Design, synthesis and activity evaluation of pyranopyrimidine heterocyclic p97 / VCP inhibitor

Abstract

The invention discloses a pyranopyrimidine heterocyclic p97 / VCP inhibitor as shown in the following formula, discloses a discovery process of the inhibitor, discloses a preparation method of the inhibitor, and discloses the activity of the inhibitor in tumor resistance, tumor metastasis resistance and p97 / VCP expression inhibition. Therefore, the invention discloses application of the compound in preparation of drugs for resisting tumors, resisting tumor metastasis and inhibiting p97 / VCP expression. # imgabs0 #

Description

[0001] manual

[0002] Pyranopyrimidine heterocyclic p97 / VCP inhibitors, their synthesis, activity and application Field of the Invention

[0003] The present invention relates to a pyranopyrimidine heterocyclic p97 / VCP inhibitor with the following structural formula, and relates to its preparation method, anti-tumor activity, anti-tumor metastasis activity and p97 / VCP inhibition activity. Therefore, the present invention relates to its use in preparing drugs with anti-tumor, anti-tumor metastasis and p97 / VCP expression inhibition. The present invention belongs to the field of biomedicine.

[0004] Background Art

[0005] In 2015, Han-Jie Zhou et al. further optimized the design and developed a novel p97 / VCP inhibitor CB-5083 based on the quinazoline skeleton of DBeQ. CB-5083 specifically acts on the D2 domain of p97 / VCP, induces the accumulation of polyubiquitinated proteins, blocks ERAD and induces UPR response, produces unsolvable protein toxicity stress, and finally activates cell apoptosis. CB-5083 has previously entered clinical phase I trials for the treatment of advanced solid tumors (NCT02243917) and lymphoid hematological malignancies (NCT02223598), but the off-target effect on phosphodiesterase 6 (PDE-6) caused the patient to become temporarily blind, and the trial was eventually terminated. After research, the inventors modified the pyrimidine ring to obtain 21 pyranopyrimidine heterocyclic p97 / VCP inhibitors, and obtained their anti-tumor, anti-tumor metastasis and p97 / VCP expression inhibition activities. Summary of the invention

[0006] The first research content of the present invention is to provide 21 pyranopyrimidine heterocyclic p97 / VCP inhibitors

[0007] The third aspect of the present invention is to evaluate the anti-tumor effect of pyranopyrimidine heterocyclic p97 / VCP inhibitors.

[0008] The fourth aspect of the present invention is to evaluate the anti-tumor metastasis effect of pyranopyrimidine heterocyclic p97 / VCP inhibitors.

[0009] The fifth aspect of the present invention is to evaluate the p97 inhibitory effect of pyranopyrimidine heterocyclic p97 / VCP inhibitors.

[0010] The pyranopyrimidine heterocyclic p97 / VCP inhibitor of the present invention can be prepared by the following method:

[0011]

[0012] i)ACN,rtor IPA,TEA,80℃,reflux or ACN,TEA,rt

[0013] ii) Pd 2 (dba) 3 ,XPHOS,CS 2 CO 3 ,100℃,reflux; or

[0014]

[0015] i)ACN,rtor IPA,TEA,80℃,reflux or ACN,TEA,rt

[0016] ii) dioxane, H 2 O,NaOH,(Boc) 2 O,rt

[0017] iii) Pd 2 (dba) 3 ,XPHOS,CS 2 CO 3 ,100℃,refluxiv)50%CF 3 COOH(DCM), rt; or

[0018]

[0019] i)ACN,rtor IPA,TEA,80℃,reflux or ACN,TEA,rt

[0020] ii) dioxane, H 2 O,NaOH,(Boc) 2 O,rt

[0021] iii) Pd 2 (dba) 3 ,XPHOS,CS 2 CO 3 ,100℃,refluxiv)50%CF 3 COOH(DCM), rt. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Tanswell migration assay cell staining image (S) Figure 2 Tanswell migration assay cell staining images (T, H) Figure 3 Tanswell invasion assay cell staining image (S) Figure 4 Tanswell invasion assay cell staining images (T, H) DETAILED DESCRIPTION

[0022] In order to further illustrate the present invention, a series of implementations are given below to specifically describe the present invention.

[0023] Implementation 1 Synthesis of Intermediate Compounds

[0024] (1) N-Benzyl-2-chloro-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (aa)

[0025]

[0026] Weigh 106.7 mg of 2,4-dichloro-5H,7H,8H-pyrano[4,3-d]pyrimidine (core) (0.5 mmol) in a 50 mL eggplant bottle, add 3 mL of benzylamine (compound a), yellow, add 3 mL of acetonitrile (ACN), react at room temperature, and monitor the reaction progress by TLC. After the reaction is completed, ACN is removed by rotation, and purified by PE / EA column chromatography to obtain 136.0 mg of light yellow solid (98.33%). ESI-MS (m / e): 276 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=7.73(t,J=5.6Hz,1H),7.47-7.11(m,5H),4.56(d,J=5.8Hz,2H),4.44(s,2H),3.88(t,J=5.5Hz,2H),2.62(t,J=5.2Hz,2H).

[0027] (2) 2-Chloro-N-phenylethyl-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (ba)

[0028]

[0029] Weigh 205.0 mg (1 mmol) of core into a 50 mL eggplant bottle, add 3 mL of phenylethylamine (compound b), it turns yellow, add 3 mL of ACN, react at room temperature, and monitor the reaction progress by TLC. After the reaction is completed, spin off ACN, and purify by PE / EA column chromatography to obtain 227.0 mg of light yellow solid (78.54%). ESI-MS (m / e): 290 [M+H] + ; 1H NMR (300 MHz, DMSO-d 6 )δ / ppm=7.26(dt,J=12.7,7.3Hz,1H),4.35(s,0H),3.86(t,J=5.5Hz,0H),3.53(d d,J=13.3,6.9Hz,2H),3.38(s,1H),2.83(t,J=7.4Hz,2H),2.61(t,J=5.3Hz,2H).

[0030] (3) 2-Chloro-N-(naphthalen-2-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (ca)

[0031]

[0032] Weigh 1.0 g (5.0 mmol) of core into a 100 mL eggplant bottle, add 1.0 g (6.0 mmol) of β-naphthylamine (compound c), which is red-yellow, add 50 mL of isopropanol (IPA), add 2.7 mL (20.0 mmol) of triethylamine (Et 3 N), heated at 80°C, and the reaction progress was monitored by TLC. After the reaction was completed, IPA was removed by rotation and purified by PE / EA column chromatography to obtain 1.2 g of light yellow solid (79.36%). ESI-MS (m / e): 312 [M+H] + ; 623[2M+H] + , 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.96(s,1H),8.07(s,1H),7.96-7.81(m,3H),7.72(dd,J=8.9,1.9Hz,1H),7.5 7-7.36(m,2H),4.67(s,2H),3.94(t,J=5.6Hz,2H),3.36(s,3H),2.74(t,J=5.4Hz,2H).

[0033] (4) N-([1,1'-biphenyl]-3-yl)-2-chloro-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (da)

[0034]

[0035] Weigh 100.0 mg (0.5 mmol) of core into a 50 mL eggplant bottle, add 99.0 mg (0.6 mmol) of 3-aminobiphenyl (compound d), which turns yellow, add 10 mL of IPA, add 270 μL (2.0 mmol) of triethylamine (Et 3N), heated at 80°C for reaction, and monitored the reaction progress by TLC. After the reaction was completed, IPA was removed by rotation and purified by PE / EA column chromatography to obtain 138.0 mg of light yellow solid (84.15%). ESI-MS (m / e): 338 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.81(s,1H),7.88(s,1H),7.64(t,J=8.7Hz,3H),7.44(ddt,J=21.9, 14.4,7.3Hz,5H),4.65(s,2H),3.93(d,J=5.6Hz,2H),2.73(t,J=5.5Hz,2H).

[0036] (5) 2-Chloro-N-(thiophen-3-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (ea)

[0037]

[0038] Weigh 100.0 mg (0.5 mmol) of core into a 50 mL eggplant bottle, add 111.0 mg (0.6 mmol) of 3-aminothiophene oxalate (compound e), which is yellow, add 10 mL of IPA, add 270 μL (2.0 mmol) of triethylamine (Et 3 N), heated at 80°C for reaction, and monitored the reaction progress by TLC. After the reaction was completed, IPA was removed by rotation and purified by PE / EA column chromatography to obtain 114.0 mg of light yellow solid (87.69%). ESI-MS (m / e): 268 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=9.10(s,1H),7.65(dd,J=3.2,1.3Hz,1H),7.48(dd,J=5.2,3.2Hz,1H),7.3 3(dd,J=5.2,1.3Hz,1H),4.60(s,2H),3.92(t,J=5.7Hz,2H),2.71(t,J=5.6Hz,2H).

