 mL of ultrapure water. A condenser was connected, and the magnetic stirrer was set to 100°C. The solution was heated to a gentle boil, and immediately 6 mL of 1% trisodium citrate solution was added. The color change was observed, from light yellow to deep yellow, then to grayish-green, until the color no longer changed. Magnetic stirring was continued, and the mixture was cooled to room temperature to obtain a silver nanoparticle dispersion. The absorbance curve of the silver nanoparticles was measured using a UV spectrophotometer. After a 3-fold dilution, the maximum absorption wavelength range was observed to be 410-430 nm, with no other impurity peaks, indicating uniform particle size. The concentration of the silver nanoparticles was calculated using the Lambert-Beer law. A=kbc (A=A) max k=3×10 11 M -1 cm -1 (b=1cm) The characterization results of AgNPs are as follows Figure 2 As shown.

[0165] 2. Preparation of magnetic nanoparticles

[0166] The Ni-NTA magnetic beads (500nm) from Shanghai Aladdin were prepared to a concentration of 0.5mg / mL, magnetically separated, and stored at 4℃ for later use.

[0167] 3. Two Raman probes related to the synthesis and screening of CA-CPSF6 interaction inhibitors.

[0168] The specific process is as follows: Figure 1 As shown:

[0169] (1) Preparation of AgNPs@CPSF6 silver probe.

[0170] To prevent AgNPs from aggregating during surface modification, concentrated silver nanoparticles (AgNPs, 0.29 nM) were mixed with 20 μL of Tween 20 for 10 min. Then, 10 μL each of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (2.5 mM) and N-hydroxysuccinimide (NHS) (2.5 mM) were added, and the mixture was stirred for 1 h. The mixture was then centrifuged at 3800 g for 6 min to remove excess activator and resuspended in ultrapure water. 2 μL of CPSF6 (0.44 mg / mL) was added to the solution, and the mixture was stirred at room temperature for 1 h. Finally, 1% BSA was added, and the mixture was reacted at room temperature for 20 min to block unbound sites. Unreacted protein was removed by centrifugation (3800 g, 6 min) and resuspended in 1 mL of ultrapure water. AgNPs@CPSF6 were stored at 4 °C for later use.

[0171] (2) Preparation of MNs@CA magnetic probe.

[0172] Take 1 mL of 0.5 mg / mL Ni-NTA magnetic beads, add 10 μL of Tween 20, mix for 10 min, then add 10 μL of CA hexamer protein (1.6 mg / mL), mix at room temperature for 30 min. Magneticly separate and wash the magnetic beads three times to remove non-specifically bound proteins, resuspend in ultrapure water, and store at 4 °C for later use.

[0173] 4. Establishment of a CA-CPSF6 interaction inhibitor screening platform

[0174] The specific process is as follows: Silver probe and magnetic probe were mixed in a 3:1 volume ratio and incubated at room temperature for 40 min. Afterward, the mixture was magnetically aggregated externally using a magnet, and the magnetic probe was washed three times. After resuspending, 1 μL of the Raman reporter molecule 4-MBA (10 mM) was added, and incubation continued for another 40 min. The magnetic probe was washed three more times, resuspended in ultrapure water, and Raman signal detection was performed. (Electron microscopy image) Figure 3 b) Demonstration: Under visible light, silver probes (small spheres) adhere to the surface of the magnetic probe (large sphere), proving that the two probes form a silver-magnetic complex due to the PPI of CA-CPSF6 and can bind to each other; unbound probes, such as Figure 3As shown in figure a. Magnetic aggregation was performed under magnetic field drive, and the complex was subjected to Raman detection (exposure time 1 s, average of 3 exposures). The detected 4-MBA signal molecule was taken at 1075 cm⁻¹. -1 Data analysis was performed on the characteristic peaks at the location.

[0175] 5. Screening of small molecule combinatorial compound libraries using SERScreen technology.

[0176] Inhibitor screening was performed in 96-well plates: 50 μL of MNs@CA and 0.3 μL of different small molecule compounds were added to each well and incubated for 1 h. Then, 150 μL of AgNPs@CPSF6 was added to each well and reacted for 40 min. After magnetic separation and washing three times, the mixture was resuspended in 200 μL of ultrapure water. 1 μL of 4-MBA (10 mM) was added to each well and reacted for 40 min. After magnetic separation and washing three times, the mixture was resuspended in 200 μL of ultrapure water. Magnetic aggregation was performed under magnetic field drive, and Raman spectroscopy was performed on the complex (exposure time 1 s, average of 3 exposures). The 4-MBA signal molecule in the homogeneous phase of each added small molecule was detected at 1075 cm⁻¹. -1 Characteristic peaks were observed at the specified locations. Each small molecule was analyzed in three batches, with each analysis performed in at least three parallel measurements. Data were processed using NGSLabSpec software, and the final spectral data for each well were processed and plotted.

