Diaryl pyrimidine derivative containing fused heterocycle as well as preparation method and application of diaryl pyrimidine derivative

Abstract

The invention discloses a diaryl pyrimidine compound containing a fused heterocyclic ring as well as a preparation method and application of the diaryl pyrimidine compound. The compound has a structure as shown in a general formula I. The invention also relates to a pharmaceutical composition containing the compound with the structure as shown in the formula I. An activity screening experiment shows that the compound has good anti-HIV and CHIKV activity, so that the invention also provides application of the compound in preparation of anti-AIDS drugs and anti-Chikungunya virus drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a derivative and a preparation method and application thereof, in particular to a diaryl pyrimidine derivative containing a fused heterocycle and a preparation method and application thereof, belonging to the technical field of medicine. Background Art

[0002] Acquired immune deficiency syndrome (AIDS) is a serious infectious disease that destroys the human immune system and is mainly caused by infection with human immunodeficiency virus type 1 (HIV-1). In the life cycle of HIV-1, reverse transcriptase (RT) is responsible for reverse transcribing single-stranded RNA carrying viral genetic information into double-stranded DNA, and is a preferred target for the design of anti-AIDS drugs. As an important component of combined antiretroviral therapy (cART), non-nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit the activity of HIV-1RT through allosteric binding. They have the advantages of high activity, strong selectivity, and low toxicity, and are a hot topic in the research of anti-AIDS drugs.

[0003] Etravirine (ETR) and rilpivirine (RPV) are new generation diarylpyrimidine NNRTIs. However, with their widespread clinical application, a variety of mutants that are resistant to ETR and RPV have emerged, such as E138A, E138K, Y181C and Y181I. In addition, this type of compound has low water solubility and oral bioavailability, large oral doses, and is prone to cause more serious toxic side effects in clinical use. Therefore, obtaining NNRTIs with high efficiency, broad spectrum resistance and good pharmacokinetic properties through reasonable structural modification is one of the important areas of current anti-AIDS drug research.

[0004] Chikungunya fever (CHIKF) is a globally widespread vector-borne infectious disease caused by the Chikungunya virus (CHIKV). Clinically, it is characterized by fever, rash, bleeding, and severe joint pain, and a few patients have persistent joint stiffness. Chikungunya disease is prone to large-scale epidemics and outbreaks in areas with high mosquito density. It is one of the vector-borne infectious diseases that seriously affects human public health security in the early 21st century. At present, there is no vaccine or antiviral drug that can be used to prevent and control CHIKV infection. Therefore, there is an urgent need to develop safe, effective, and clinically valuable small molecule drugs to treat Chikungunya fever. Summary of the invention

[0005] The present invention provides a diaryl pyrimidine derivative containing a fused heterocycle and a preparation method thereof. The present invention also provides partial activity screening results of the above-mentioned compound and uses thereof.

[0006] The technical solution of the present invention is as follows:

[0007] 1. Diarylpyrimidine derivatives containing fused heterocyclic rings

[0008] The diaryl pyrimidine derivatives containing a fused heterocycle of the present invention have a structure shown in the following general formula I:

[0009]

[0010] in,

[0011] R 1 CH 3 , CN, pC 4 H 6 -CN or CH=CHCN;

[0012] R 2 For H, CH 3 , CN, CF 3 , COCH 3 , NH 2 , NO 2 , F, Br, I, Cl, PO(CH 3 ) 2 ,PO(OCH 3 ) 2 ,SO 2 CH 3 , SOCH 3 , OCH 3 , SCH 3 , NHCH 3 , NHCOCH 3 or NHSO 2 CH 3;

[0013] R 3 For H, CH 3 , CN, CF 3 , COCH 3 , NH 2 , NO 2 , F, Br, I, Cl, PO(CH 3 ) 2 ,PO(OCH 3 ) 2 ,SO 2 CH 3 , SOCH 3 , OCH 3 , SCH 3 , NHCH 3 , NHCOCH 3 or NHSO 2 CH 3 ;