[0039] (6) 2-Chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (fa)

[0040]

[0041] Weigh 1.0 g (5.0 mmol) of core into a 100 mL eggplant bottle, add 940 μL (6.0 mmol) of 4-aminomethyltetrahydropyran (compound f), which is light yellow, add 50 mL of IPA, add 2 mL (15.0 mmol) of triethylamine (Et 3 N), react at room temperature, and monitor the reaction progress by TLC. After the reaction is completed, IPA is removed by rotation and purified by PE / EA column chromatography to obtain 1.2 g of light yellow solid (84.88%). ESI-MS (m / e): 284 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=7.12(s,1H),4.39(s,2H),3.85(dd,J=11.2,6.1Hz,4H),3.27(dd,J=22.5,10.0Hz,4H), 2.60(s,2H),1.82(dd,J=9.1,5.4Hz,1H),1.57(d,J=12.6Hz,2H),1.17(qd,J=12.3,4.0Hz,2H).

[0042] (7) 2-Chloro-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (ga)

[0043]

[0044] Weigh 1.0 g (5.0 mmol) of core into a 100 mL eggplant bottle, add 1.1 mL (7.5 mmol) of 4-(2-aminoethyl)tetrahydropyran (compound g), which turns light yellow, add 50 mL of IPA, add 2 mL (15.0 mmol) of triethylamine (Et 3 N), react at room temperature, and monitor the reaction progress by TLC. After the reaction is completed, IPA is removed by rotation and purified by PE / EA column chromatography to obtain 1.2 g of light yellow solid (85.68%). ESI-MS (m / e): 298 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=7.08(s,1H),4.37(s,2H),3.85(dd,J=14.1,8.6Hz,4H),3.43-3.17(m,5H),2.60(s,2H),1.54(dd,J=46.6,9.7Hz,5H),1.15(dd,J=20.6,8.7Hz,2H). Synthesis of 2S series compounds

[0045] (1) N2, N4-diphenylmethyl-7, 8-dihydro-5H-pyrano[4, 3-d]pyrimidine-2, 4-diamine (Sa)

[0046]

[0047] 1mL compound a and 2.0g (5.4mmol) CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 173.0 mg (0.54 mmol) of X-Phos, 333.0 mg (0.54 mmol) of Pd 2 (dba) 3 and 500.0 mg (1.8 mmol) of compound aa were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound aa disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 401.2 mg of light yellow solid (63.89%). HPLC: 99.66%; mp136.2-136.7°C; IR (cm -1 ):3439,3237,3056,2958,2827,1592,1537,1514,1462,1449,1411,1397,1353,12 35,1144,1093,1026,985,948,864,782,740,715,693,604; ESI-MS(m / e):347[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.21(s,1H),7.23(d,J=5.6Hz,30H),4.52(d,J=4.9Hz,5H),4.39(d,J=10.0Hz,10H),3.81(d,J=4.9Hz,5H).

[0048] (2) N2,N4-diphenylethyl-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine-2,4-diamine (Sb)

[0049]

[0050] 1mL compound b and 2.3g (6.9mmol) CS 2 CO 3Dissolve in ACN and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 335.2 mg (0.69 mmol) of X-Phos, 650.3 mg (0.69 mmol) of Pd 2 (dba) 3 and 664.0 mg (2.3 mmol) of compound ba were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ba disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 647.2 mg of a yellow solid (75.31%). HPLC: 93.61%; mp 108.1-109.2°C; IR (cm -1 ):3433,3248,3169,3083,3020,2963,2842,1599,1573,1518,1495,1464,137 4,1352,1298,1146,1098,975,870,783,747,724,694; ESI-MS(m / e):375[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.22(s,1H),7.23(d,J=11.2Hz,6H),4.31(s,1H),3.82(t,J=5.1Hz,1H),3.52(dd,J=15.9,7.6Hz,2H),2.85(dd,J=14.7,7.2Hz,2H).

[0051] (3) N2, N4-di(naphthalene-2-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine-2,4-diamine (Sc)

[0052]

[0053] Weigh 63.0 mg (0.4 mmol) of compound c and 350.0 mg (0.9 mmol) of CS 2 CO 3 Dissolve in ACN and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 30.0 mg (0.1 mmol) of X-Phos, 60.0 mg (0.1 mmol) of Pd 2 (dba) 3and 100.0 mg (0.3 mmol) of compound ca were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ca disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 70.0 mg of a red-yellow solid (51.85%). HPLC: 93.24%; mp 174.9-176.2°C; IR (cm -1 ):3433,3306,3051,2927,1722,1632,1574,1537,1493,1428,1398,1372, 1233,1214,1101,978,846,808,774,743,697,618; ESI-MS(m / e):419[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=9.43(s,1H),8.52(s,1H),8.31(s,1H),8.21(s,1H),7.92(d,J=8.5Hz,3H),7.84-7.60(m, 7H),7.46(d,J=2.8Hz,3H),7.29-7.10(m,3H),6.97(s,2H),4.70(s,3H),3.98(s,3H),2.72(s,3H).

[0054] (4) N2,N4-di([1,1'-biphenyl]-3-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine-2,4-diamine (Sc)

[0055]

[0056] Weigh 73.0 mg (0.48 mmol) of compound d and 348.0 mg (1.05 mmol) of CS 2 CO 3 Dissolve in ACN and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 34.0 mg (0.1 mmol) of X-Phos, 66.0 mg (0.1 mmol) of Pd 2 (dba) 3and 120.0 mg (0.35 mmol) of compound da were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound da disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 107.0 mg of a red-yellow solid (62.94%). HPLC: 95.96%; mp110.0-111.5; IR (cm -1 ):3389,3028,2926,2850,1716,1572,1519,1475,1445,1406,1384,1307,1239,1211,1100,981,860,753,694,614; ESI-MS(m / e):471[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=9.22(s,1H),8.29(s,0H),7.90(s,1H),7.72(s,2H),7.57(d,J=7.5Hz ,2H),7.47-7.23(m,11H),7.06(s,2H),4.65(s,2H),3.95(s,2H),2.68(s,2H).

[0057] (5) N2,N4-di(thiophen-3-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine-2,4-diamine (Se)

[0058]

[0059] Weigh 553.0 mg (0.48 mmol) of compound e and 2.4 g (7.2 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 223.0 mg (0.72 mmol) of X-Phos, 446.0 mg (0.72 mmol) of Pd 2 (dba) 3and 640.0 mg (2.4 mmol) of compound ea were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ea disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 395.0 mg of a yellow solid (81.95%). HPLC: 97.96%; mp81.6-82.2°C; IR (cm -1 ):3343,3097,2929,1715,1589,1538,1505,1449,1428,1414,1382,1332,124 8,1217,1159,1098,984,952,861,836,764,621,517; ESI-MS(m / e):331[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=9.43(s,1H),8.55(s,1H),7.90(s,1H),7.59-7.27(m,4H),7.15(d, J=4.7Hz,1H),4.58(s,2H),3.92(d,J=5.1Hz,2H),2.67(s,2H),1.99(s,1H).

[0060] (6) N2,N4-di((tetrahydro-2H-pyran-4-yl)methyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine-2,4-diamine (Sf)

[0061]

[0062] Take 55 μL (0.42 mmol) of compound f and weigh 346 mg (1.05 mmol) of CSbCO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 34.0 mg (0.1 mmol) of X-Phos, 65.0 mg (0.1 mmol) of Pd 2 (dba) 3and 100.0 mg (0.35 mmol) of compound fa were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound fa disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 64.0 mg of light yellow solid (50.00%). HPLC: 95.91%; mp152.6-153.1°C; IR (cm -1 ):3364,32775,2913,1838,1591,1514,1464,1421,1382,1360,1288,1230,1088,869,787,650; ESI-MS(m / e):363[M+H] + : 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=6.35(d,J=24.9Hz,1H),4.30(s,1H),3.81(dd,J=14.2,8.6Hz,4H),3.32-3.18(m,3H),3.08(t,J=6.4H z,1H),2.41(t,J=5.5Hz,1H),1.88-1.72(m,1H),1.56(d,J=12.7Hz,2H),1.17(ddd,J=17.4,12.3,4.7Hz,2H).

[0063] (7) N2,N4-di(2-(tetrahydro-2H-pyran-4-yl)ethyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine-2,4-diamine (Sg)

[0064]

[0065] Take 400 μL (2.2 mmol) of compound g and weigh 1.8 g (5.4 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 174.0 mg (0.54 mmol) of X-Phos, 334.0 mg (0.54 mmol) of Pd 2 (dba) 3and 540.0 mg (1.8 mmol) of compound ga were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ga disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to finally obtain 290.9 mg of light yellow solid (40.88%). HPLC: 96.35%; mp150.6-151.3°C; IR (cm -1 ):3335,3254,2921,2842,2756,1593,1548,1519,1464,1417,1369,1233,1090,977,861,815,785,700,596; ESI-MS(m / e):391[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=6.14(s,1H),4.29(s,1H),3.81(d,J=6.3Hz,4H),3.44-3.12(m,6H),2.41(s,1H),1.66-1.34(m,7H),1.16(d,J=4.6Hz,3H).