[0177] The specific process is as follows: (1) Blank control: Raman probes were prepared fresh for use. In a 96-well plate, 150 μL of silver probe and 50 μL of magnetic probe were mixed together, and 0.3 μL of DMSO was added. After incubation for 40 min, Raman reporter molecules were added, and incubation was continued for another 40 min before Raman signal detection was performed.

[0178] (2) Experimental group:

[0179] Add 50 μL of magnetic probe and 0.3 μL of different small molecule compounds, and mix for 1 h. Then add 150 μL of silver probe to the wells and react for 40 min. After magnetic separation and washing three times, incubate for 40 min, add Raman reporter molecules, and continue incubation for 40 min. After magnetic separation and washing three times, perform Raman signal detection in a 96-well plate. Set the exposure time to 1 s and the number of exposures to 3, and take the average value to determine whether the compound has an inhibitory effect on binding. If the Raman signal weakens, it may have an inhibitory effect on PPI. From the compound library, select potential small molecules as candidate small molecules for subsequent experiments, and finally screen to obtain new small molecule inhibitors.

[0180] Each group was added with a final concentration of 11.2 ng / mL of the small molecule compound to be screened. After incubation with the probe for 40 minutes, Raman detection was performed. The sequence number of the small molecule that caused a significant reduction in the Raman signal was recorded. The screening results showed that some small molecules had good inhibitory effects and were potential small molecule inhibitors targeting the CA-CPSF6 interaction.

[0181] Example 11: Results of in vitro and in vivo antiviral assays of small molecules

[0182] Materials used: Unless otherwise specified, all conventional materials and consumables used in this experiment were obtained through commercial channels; the cell lines and virus strains used were obtained from authoritative resource collection institutions such as the American Type Culture Collection (ATCC).

[0183] The experimental methods described in this example are standard operating procedures in the fields of molecular biology, cell biology, and virology, and researchers in these fields should be able to understand and replicate them smoothly.

[0184] 1. Antiviral assay in MT-4 cells

[0185] Previous studies have shown that MT-4 cells are a natural target cell line for HIV. Therefore, this invention identified the inhibitory effects of a series of small molecules on HIV in this cell line. The specific procedures are as follows: A series of small molecules were prepared into final concentration gradients using cell growth medium containing 2% FBS (RPMI 1640, 2% FBS, 1% penicillin-streptomycin, 1% L-glutamine, 1% HEPES). The above-mentioned media solutions containing the compounds were added to 24-well plates (three independent replicate wells for each concentration of each compound).

[0186] At 37°C, 1 mL aliquots of MT-4 cells were infected for 4 hours with either 25 μL of cell growth medium (simulated infection) or 10 μL of fresh HIV-1NL4-3 concentrate (MOI=0.1). After 4 hours, the virus solution in the cell culture medium was discarded by centrifugation at 1000 rpm for 5 minutes. Infected and uninfected cells were resuspended and diluted in cell growth medium, and 500 μL (1×10⁻⁶) of the culture medium was added. 5 (Number) cells were added to each well of the assay plate.

[0187] The assay plate was then incubated at 37°C in a humidified 5% CO2 incubator. After 48 hours of incubation, the cell culture supernatant was collected and centrifuged at 12,000 rpm for 5 minutes to remove cell debris. 60 μL of the culture supernatant was seeded into 24-well plates, with three independent replicates for each concentration of each small molecule. 1 × 10⁻⁶ cells were seeded into each well. 5 One TZM-bl cell.

[0188] After incubating the assay plate at 37°C in a humid 5% CO2 incubator for 48 hours, discard the culture medium, add 500 μL of 0.25% trypsin to each well, and place the plate in a 37°C, 5% CO2 incubator to digest the cells for 5 minutes. Add 1 mL of complete culture medium to terminate the digestion reaction, centrifuge at 12000 rpm for 5 minutes, and harvest the TZM-bl cell pellet.