[0066] Implementation of the synthesis of 3T series compounds

[0067] (1) tert-Butyl (2-(1H-indol-3-yl)ethyl)carbamate (BT)

[0068]

[0069] Weigh 2.0 g (12.5 mmol) of tryptamine in a 200 mL eggplant bottle, add 20 mL of UP water, 10 mL of dioxane and 10 mL of 2N sodium hydroxide solution to obtain reaction solution A, weigh 3.3 g (15.0 mmol) (Boc) 2 O in a small beaker, add 20 mL of dioxane to dissolve to obtain reaction solution B, mix reaction solution B with reaction solution A, pH of the solution is 9, react at room temperature. TLC monitors the reaction progress. After the reaction is completed, saturated KHSO 4 Adjust pH to 7, concentrate to dryness under reduced pressure, add UP water, saturated KHSO 4 Adjust pH = 2, extract with a large amount of ethyl acetate (EA), take the EA layer, anhydrous Na 2 SO 4 Dry, filter and collect the filtrate, and concentrate to dryness under reduced pressure to obtain 3.0 g of brown solid (90.97%). ESI-MS (m / e): 278 [M+H]+ ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=10.77(s,1H),7.50(d,J=7.8Hz,1H),7.32(d,J=8.1Hz,1H),7.11(s,1H),7.00(dt,J=14.7,7.1Hz,2H ),6.86(t,J=5.4Hz,1H),3.36(d,J=6.8Hz,1H),3.19(dd,J=14.2,6.6Hz,2H),2.84-2.75(m,2H),1.37(s,9H).

[0070] (2) tert-Butyl (2-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTa)

[0071]

[0072] Weigh 300.0 mg (1.2 mmol) of compound BT and 1.0 g (3.0 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 260.0 mg (0.2 mmol) of X-Phos, 754.0 mg (0.2 mmol) of Pd 2 (dba) 3 and 280.0 mg (1.0 mmol) of compound aa were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound aa disappeared, post-treatment was performed: hot filtration, DCM was eluted for multiple times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by PE / EA column chromatography, and concentrated to dryness under reduced pressure to obtain 648.0 mg of light yellow solid BTa (90.00%). ESI-MS (m / e): 500 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.28(d,J=1.1Hz,1H),7.73(t,J=5.8Hz,1H),7.53(s,1H),7.34(dt,J=12.9,7.3Hz,5H),7.26-7.18(m,1H),6.85(t,J=5.5Hz,1H),4 .64(d,J=5.7Hz,2H),4.50(s,2H),3.92(t,J=5.6Hz,2H),3.18(dd,J=13.1,6.9Hz,2H),2.63(dt,J=14.5,6.3Hz,4H),1.36(d,J=6.3Hz,10H).

[0073] (3) 2-(3-(2-aminoethyl)-1H-indol-1-yl)-N-benzyl-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Ta)

[0074]

[0075] Weigh 600.0 mg (1.5 mmol) BTa into a 50 mL eggplant bottle and add 10 mL 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BTa: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain 520.0 mg of light yellow solid Ta (80.00%). HPLC: 97.91%; mp 116.5-117.9℃; IR (cm -1 ):3267,2922,1580,1521,1494,1451,1421,1403,1355,1286,1101,873,779,743,698; ESI-MS(m / e):400[M+H] + ; 1 H NMR(300MHz, DMSO-d6)δ / ppm=8.47(d,J=5.2Hz,1H),7.96(s,1H),7.67-7.51(m,2H),7.47-7.29(m,5H),7.28-7.18(m,2H),7.16-7.09(m ,2H),6.92(d,J=9.9Hz,2H),4.72(d,J=5.0Hz,2H),4.56(s,1H),3.96(s,2H),3.23(d,J=6.1Hz,2H),2.82(t,J=7.0Hz,2H),2.73(s,2H).

[0076] (4) tert-Butyl (2-(1-(4-(phenylethylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTb)

[0077]

[0078] Weigh 1.1 g (4.2 mmol) of compound BT and 3.8 g (10.5 mmol) of CS 2 CO 3 Dissolve in ACN and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 335.0 mg (0.7 mmol) of X-Phos, 645.0 mg (0.7 mmol) of Pd 2 (dba) 3 and 1.0 g (3.5 mmol) of compound ba were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ba disappeared, post-treatment was performed: hot filtration, DCM was used to rinse the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and the solid was purified by PE / EA column chromatography and concentrated to dryness under reduced pressure to obtain 1.8 g of light yellow solid BTb (91.00%). ESI-MS (m / e): 514 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.76(d,J=7.7Hz,1H),8.08(s,1H),7.60(d,J=6.6Hz,1H),7.32(t,J=8.1Hz,4H),7.25-7.15(m,3H),7.10(s,1H) ,6.93(s,1H),4.44(s,2H),3.93(s,2H),3.72(s,2H),3.03-2.93(m,2H),2.86(t,J=6.8Hz,2H),2.72(s,2H),1.38(s,9H).

[0079] (5) 2-(3-(2-aminoethyl)-1H-indol-1-yl)-N-phenethyl-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Tb)

[0080]

[0081] Weigh 500.0 mg (1.0 mmol) BTb into a 50 mL eggplant bottle and add 10 mL 50% CF 3COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BTb: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain 421.0 mg of light yellow solid Tb (84.00%). HPLC: 93.26%; mp 139.1-140.5℃; IR (cm -1 ):3298,2924,2603,2496,1582,1523,1496,1449,1398,1356,1287,1069,874,779,742,697,519; ESI-MS(m / e):414[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.77(d,J=6.9Hz,1H),8.11(s,1H),7.64(d,J=6.1Hz,1H),7.41-7.08( m,9H),4.45(s,3H),3.93(s,2H),3.71(s,2H),3.03-2.95(m,4H),2.72(s,1H).

[0082] (6) tert-Butyl (2-(1-(4-(naphthalen-2-ylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTc)

[0083]

[0084] Weigh 380.0 mg (1.4 mmol) of compound BT and 724.0 mg (2.1 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 70.6 mg (2.1 mmol) of X-Phos, 135.8 mg (2.1 mmol) of Pd 2 (dba) 3 and 230.0 mg (0.7 mmol) of compound ca were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ca disappeared, post-treatment was performed: hot filtration, DCM was used to rinse the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by PE / EA column chromatography, and concentrated to dryness under reduced pressure to obtain 326.5 mg of light yellow solid BTc (82.28%). ESI-MS (m / e): 536 [M+H] + ; 1H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.84(s,1H),8.49(d,J=8.4Hz,1H),8.22(s,1H),8.04-7.93(m,3H),7.87(d,J=8.2Hz,1H),7.77(dd,J=8.8,2.1Hz,1H),7.58-7.44( m,4H),7.11(t,J=7.1Hz,1H),6.99-6.86(m,2H),4.74(s,2H),4.02(t,J=5.6Hz,2H),3.26-3.21(m,2H),2.83(d,J=3.8Hz,3H),1.36(s,8H).

[0085] (7) 2-(3-(2-aminoethyl)-1H-indol-1-yl)-N-(naphthalen-2-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Tc)

[0086]

[0087] Weigh 283.0 mg (0.5 mmol) of BTc into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BTc: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times, and obtain 220.0 mg of light yellow solid Tc (95.57%). HPLC: 99.20%; mp 159.1-160.0℃; IR (cm -1 ):2924,1681,1572,1499,1524,1454,1428,1396,1340,1219,1176,1143,1101,1016,984,874,854,775,742; ESI-MS(m / e):436[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.85(s,1H),8.51(d,J=8.3Hz,1H),8.22(s,1H),7.97(dd,J=16.5,7.9Hz,4H),7.86(d,J=7.9Hz,2H),7.77(dd,J=8.8,1.8Hz, 1H),7.51(dt,J=16.6,7.6Hz,4H),7.10(t,J=7.4Hz,2H),6.91(t,J=7.7Hz,1H),4.74(s,2H),4.01(t,J=5.4Hz,3H),2.95-2.77(m,8H).

[0088] (8) tert-Butyl (2-(1-(4-([1,1'-biphenyl]-3-ylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTd)

[0089]

[0090] Weigh 214.0 mg (0.8 mmol) of compound BT and 400.0 mg (1.2 mmol) of CS 2 CO 3 Dissolve in ACN and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 39.0 mg (0.12 mmol) of X-Phos, 75.0 mg (0.12 mmol) of Pd 2 (dba) 3 and 138.0 mg (0.4 mmol) of compound da were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound da disappeared, post-treatment was performed: hot filtration, DCM was used to rinse the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by PE / EA column chromatography, and concentrated to dryness under reduced pressure to obtain 206.0 mg of light yellow solid BTd (77.44%). ESI-MS (m / e): 562 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.70(s,1H),8.49(d,J=8.3Hz,1H),7.97(s,2H),7.74-7.67(m,3H),7.6 1(dt,J=7.4,1.8Hz,1H),7.49(ddd,J=14.8,10.6,7.5Hz,6H),7.40-7.34(m,1H) ,7.08(t,J=7.1Hz,1H),6.91(dd,J=9.8,5.7Hz,3H),4.72(s,2H),4.01(t,J=5.6 Hz,2H),3.18(dd,J=13.1,6.8Hz,3H),2.88-2.76(m,5H),1.38(d,J=9.7Hz,13H).

[0091] (9) N-([1,1'-biphenyl]-3-yl)-2-(3-(2-aminoethyl)-1H-indol-1-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Td)

[0092]

[0093] Weigh 132.0 mg (0.3 mmol) BTd into a 50 mL eggplant bottle and add 10 mL 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BTd: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain 128.9 mg of light yellow solid Td (97.30%). HPLC: 99.67%; mp 139.4-140.0℃; IR (cm -1 ):3426,2927,1678,1572,1502,1524,1456,1446,1409,1382,1341,1182,1125,873,749,721,696,588; ESI-MS(m / e):462[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.74(s,1H),8.50(s,1H),7.99(s,2H),7.70(s,2H),7.46(d,J=30. 5Hz,7H),7.09(s,1H),6.93(s,1H),4.73(s,2H),4.02(s,1H),2.84(s,3H).

[0094] (10) tert-Butyl (2-(1-(4-(thiophen-3-ylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTe)

[0095]

[0096] Weigh 395.0 mg (1.5 mmol) of compound BT and 988.0 mg (3.0 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 95.0 mg (0.2 mmol) of X-Phos, 186.0 mg (0.2 mmol) of Pd 2 (dba) 3 and 270.0 mg (1.0 mmol) of compound ea were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ea disappeared, post-treatment was performed: hot filtration, DCM was eluted for multiple times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by PE / EA column chromatography, and concentrated to dryness under reduced pressure to obtain 100.0 mg of brown-yellow solid BTe (80.65%). ESI-MS (m / e): 492 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.88(s,0H),8.62(dd,J=5.9,3.3Hz,1H),7.99(s,1H),7.66-7.51(m,2H),7.35(dd,J=5.2,1.3Hz,1H),7.22-7.12(m, 1H), 6.93 (t, J = 5.4Hz, 0H), 4.66 (s, 2H), 3.99 (t, J = 5.6Hz, 1H), 3.28-3.20 (m, 1H), 2.87-2.77 (m, 2H), 1.38 (d, J = 10.5Hz, 5H).

[0097] (11) 2-(3-(2-aminoethyl)-1H-indol-1-yl)-N-(thiophen-3-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Te)

[0098]

[0099] Weigh 294.0 mg (0.5 mmol) BTe into a 50 mL eggplant bottle and add 10 mL 50% CF 3COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the compound BTe disappears: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times, and obtain 216.1 mg of light yellow solid Te (92.27%). HPLC: 99.22%; mp 179.9-180.9℃; IR (cm -1 ):3261,2924,2851,1589,1550,1518,1455,1417,1398,1294,1222,1099,746,618; ESI-MS(m / e):392[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=9.02(s,0H),8.62(d,J=6.9Hz,1H),8.00(s,1H),7.74-7.50(m,2H),7.45-7. 32(m,1H),7.27-7.09(m,1H),4.68(s,2H),3.99(t,J=5.3Hz,2H),2.96-2.77(m,6H).

[0100] (12) tert-Butyl (2-(1-(4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTf)

[0101]

[0102] Weigh 670.0 mg (2.6 mmol) of compound BT and 1.7 g (5.1 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 170.0 mg (3.4 mmol) of X-Phos, 324.0 mg (3.4 mmol) of Pd 2 (dba) 3 and 500.0 mg (1.7 mmol) of compound fa were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound fa disappeared, post-treatment was performed: hot filtration, DCM was used to rinse the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and the solid was purified by PE / EA column chromatography and concentrated to dryness under reduced pressure to obtain 857.0 mg of light yellow solid BTf (97.799%). ESI-MS (m / e): 508 [M+H] + ; 1H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.73(d,J=8.1Hz,0H),8.05(s,1H),7.60(d,J=7.3Hz,1H),7.31-7.10(m,1H ),7.02(t,J=5.6Hz,1H),6.90(t,J=5.3Hz,0H),4.47(s,2H),3.97-3.83(m,2H),3.41 (t,J=6.1Hz,1H),3.25(dd,J=13.7,8.2Hz,2H),2.85(t,J=7.1Hz,1H),2.71(t,J=5.4 Hz,1H),2.01(dt,J=10.9,4.7Hz,1H),1.68(d,J=11.4Hz,1H),1.39(d,J=5.5Hz,5H).

[0103] (13) 2-(3-(2-aminoethyl)-1H-indol-1-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Tf)

[0104]

[0105] Weigh 800.0 mg (0.5 mmol) BTf into a 50 mL eggplant bottle and add 10 mL 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BTf: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times, and obtain 710.0 mg of light yellow solid Tf (86.48%). HPLC: 99.95%; mp 128.8-129.7℃; IR (cm -1 ):3290,2920,2847,1681,1582,1522,1450,1406,1364,1232,1138,1090,1015,982,874,849,779,743:ESI-MS(m / e):408[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.75(d,J=8.1Hz,1H),8.10(s,1H),7.64(d,J=7.2Hz,1H),7.34-7.09 (m,3H),5.07(s,3H),4.48(s,2H),4.00-3.84(m,2H),3.41(t,J=6.2Hz,1H),3 .29-3.17(m,1H),3.09-2.93(m,2H),2.71(t,J=5.4Hz,1H),2.02(ddd,J=11.1 ,7.4,4.0Hz,0H),1.69(d,J=11.1Hz,1H),1.28(ddd,J=24.7,12.2,4.1Hz,1H).

[0106] (14) tert-Butyl (2-(1-(4-((2-(tetrahydro-2H-pyran-4-yl)ethyl)amino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-indol-3-yl)ethyl)carbamate (BTg)

[0107]

[0108] Weigh 657.0 mg (2.1 mmol) of compound BT and 1.6 g (4.2 mmol) of CS 2 CO 3 Dissolve in ACN and reflux at 100°C for 30 min to obtain reaction solution A; weigh 160.0 mg (2.8 mmol) of X-Phos, 308.0 mg (2.8 mmol) of Pd 2 (dba) 3 and 427.0 mg (1.4 mmol) of compound ga were suspended with ACN to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ga disappeared, post-treatment was performed: hot filtration, DCM was used to rinse the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by PE / EA column chromatography, and concentrated to dryness under reduced pressure to obtain 732.0 mg of light yellow solid BTg (97.73%). ESI-MS (m / e): 522 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.34(d,J=1.1Hz,1H),7.57(s,1H),7.06(t,J=5.3Hz,1H),6.86(t,J=5.5Hz,1H),4.42(s,2H),4.01-3.77(m,2H),3 .49-3.40(m,1H),3.37-3.10(m,3H),2.64(dd,J=14.1,6.5Hz,2H),1.69-1.48(m,3H),1.43-1.35(m,5H),1.31-1.12(m,1H).

[0109] (15) 2-(3-(2-aminoethyl)-1H-indol-1-yl)-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Tg)

[0110]

[0111] Weigh 730.0 mg (0.5 mmol) BTg into a 50 mL eggplant bottle and add 10 mL 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BTg: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times, and obtain 626.0 mg of light yellow solid Tg (83.58%). HPLC: 99.87%; mp119.9-120.8℃; IR (cm -1 ):3321,2924,2850,1678,1584,1524,1451,1407,1369,1199,1129,1093,1015,977,874,798,744,720; ESI-MS(m / e):422[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.76(d,J=7.7Hz,1H),8.10(d,J=21.4Hz,1H),7.72-7.57(m,1H) ,7.24(dtd,J=17.2,7.3,1.1Hz,2H),7.07(t,J=5.5Hz,1H),4.46(s,2H),4 .00-3.79(m,2H),3.58-3.47(m,1H),3.30(t,J=11.0Hz,1H),3.09(dd,J=1 3.9,6.1Hz,2H),2.75-2.55(m,2H),1.65-1.55(m,2H),1.31-1.14(m,1H).