[0189] The harvested TZM-bl cell pellet was lysed with 60 μL of the cell lysis buffer from the Promega Luciferase Reporter System Kit (E1910) at room temperature for 30 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. 4 μL of the supernatant was collected and 20 μL of the substrate reaction buffer from the kit was added. The chemiluminescence was rapidly read using a GloMax 20 / 20 luminometer (Promega). EC... 50 The values ​​were calculated as the concentration of small molecules that would reduce the luminescence signal by 50%, where the luminescence signal is a measure of HIV-1 replication. Compared to LEN, the series of small molecules of this invention all showed varying degrees of antiviral activity, as shown in Table 1.

[0190] 2. Cytotoxicity test

[0191] A series of small molecule cytotoxicities and corresponding CC were determined using the protocol described in the antiviral assay. 50 The values ​​differ in that they use uninfected cells, as shown in Table 1 below.

[0192] Table 1 EC of compounds 50 and CC 50

[0193] 3. In vitro prophylaxis test

[0194] MT-4 cells, in aliquoted into 1 mL portions, were seeded into 24-well plates and incubated at 37°C in a 5% CO2 incubator. After seeding, the cells were pretreated with small molecules at different concentration gradients and then incubated at 37°C in a 5% CO2 incubator. The cells were incubated for 24 h under conditions simulating pre-exposure drug administration. Subsequently, HIV-1 NL4-3 virus was added to the cells for infection (MOI = 0.02), and the cells were incubated at 37°C and 5% C for 24 h. After incubation for another 4 hours under the specified conditions, the virus solution in the cell culture medium was discarded by centrifugation at 1000 rpm for 5 minutes. The cells were then resuspended and cultured again, and the presence of small molecules was maintained during subsequent culture.

[0195] Forty-eight hours after infection, the cell culture supernatant was collected and centrifuged at 12,000 rpm for 5 minutes to remove cell debris. 60 μL of the culture supernatant was seeded into 24-well plates, with three independent replicates for each concentration of each small molecule. 1 × 10⁶ cells were seeded into each well. 5 One TZM-bl cell.

[0196] After incubating the assay plate at 37°C in a humid 5% CO2 incubator for 48 hours, discard the culture medium, add 500 μL of 0.25% trypsin to each well, and place the plate in a 37°C, 5% CO2 incubator to digest the cells for 5 minutes. Add 1 mL of complete culture medium to terminate the digestion reaction, centrifuge at 12000 rpm for 5 minutes, and harvest the TZM-bl cell pellet.

[0197] The harvested TZM-bl cell pellet was lysed with 60 μL of the cell lysis buffer from the Promega Luciferase Reporter System Kit (E1910) at room temperature for 30 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. 4 μL of the supernatant was collected and 20 μL of the substrate reaction buffer from the kit was added. The chemiluminescence was rapidly read using a GloMax 20 / 20 luminometer (Promega). EC... 95 The values ​​are calculated as the concentration of small molecules that reduces the luminescence signal by 95%, and the luminescence signal is a measure of HIV-1 replication. Some of the small molecules in this invention exhibit antiviral activity, as shown in Table 2.

[0198] Table 2 Activity Data

[0199] 4. Antiviral assays in humanized mouse models

[0200] To further identify the anti-HIV-1 activity of the series of small molecules screened in this invention in vivo, we selected CD34. + A stem cell-derived humanized mouse model was used for testing. The specific procedures are as follows: Reagent formulation: Prepare a solvent mixture of 5% DMSO, 40% PEG300, 5% Tween80, and 50% (20% SED in saline). Dissolve LEN and the 4c ​​small molecule in the above solvent at a concentration of 2.25 mg / mL.

[0201] Laboratory animals: Eighteen female NPG-RF mice aged 4-6 weeks (a strain of severely immunodeficient NPG mice in which amino acid 831 of the c-kit protein is mutated from valine to methionine; this strain lacks T, B, and NK cells, and hematopoietic stem cell function is suppressed) were selected. At the time of infection, each animal weighed between 18.0 and 21.0 g.

[0202] Constructing a humanized mouse model: Each NPG-RF mouse was injected via tail vein with 100 μL containing 1×10 5 CD34 + Culture medium for stem cells. After 10-12 weeks, 50 μL of blood was collected from the tail vein, and the red blood cells were lysed using erythrocyte lysis buffer (BDbiosciences, #555899). The cells were stained at 4°C in the dark for 30 minutes using anti-mouse CD45 antibody, anti-human CD45 antibody, anti-human CD3 antibody, anti-human CD4 antibody, and anti-human CD8 antibody. The degree of humanization of the mice was detected by flow cytometry.