[0112] Implementation of the synthesis of 4H series compounds

[0113] (1) tert-Butyl (2-(1H-imidazol-4-yl)ethyl)carbamate (BH)

[0114]

[0115] Weigh 500.0 mg (4.5 mmol) of histamine in a 100 mL eggplant bottle, add 10 mL of UP water, 50 mL of dioxane and 50 mL of 2N sodium hydroxide solution to obtain reaction solution A, weigh 1.2 g (5.4 mmol) (Boc) 2 O in a small beaker, add 10 mL of dioxane to dissolve to obtain reaction solution B, mix reaction solution B with reaction solution A, pH of the solution is 9, react at room temperature. TLC monitors the reaction progress. After the reaction is completed, saturated KHSO 4 Adjust pH=7, concentrate to dryness under reduced pressure, add a large amount of ethyl acetate (EA), filter and collect the filtrate, anhydrous Na 2 SO 4 The mixture was dried and concentrated to dryness under reduced pressure to obtain 876.0 mg of a white solid (92.21%). ESI-MS (m / e): 212

[0116] [M+H] + ,423[2M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=11.77(s,1H),7.50(s,1H),6.85(s,2H),3.35(s,1H),3.19-3.06(m,2H),2.59(t,J=6.8Hz,2H),1.37(s,9H).

[0117] (2) tert-Butyl (2-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHa)

[0118]

[0119] Weigh 230.0 mg (1.1 mmol) of compound BH and 886.0 mg (2.7 mmol) of CS 2 CO 3 Dissolve in IPA and reflux at 100°C for 30 min to obtain reaction solution A; weigh 113.0 mg (1.8 mmol) of BINAP, 166.0 mg (1.8 mmol) of Pd 2 (dba)3 and 250.0 mg (0.9 mmol) of compound aa were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound aa disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 326.5 mg of light yellow solid BHa (82.28%). ESI-MS (m / e): 451 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.28(d,J=1.2Hz,1H),7.73(t,J=5.8Hz,1H),7.53(s,1H),7.41-7.3 5(m,2H),7.35-7.27(m,3H),7.22(ddd,J=7.1,3.8,1.4Hz,1H),6.85(t,J=5.5 Hz,1H),4.64(d,J=5.7Hz,2H),4.50(s,2H),3.92(t,J=5.6Hz,2H),3.18(dd,J =13.1,6.9Hz,2H),2.67(t,J=5.5Hz,2H),2.60(t,J=7.3Hz,2H),1.37(s,9H).

[0120] (3) 2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-N-benzyl-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Ha)

[0121]

[0122] Weigh 450.0 mg (1.0 mmol) of BHa into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after compound BHa disappears: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a light yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 300.0 mg of light yellow solid Ha (88.50%). HPLC: 94.02%; mp 157.7-140.7°C; IR (cm -1):3226,3144,3061,2924,2854,1602,1533,1484,1431,1349,1282,1140,1112,1039,878,778,695,618; ESI-MS(m / e):351[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.29(d,J=1.0Hz,0H),7.79(t,J=5.8Hz,0H),7.55(s,0H),7.42-7.36(m,1H),7.31(dd,J=8.1,6.7Hz,1H),7.22(ddd,J =7.1,3.8,1.4Hz,1H),4.64(d,J=5.7Hz,2H),4.51(s,2H),3.92(t,J=5.6Hz,2H),2.89(t,J=7.1Hz,2H),2.65(t,J=6.0Hz,3H).

[0123] (4) tert-Butyl (2-(1-(4-(phenylethylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHb)

[0124]

[0125] Weigh 584.0 mg (2.8 mmol) of compound BH and 1.3 g (4.2 mmol) of CS 2 CO 3 Dissolve in IPA and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 172.0 mg (0.28 mmol) of BINAP, 253.0 mg (0.28 mmol) of Pd 2 (dba) 3 and 400.0 mg (1.4 mmol) of compound ba were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ba disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 511.0 mg of light yellow solid BHb (79.60%). ESI-MS (m / e): 465 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.39(d,J=1.0Hz,1H),7.59(s,1H),7.35-7.15(m,7H),6.87(t,J=5.4Hz,1H),4.40(s,2H),3.90(t, J=5.6Hz,2H),3.71-3.56(m,2H),3.24-3.15(m,2H),2.96-2.79(m,3H),2.66(t,J=5.8Hz,4H),1.37(s,9H).

[0126] (5) 2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-N-phenethyl-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Hb)

[0127]

[0128] Weigh 311.0 mg (0.6 mmol) of BHb into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after compound BHb disappears: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 200.0 mg of light yellow solid Hb (82.30%). HPLC: 94.00%; mp 137.0-138.0℃; IR (cm -1 ):3259,3162,3025,2967,2938,2865,1606,1529,1454,1419,1354,1283,1144,981,880,749; ESI-MS(m / e):365[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.36(s,1H),7.58(s,1H),7.42-7.08(m,6H),4.41(s,2H),3.90(t,J=5.5Hz ,2H),3.64(dd,J=13.9,6.3Hz,2H),2.88(dd,J=11.1,7.3Hz,4H),2.76-2.58(m,4H).

[0129] (6) tert-Butyl (2-(1-(4-(naphthalen-2-ylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHc)

[0130]

[0131] Weigh 474.0 mg (2.2 mmol) of compound BH and 1.1 g (3.3 mmol) of CS 2 CO 3 Dissolve in IPA and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 140.0 mg (0.22 mmol) of BINAP, 205.0 mg (0.22 mmol) of Pd 2 (dba) 3 and 350.0 mg (1.1 mmol) of compound ca were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ca disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 490.0 mg of light yellow solid BHc (89.74%). ESI-MS (m / e): 487 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.84(s,1H),8.49(d,J=8.4Hz,1H),8.22(s,1H),7.97(dd,J=18.3 ,9.3Hz,3H),7.87(d,J=8.2Hz,1H),7.77(dd,J=8.8,2.1Hz,1H),7.5-7.43 (m,3H),7.11(t,J=7.1Hz,1H),6.98-6.86(m,2H),4.74(s,2H),4.02(t,J= 5.6Hz, 2H), 3.24 (dd, J=13.0, 6.7Hz, 2H), 2.90-2.76 (m, 4H), 1.36 (s, 8H).

[0132] (7) 2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-N-(naphthalen-2-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Hc)

[0133]

[0134] Weigh 327.0 mg (1.0 mmol) of BHc into a 50 mL eggplant bottle and add 10 mL of 50% CF 3COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after compound BHc disappears: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 209.0 mg of light yellow solid Hc (80.69%). HPLC: 96.15%; mp 135.8-136.5℃; IR (cm -1 ):2930,1584,1429,1404,1289,1174,1131,1047,986,880,773,742,613; ESI-MS(m / e):387[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.29(d,J=0.9Hz,1H),8.19(d,J=1.7Hz,1H),7.94-7.82(m,4H),7.55-7.37(m,3H),4 .72(s,2H),3.98(t,J=5.5Hz,3H),2.90(s,1H),2.79(t,J=5.3Hz,2H),2.67(t,J=7.1Hz,2H).

[0135] (8) tert-Butyl (2-(1-(4-([1,1'-biphenyl]-3-ylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHd)

[0136]

[0137] Weigh 500.0 mg (2.4 mmol) of compound BH and 1.2 g (3.6 mmol) of CS 2 CO 3 Dissolve in IPA and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 147.0 mg (0.24 mmol) of BINAP, 217.0 mg (0.24 mmol) of Pd 2 (dba) 3and 400.0 mg (1.2 mmol) of compound da were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound da disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 479.0 mg of light yellow solid BHd (78.40%). ESI-MS (m / e): 513 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.75(s,1H),8.26(d,J=1.1Hz,1H),8.00(s,1H),7.75-7.66(m,3H),7.51(dt,J=7.9,4.1Hz,4H),7.46-7.36(m,2H),6.86(t,J= 5.6Hz,1H),4.70(s,2H),3.98(t,J=5.6Hz,2H),3.18(dd,J=13.3,6.9Hz,2H),2.78(t,J=5.4Hz,2H),2.61(t,J=7.3Hz,2H),1.37(s,9H).

[0138] (9) N-([1,1'-biphenyl]-3-yl)-2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Hd)

[0139]

[0140] Weigh 180.0 mg (0.35 mmol) of BHd into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BHd: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 100.0 mg of light yellow solid Hd (68.97%). HPLC: 99.14%; mp 100.2-101.3℃; IR (cm -1 ):2930,1673,1587,1447,1420,1199,1175,1125,774,756,697,614; ESI-MS(m / e):413[M+H] + ; 1 HNMR (300 MHz, DMSO-d6 )δ / ppm=8.26(d,J=1.0Hz,1H),8.03(s,1H),7.70(dd,J=7.4,6.0Hz,3H),7.50(dd,J=11.4,4.2Hz,4H) ,7.44-7.38(m,2H),4.70(s,2H),3.97(t,J=5.5Hz,2H),2.79(t,J=6.6Hz,5H),2.58(d,J=6.9Hz,2H).

[0141] (10) tert-Butyl (2-(1-(4-(thiophen-3-ylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHe)

[0142]

[0143] Weigh 395.0 mg (1.8 mmol) of compound BH and 912.0 mg (2.7 mmol) of CS 2 CO 3 Dissolve in IPA and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 116.0 mg (0.18 mmol) of BINAP, 171.0 mg (0.18 mmol) of Pd 2 (dba) 3 and 250.0 mg (0.9 mmol) of compound ea were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ea disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 329.0 mg of light yellow solid BHe (79.66%). ESI-MS (m / e): 443 [M+H] + ;1H NMR (300MHz, DMSO-d6) δ / ppm=8.88(s,0H),8.62(dd,J=5.9,3.3Hz,1H),7.99(s,1H),7.66-7.51(m,2H),7.35(dd,J=5.2,1.3Hz,1H),7.2 2-7.12(m,1H),6.93(t,J=5.4Hz,0H),4.66(s,2H),3.99(t,J=5.6Hz,1H),3.28-3.20(m,1H),2.87-2.77(m,2H),1.38(d,J=10.5Hz,5H).

[0144] (11) 2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-N-(thiophen-3-yl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (He)

[0145]

[0146] Weigh 367.0 mg (0.8 mmol) of BHe into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BHe: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 155.0 mg of light yellow solid He (54.77%). HPLC: 94.39%; mp 127.4-128.2℃; IR (cm -1 ):3268,3153,2920,1592,1551,1455,1422,762,618; ESI-MS(m / e):343[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.37(d,J=1.1Hz,1H),7.72(dd,J=3.1,1.3Hz,1H),7.58(s,1H),7.51(dd,J=5.2,3.2Hz,1H),7.36(dd,J=5.2 ,1.3Hz,1H),4.64(s,2H),3.95(t,J=5.6Hz,2H),2.85(d,J=7.1Hz,2H),2.75(d,J=5.4Hz,2H),2.62(t,J=7.0Hz,2H).

[0147] (12) tert-Butyl (2-(1-(4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHf)

[0148]

[0149] Weigh 365.0 mg (1.8 mmol) of compound BH and 1.1 g (3.6 mmol) of CS 2 CO 3Dissolve in IPA and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 143.0 mg (0.24 mmol) of BINAP, 211.0 mg (0.24 mmol) of Pd 2 (dba) 3 and 327.0 mg (1.2 mmol) of compound fa were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound fa disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 329.0 mg of light yellow solid BHf (62.19%). ESI-MS (m / e): 459 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.39(d,J=1.0Hz,1H),7.59(s,1H),7.13(t,J=5.5Hz,1H),6.86(t,J=5.5Hz,1H),4.43(s,2H),4.03-3.71(m,4H),3.35(d,J=6.1Hz,2H ),3.20(dd,J=13.3,6.7Hz,3H),2.69-2.58(m,4H),1.86(ddd,J=11.2,7.4,4.1Hz,1H),1.58(s,2H),1.39(d,J=6.5Hz,10H),1.29-1.18(m,3H).

[0150] (13) 2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Hf)

[0151]

[0152] Weigh 181.0 mg (0.4 mmol) of BHf into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BHf: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 107.0 mg of light yellow solid Hf (57.69%). HPLC: 99.65%; mp 135.2-133.6℃; IR (cm -1):3253,3151,2920,2846,1599,1479,1417,1109,983,880,779; ESI-MS(m / e):359[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6 )δ / ppm=8.36(d,J=1.1Hz,1H),7.56(s,1H),7.14(t,J=5.7Hz,1H),4.43(s,2H),3.86(ddd,J=11.2,6.8,3.3Hz,4H),3.38-3.16( m,5H),2.84(d,J=7.1Hz,2H),2.69-2.58(m,4H),1.87(ddd,J=11.0,7.2,3.9Hz,1H),1.61(d,J=12.9Hz,2H),1.32-1.17(m,3H).

[0153] (14) tert-Butyl (2-(1-(4-((2-(tetrahydro-2H-pyran-4-yl)ethyl)amino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-1H-imidazol-4-yl)ethyl)carbamate (BHg)

[0154]

[0155] Weigh 571.0 mg (2.6 mmol) of compound BH and 1.8 g (5.1 mmol) of CS 2 CO 3 Dissolve in IPA and activate under reflux at 100°C for 30 min to obtain reaction solution A; weigh 224.0 mg (0.34 mmol) of BINAP, 330.0 mg (0.34 mmol) of Pd 2 (dba) 3 and 500.0 mg (1.7 mmol) of compound ga were suspended with IPA to obtain reaction solution B, reaction solution B was mixed with reaction solution A, and refluxed at 100°C for reaction. TLC monitored the reaction progress, and after compound ga disappeared, post-treatment was performed: hot filtration, DCM was rinsed the filter cake several times until the filter cake was an off-white solid, the filtrate was taken, the solvent was dried to obtain a red-black solid, and purified by DCM / MeOH column chromatography, and concentrated to dryness under reduced pressure to obtain 600.0 mg of light yellow solid BHg (80.51%). ESI-MS (m / e): 473 [M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.34(d,J=1.1Hz,1H),7.57(s,1H),7.06(t,J=5.3Hz,1H),6.86(t,J= 5.5Hz,1H),4.42(s,2H),3.91(d,J=5.5Hz,2H),3.83(dd,J=11.3,3.1Hz,2H), 3.54-3.44(m,2H),3.35-3.15(m,5H),2.64(dd,J=14.1,6.5Hz,4H),1.64(d,J =13.4Hz,2H),1.52(t,J=6.5Hz,3H),1.37(d,J=6.1Hz,9H),1.26-1.12(m,3H).

[0156] (15) 2-(4-(2-aminoethyl)-1H-imidazol-1-yl)-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine (Hg)

[0157]

[0158] Weigh 432.0 mg (0.8 mmol) of BHg into a 50 mL eggplant bottle and add 10 mL of 50% CF 3 COOH (DCM), add a drying tube, react at room temperature in a fume hood, monitor the reaction progress by TLC, and perform post-treatment after the disappearance of compound BHg: drain the solvent under reduced pressure in a warm water bath, dissolve in DCM and drain again, repeat 3 times to obtain a yellow solid, purify by DCM / MeOH column chromatography, and concentrate to dryness under reduced pressure to obtain 323.0 mg of light yellow solid Hg (72.58%). HPLC: 93.06%; mp 140.0-140.7℃; IR (cm -1 ):3344,3275,3149,2907,2841,2604,2530,2108,1610,1530,1448,1385,993,836,775; ESI-MS(m / e):373[M+H] + ; 1 H NMR (300 MHz, DMSO-d 6)δ / ppm=8.35(d,J=1.0Hz,1H),7.58(s,1H),7.11(t,J=5.4Hz,1H),4.42(s ,2H),3.90(t,J=5.6Hz,2H),3.83(dd,J=11.3,3.1Hz,3H),3.46(dd,J=12.7 ,6.5Hz,3H),3.31-3.20(m,2H),2.90(t,J=7.1Hz,2H),2.66(t,J=6.7Hz,4H ),1.64(d,J=13.4Hz,2H),1.59-1.49(m,3H),1.20(dt,J=17.7,7.6Hz,3H).

[0159] Example 5 Evaluation of ATPase activity of pyranopyrimidine heterocyclic p97 / VCP inhibitors

[0160] Using ADP-Glo TM Kinase Assay method, a universal, homogeneous, high-throughput screening method, measures kinase activity by quantifying the amount of ADP produced during the kinase reaction and determines the IC of target compounds against p97 / VCP 50 The inhibitory activity of the target compound on p97 / VCP was determined by the kinetic analysis.

[0161] ADP-Glo TM The Kinase Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during the kinase reaction. TM The KinaseAssay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using up to 1 mM ATP. ADP-Glo TM The kinase assay is performed in a multi-well plate and can measure kinase activity in reaction volumes as low as 5 μL. The assay is performed in two steps; first, after the kinase reaction, an equal volume of ADP-Glo TM The first step is to add a kinase assay reagent to terminate the kinase reaction and deplete the remaining ATP. Second, a kinase assay reagent is added to simultaneously convert ADP to ATP and allow measurement of newly synthesized ATP using the luciferase / luciferin reaction.