[0203] HIV infection in mice: Each mouse was injected with 100 μL of HIV-JRCSF virus (MOI=0.1) via the tail vein. Two weeks after infection, blood was collected from the submandibular region, and the viral load in the plasma was measured using an HIV viral load assay kit (DaAn Gene, DA0331). Simultaneously, CD4 counts were measured using the flow cytometry method described above (mentioned in the section on constructing humanized mouse models). + Cell proportions. Mice were divided into three groups of six mice each, based on their viral load. All viral infection experiments were performed in the ABSL-3 laboratory.

[0204] Administration: The prepared LEN, 4c small molecule solutions and solvents were administered to mice via subcutaneous injection (200 μL each). The dosage of LEN and 4c was 30 mg / kg. The negative control group received an equal volume of solvent. Treatment continued for 14 days, followed by a 14-day break, and then once every two weeks thereafter.

[0205] Sample collection: After administration, approximately 50 μL of blood was collected from each mouse per week via the submandibular region. The blood samples were collected into blood collection tubes containing 0.5 MEDTA as an anticoagulant and plasma was obtained by centrifugation at 1600 rpm for 8 minutes for viral load determination.

[0206] Determination of viral load in plasma: First, plasma RNA was extracted. 800 μL of Trizol solution was added to mouse plasma, quickly dispersed by agitation, followed by 200 μL of chloroform. The mixture was thoroughly mixed and centrifuged at 12000 × 10⁻⁶ rpm in a pre-cooled centrifuge at 4°C. g Centrifuge for 20 minutes. Transfer the supernatant to a new 1.5 mL EP tube, add an equal volume of isopropanol, and allow to precipitate for 30 minutes. Centrifuge at 12000 × 10⁻⁶ rpm in a pre-cooled centrifuge at 4°C. g Centrifuge for 20 minutes, discard the supernatant, and add 1 mL of 70% ethanol at 12000× 10000 rpm. gCentrifuge for 10 minutes. Discard the supernatant, add 30 μL of ultrapure water to the precipitate, and obtain mouse plasma RNA. Perform reverse transcription using a reverse transcription kit (Mona, MR05101M) to obtain cDNA.

[0207] According to the instructions of the Human Immunodeficiency Virus Type 1 Nucleic Acid Assay Kit (DaAn Gene, DA0331), a PCR system was prepared including standards, blank controls, positive controls, and test samples, and the PCR program was set up. Three replicates were prepared for each sample. The viral load of each mouse was obtained based on the PCR results. The small molecule 4c screened in this invention showed superior antiviral activity compared to LEN in inhibiting HIV-1 viral load in mouse plasma. The mouse plasma viral load data are summarized as follows: Figure 5 .

[0208] As can be seen from the above embodiments, the present invention provides a novel isoxazolopyrimidine derivative, a pharmaceutical composition, and its application. The present invention employs SERScreen technology, a surface-enhanced Raman spectroscopy technique amplified by a magnetic field in a homogeneous phase, to screen for small molecule inhibitors capable of inhibiting the binding of HIV CA to the host factor CPSF6. CA and CPSF6 proteins are respectively bound to Raman probes linked to Raman reporter molecules (…). Figure 1 a) and magnetic probe ( Figure 1 b) On the surface, when the two proteins bind to the magnetic probe through protein-protein interactions (PPI), the magnetic field amplifies the plasmonic coupling effect on the magnetic probe surface during excitation, resulting in a strong Raman signal from the Raman reporter molecule adsorbed on the magnetic probe under SERS. However, the presence of a small molecule inhibitor in the system hinders the PPI of the two proteins, causing the Raman probe with the reporter molecule attached to detach from the magnetic probe surface, thus resulting in a weaker Raman signal detected under SERS. Figure 1 c). This method successfully constructed a small molecule capable of screening for the interaction between CA and CPSF6, and through screening from a combinatorial compound library, a small molecule 4c that inhibits the interaction between CA and CPSF6 was obtained.

[0209] The above description is only a preferred embodiment of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of an isoxazolopyrimidine derivative in the preparation of a drug for the prevention or treatment of human immunodeficiency virus infection, characterized in that, The isoxazolopyrimidine derivative is selected from one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； 、 、 、 、 、 、 ； 、 、 、 、 、 、 、 、 ； The human immunodeficiency virus mentioned is HIV-1 virus.

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