[0162] Table 1 Inhibitory activity of compounds against p97 / VCP

[0163]

[0164]

[0165] Example 6 Evaluation of the in vitro anti-tumor cell proliferation activity of pyranopyrimidine heterocyclic p97 / VCP inhibitors

[0166] 1) PBS and culture medium were purchased from Jiangsu Keygene Biotechnology, fetal bovine serum (FBS) was purchased from CORNING, dimethyl sulfoxide (DMSO) was purchased from Shanghai Aladdin, and tetrathiazolium blue (MTT) was purchased from Solarbio: dissolved in PBS solution to make a 5 mg / mL solution, filtered and sterilized before use, and stored in the dark; Cell-Counting-Kit-8 (CCK8) was purchased from Dojindo.

[0167] 2) Test cell lines: A549 (human lung cancer cells, RPMI 1640 complete medium), LLC (mouse lung cancer cells, DMEM complete medium), HCT116 (human colon cancer cells, DMEM complete medium), MC38 (mouse colorectal cancer cells, RPMI 1640 complete medium), HepG 2 (human liver cancer cells, DMEM complete medium), NB4 (human acute promyelocytic leukemia cells, RPMI 1640 complete medium), B16F10 (mouse melanoma cells, RPMI 1640 complete medium), MCF-7 (human breast cancer cells, DMEM complete medium)

[0168] 3) Adherent cells (MTT method): The adherent cells in good growth state and in the logarithmic growth phase were washed three times with PBS, digested with trypsin until most of the cells fell off the flask wall, added the corresponding culture medium to terminate the digestion, pipetted until the cells were completely detached, transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 3 min, discarded the supernatant, added culture medium to pipet and resuspend, and counted. The cell density was 4-5×10 4 / mL, inoculated into a 96-well plate (Corning 3599), and 100 μL was added to each well (100 μL PBS was added to each well of the 96-well plate to seal the outer periphery); the conditions were 37°C, 5% CO 2 Incubate in an incubator for 6-12 hours, observe the cell adhesion, and administer the drug when the adhesion rate reaches more than 50%. Add 25μL of the test sample solution to each well for drug administration, and gently shake it evenly (blank and positive controls must be set up for each plate); after 48 hours of incubation, add 25μL of 5mg / mL MTT solution to each well, incubate in the incubator for 4 hours, remove the supernatant, add 100μL DMSO to each well, shake for 15 minutes to fully dissolve the precipitate, and use an enzyme reader to measure the OD value (absorbance) of each well at a wavelength of 570nm and 490nm within 5 minutes; calculate the inhibition rate according to the following formula, set up 9 replicate wells in the experiment, plot the inhibition rate against the concentration of the test compound, and calculate the IC of the test compound 50 (half effective inhibitory concentration) value.

[0169] Inhibition rate = [(OD average of blank - OD average of compound group) / OD value of blank] × 100%

[0170] Suspension cells (CCK8 method): transfer the well-growing suspension cells in the logarithmic growth phase to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add culture medium, resuspend by pipetting, and count the cells. The cell density is 6-8×10 4 / mL, inoculated in a 96-well plate (Corning 3799), 100μL was added to each well (100μL PBS was added to each well of the 96-well plate to seal); the drug can be administered immediately after cell inoculation. Add 25μL of the test sample solution to each well for drug administration, and gently shake it evenly (a blank and positive control must be set for each plate); after 48h of incubation, 10μL of CCK8 solution was added to each well, incubated in the incubator for 5h, and the OD value (absorbance) of each well was measured at a wavelength of 450nm using an enzyme reader; the inhibition rate was calculated according to the following formula, and 9 replicate wells were set up in the experiment. The inhibition rate was plotted against the concentration of the test compound to calculate the IC of the test compound 50 (half effective inhibitory concentration) value.

[0171] Inhibition rate = [(OD average of blank - OD average of compound group) / OD value of blank] × 100%

[0172] 4) The results are shown in the table

[0173] Table 2 Cytotoxic activity (IC 50 / μM)

[0174]

[0175] Table 3 Cytotoxic activity of compounds (IC 50 / μM)

[0176]

[0177]

[0178] Table 4H Cytotoxic activity of compounds (IC 50 / μM)

[0179]

[0180] In vivo anti-tumor cell proliferation activity of p97 / VCP inhibitors

[0181] The experiment was evaluated using the mouse MC38 colorectal cancer model.

[0182] C57 BL / 6 female mice (20 ± 2 g): SPF grade, Vital River, purchased from the Experimental Animal Center of Capital Medical University. Animal welfare and animal ethics were approved by the Animal Experiment Ethics Review of the Department of Laboratory Animal Science of Capital Medical University (approval number: AEEI-2021-202). All experimental protocols were reviewed and approved by the Ethics Committee of Capital Medical University.

[0183] 1) Model establishment: MC38 cells were revived and passaged. When the cells were in good growth state and in the logarithmic growth phase, the cells were digested and the live cell concentration was suspended with NS to 7.5×10 6 / mL. Take inbred C57BL / 6 female mice, fix the mice with the left hand, disinfect the skin of the right forelimb axilla of the mice with 75% EtOH, hold a 1mL sterile syringe in the right hand and inject 0.25mL / mouse of tumor cell suspension subcutaneously into the axilla of the mice.

[0184] 2) Drug administration: Two days after inoculation, a rice-sized mass can be felt in the mice, and drug administration can be started. The mice are randomly divided into groups and drug administration is started for 7 days. The body weight is measured every day, and the tumor volume is recorded starting from the second day of drug administration.

[0185] 3) Treatment: The mice were killed by cervical dislocation, and the solid tumors in the right armpit of the mice were bluntly dissected and weighed according to the formula: tumor inhibition rate = [(average tumor weight of blank control group - average tumor weight of test group) / average tumor weight of blank control group] × 100%.

[0186] All experimental data were processed according to the characteristics of the samples. Data with equal variance were statistically analyzed using one-way analysis of variance (ANOVA one-way, LSD), and data with unequal variance were statistically analyzed using Student's ttest (T-test). The analysis software was SPSS22.0. The P value was determined by the analysis software with a confidence interval of 95%. When P<0.05, the results were considered to be different and statistically significant.

[0187] Table 5 Effects of compounds on tumor weight in MC38 tumor-bearing mice

[0188]

[0189] Dosage: 90 μmol / kg / dayn≥7; Compared with the NS group by one-way ANOVA: a means P<0.05, b means P<0.01, c means P<0.001. Implement 8 Evaluation of the in vitro anti-migration activity of pyranopyrimidine heterocyclic p97 / VCP inhibitors

[0190] Transwell method

[0191] 1) Cell inoculation: The experiment was conducted using cells in the logarithmic growth phase. The original culture medium in the bottle was discarded, and 1 mL of PBS buffer was added to wash the residual culture medium twice. Then, 1 mL of trypsin-EDTA digestion solution was added and the cells were digested in an incubator. The cell morphology was observed under an inverted microscope until most of the cells became round and could fall off the bottle wall. Then, 1 mL of culture medium was added to terminate the digestion. The cells were repeatedly blown along the wall to make them fall off and disperse. The cells were transferred to a sterilized 15 mL centrifuge tube and centrifuged at 1500 rpm for 3 min. After the centrifugation, the supernatant was discarded, 5 mL of incomplete culture medium was added, and the cells were blown with a pipette to make them evenly dispersed. The cells were centrifuged at 1500 rpm for 3 min. After the centrifugation, the supernatant was discarded, and 5 mL of incomplete culture medium was added. The cells were blown with a pipette to make them evenly dispersed. 15 μL of cell suspension was aspirated onto a red blood cell counting plate and counted under an inverted microscope. Finally, the cell suspension was diluted to make it evenly dispersed so that the cell concentration was 5×10 5 / mL; add 100 μL of cell solution to each well in the upper chamber and 600 μL of complete culture medium to each well in the lower chamber;

[0192] 2) Drug-addition culture: Add 25 μL of the corresponding drug to the corresponding upper chamber (two replicate wells, incomplete culture medium configuration) and incubate in an incubator for 40 h;

[0193] 3) Fixation and staining: Aspirate the liquid in the upper chamber, add 1 mL of PBS to wash, repeat twice, add 1 mL of 4% paraformaldehyde fixative to fix the cells at room temperature for 20 min, aspirate the liquid, wash with PBS, add 1 mL of crystal violet solution, and stain at room temperature for 15 min; after staining, recover the crystal violet, wash with distilled water and dry;

[0194] 4) Photographing and counting: In each chamber, 5 fields of view with uniform cell numbers were selected for photographing, and the number of cells in the 5 fields of view was counted using ImageJ to obtain the average number of migrated cells and SD, and data statistics were performed.

[0195] 5) The experimental results are as follows:

[0196] Table 6 Effects of compounds on the migration ability of A549 cells (S, n = 5)

[0197]

[0198] n=5; by one-way ANOVA, compared with the NS group: a means P < 0.05, b means P < 0.01, c means P < 0.001; compared with the DBeQ group: d means P < 0.05, e means P < 0.01, f means P < 0.001

[0199] Table 7 Effect of compounds on cell counts of A549 cell migration ability (T&H, n=5)

[0200]

[0201] n=5; by one-way ANOVA, compared with the NS group: a means P < 0.05, b means P < 0.01, c means P < 0.001; compared with the DBeQ group: d means P < 0.05, e means P < 0.01, f means P < 0.001

[0202] Example 9 Evaluation of the in vitro anti-invasive activity of pyranopyrimidine heterocyclic p97 / VCP inhibitors

[0203] Transwell method

[0204] 1) Coating with matrigel: Take matrigel from -20℃ and place at 4℃ to liquid; take 150μL matrigel, add 1350μL incomplete culture medium to dilute, add 100μL diluent to each well, and place the Transwell plate in 37℃, 5% CO 2 Culture in an incubator for 5 h;

[0205] 2) Hydrate the basement membrane: remove the liquid in the upper chamber, add 50 μL of incomplete culture medium, and place the Transwell plate in a 37°C, 5% CO 2 Incubate in an incubator for 30 min;

[0206] 3) Cell inoculation: The experiment was conducted using cells in the logarithmic growth phase. The original culture medium in the bottle was discarded. After adding 1 mL of PBS buffer to each bottle to wash the residual culture medium twice, 1 mL of trypsin-EDTA digestion solution was added to the bottle and digested in an incubator. The cell morphology was observed under an inverted microscope until most of the cells became round and could fall off the bottle wall. 1 mL of culture medium was added to terminate the digestion. The cells were repeatedly blown along the wall to make them fall off and disperse. The tubes were transferred to a sterilized 15 mL centrifuge tube and centrifuged at 1500 rpm for 3 min. After the centrifugation, the supernatant was discarded, 5 mL of incomplete culture medium was added, and the cells were blown with a pipette to make them evenly dispersed. The tubes were centrifuged at 1500 rpm for 3 min. After the centrifugation, the supernatant was discarded, 5 mL of incomplete culture medium was added, and the cells were blown with a pipette to make them evenly dispersed. 15 μL of cell suspension was aspirated onto a red blood cell counting plate and counted under an inverted microscope. Finally, the cell suspension was diluted to make it evenly dispersed so that the cell concentration was 4×10 5 / mL; add 100 μL of cell solution to each well in the upper chamber and 600 μL of complete culture medium to each well in the lower chamber;

[0207] 4) Drug addition culture: Add 25 μL of the corresponding drug to the corresponding upper chamber (two replicate wells, incomplete culture medium configuration), and incubate in an incubator for 15-18 hours;

[0208] 5) Fixation and staining: Aspirate the liquid in the upper chamber, add 1 mL of PBS to wash, repeat twice, add 1 mL of 4% paraformaldehyde fixative to fix the cells at room temperature for 20 minutes, aspirate the liquid, wash with PBS, add 1 mL of crystal violet solution, and stain at room temperature for 15 minutes; after staining, recover the crystal violet, wash with distilled water and dry;

[0209] 6) Photographing and counting: In each chamber, 5 fields of view with uniform cell numbers were selected for photographing, and the number of cells in the 5 fields of view was counted using ImageJ to obtain the average number of migrated cells and SD, and data statistics were performed.

[0210] 7) The results are as follows:

[0211] Table 8 Effects of compounds on the invasion ability of A549 cells (S, n = 5)

[0212]

[0213]

[0214] n=5; by one-way ANOVA, compared with the NS group: a means P<0.05, b means P<0.01, c means P<0.001; compared with the DBeQ group: d means P<0.05, e means P<0.01, f means P<0.001 Table 9 Effects of compounds on the invasion ability of A549 cells (T&H, n=5)

[0215]

[0216] n=5; by one-way ANOVA, compared with the NS group: a means P < 0.05, b means P < 0.01, c means P < 0.001; compared with the DBeQ group: d means P < 0.05, e means P < 0.01, f means P < 0.001

[0217] Example 10 Evaluation of the in vivo anti-tumor metastasis activity of pyranopyrimidine heterocyclic p97 / VCP inhibitors

[0218] The Lewis lung cancer metastasis model was used for evaluation.

[0219] C57 BL / 6 male mice (20 ± 2 g): SPF grade, Vital River, purchased from the Experimental Animal Center of Capital Medical University. Animal welfare and animal ethics were approved by the Animal Experiment Ethics Review of the Department of Laboratory Animal Science of Capital Medical University (approval number: AEEI-2021-202). All experimental protocols were reviewed and approved by the Ethics Committee of Capital Medical University.

[0220] 1) Lewis tumor source establishment: LLC cells were revived and passaged. When the cells were in good growth state and in the logarithmic growth phase, the cells were digested. The concentration of live cells in NS suspension was 2×10 7 Take an inbred C57BL / 6 male mouse, fix the mouse with the left hand, disinfect the skin of the right forelimb armpit of the mouse with 75% EtOH, and hold a 1mL sterile syringe in the right hand to inject 0.2mL of tumor cell suspension into the mouse's armpit subcutaneously.

[0221] 2) Model establishment: 15-day-old Lewis lung cancer-bearing mice were killed by cervical dislocation, and then disinfected by soaking in 75% EtOH for 5 min. The tumor was removed on a clean bench, and the well-growing tumor tissue was selected. A small amount of physiological saline was added to a sterile plate to rinse off the floating blood, and the tissue was cut into pieces and placed in a glass tissue homogenizer. 4°C precooled physiological saline was added and gently ground to make a cell suspension. The cell suspension was passed through a 200-mesh nylon mesh twice to make a single-cell suspension. The trypan blue exclusion test showed the number of live cells. The live cells were suspended with physiological saline to a concentration of 2×10 7 / mL. Take an inbred C57 BL / 6 male mouse, fix the mouse with the left hand, disinfect the skin of the right forelimb armpit of the mouse with 75% EtOH, hold a 1mL sterile syringe in the right hand and inject 0.2mL of tumor cell suspension subcutaneously into the mouse armpit.

[0222] 3) Grouped administration: 6-7 days after inoculation, a tumor with a diameter of about 3-4 mm can be grown. The tumor volume is measured and the experiment is carried out in groups according to the average tumor volume. Each group is administered for 10-12 days in succession, and the tumor volume is measured every day.

[0223] 4) Treatment: After weighing and measuring the tumor volume, the mice were killed by cervical dislocation, and the solid tumor in the right armpit of the mouse was bluntly dissected and weighed. After the solid tumor in the mouse body was dissected, the heart, lung, liver, spleen, kidney, brain and thymus were dissected and weighed, and the tumor lung metastasis rate and the number of metastatic tumor nodes were recorded.

[0224] Table 10 Effects of compounds on the number of metastatic tumor nodes in Lewis lung cancer mice

[0225]

[0226]

[0227] n≥7; compared with the NS group by one-way ANOVA: a means P<0.05, b means P<0.01, c means P<0.001 Table 11 Effects of compounds on the number of metastatic tumor nodes in Lewis lung cancer mice

[0228]

[0229] n≥7; compared with the NS group by one-way analysis of variance: a means P<0.05, b means P<0.01, c means P<0.001.

Claims

1. A pyranopyrimidine heterocyclic p97 / VCP inhibitor, characterized in that: Its structure is as follows:

2. A method for preparing the pyranopyrimidine heterocyclic p97 / VCP inhibitor according to claim 1, characterized in that: The pyranopyrimidine heterocyclic p97 / VCP inhibitor is prepared by the following steps: 1) i)ACN,rtor IPA,TEA,80℃,reflux or ACN,TEA,rt ii) Pd2(dba)3, XPHOS, CS2CO3, 100℃, reflux; or 2) i)ACN,rtor IPA,TEA,80℃,reflux or ACN,TEA,rt ii)dioxane,H2O,NaOH,(Boc)2O,rt iii)Pd2(dba)3,XPHOS,CS2CO3,100℃,reflux iv) 50% CF3COOH (DCM), rt; or 3) i)ACN,rtor IPA,TEA,80℃,reflux or ACN,TEA,rt ii)dioxane,H2O,NaOH,(Boc)2O,rt iii)Pd2(dba)3,XPHOS,CS2CO3,100℃,reflux iv) 50% CF3COOH (DCM), rt.

3. Use of the pyranopyrimidine heterocyclic p97 / VCP inhibitor as claimed in claim 1 in the preparation of anti-tumor drugs.

4. Use of the pyranopyrimidine heterocyclic p97 / VCP inhibitor as claimed in claim 1 in the preparation of anti-tumor metastasis drugs.

5. Use of the pyranopyrimidine heterocyclic p97 / VCP inhibitor according to claim 1 in the preparation of a p97 inhibitor.

Images