[0014] X is

[0015]

[0016] According to the present invention, the diaryl pyrimidine derivative containing a fused heterocyclic ring is preferably one of the following:

[0017]

[0018]

[0019] 2. Preparation method of diaryl pyrimidine derivatives containing fused heterocycles

[0020] A method for preparing a diaryl pyrimidine derivative containing a fused heterocycle, comprising the following steps: first, using 2,4-dichloropyrimidine 1 as a raw material, reacting it with (E)-3,5-dimethyl-4-hydroxyphenyl acrylonitrile in an N,N-dimethylformamide solution to generate an intermediate 2; the intermediate 2 and the fused heterocycle aniline undergo a coupling reaction under the catalysis of palladium acetate to obtain a target compound 3(ax) or an intermediate 4 or 7; then, removing the Boc group from the intermediate 4 or 7 to obtain an intermediate 5 or 8, and the intermediate 5 or 8 reacts with different sulfonyl chlorides, phosphoryl chlorides, acyl chlorides or alkyl halides to obtain a final product 6(ae) or 9(ae);

[0021]

[0022] Reagents and conditions: (i) (E)-3,5-dimethyl-4-hydroxybenzene acrylonitrile, N,N-dimethylformamide, potassium carbonate, 50°C; (ii) palladium acetate, 4,5-bis(diphenylphosphino-9,9-dimethylxanthene), cesium carbonate, nitrogen, 1,4-dioxane, 90°C; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) sulfonyl chloride, phosphorus oxychloride, acyl chloride or alkyl halide, triethylamine, 0°C.

[0023] 3. Application of diaryl pyrimidine derivatives containing fused heterocyclic rings

[0024] The present invention discloses anti-HIV activity screening results and anti-CHIKV results of diaryl pyrimidine compounds containing fused heterocycles, and the first application of the compounds as HIV inhibitors and anti-chikungunya virus inhibitors. Experiments prove that the diaryl pyrimidine compounds containing fused heterocycles provided by the present invention can be used to prepare anti-HIV drugs and anti-chikungunya virus drugs. The present invention also provides the application of the compounds in the preparation of anti-HIV drugs and anti-chikungunya virus drugs.

[0025] Therefore, the diaryl pyrimidine compounds containing fused heterocycles provided by the present invention can be used as anti-HIV inhibitors and anti-Chikungunya virus inhibitors, and can be used to prepare anti-HIV drugs and anti-Chikungunya virus drugs.

[0026] A pharmaceutical composition for resisting HIV and Chikungunya virus contains the above-mentioned diaryl pyrimidine compound containing a fused heterocycle and a pharmaceutically acceptable salt thereof and pharmaceutical excipients, and is prepared into medicines of different dosage forms. DETAILED DESCRIPTION

[0027] The following examples are provided to help understand the present invention, but they are not intended to limit the present invention.

[0028] Example 1: Preparation of (E)-3-(4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (2)

[0029]

[0030] 2,4-Dichloropyrimidine (1.49 g, 0.01 mol) and potassium carbonate (1.66 g, 0.012 mol) were dissolved in N,N-dimethylformamide (20 mL), (E)-3,5-dimethyl-4-hydroxybenzene acrylonitrile (1.73 g, 0.01 mol) was added to the solution, and stirred at 50 ° C for 4 h until the reaction was completed. Ice water (200 mL) was added and extracted with ethyl acetate (3×50 mL). The combined organic layer was washed with saturated brine and anhydrous Na 2 SO 4Dry, filter and concentrate under reduced pressure, and finally recrystallize using ethyl acetate and petroleum ether to provide the pure intermediate 4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile 2. White solid, yield: 84.7%. 1 H NMR (400 MHz, DMSO-d 6 )δ8.67(d,J=5.7Hz,1H,pyrimidine-H),7.62(d,J=16.7Hz,1H,CH=),7.52(s,2H,Ph-H ),7.25(d,J=5.7Hz,1H,pyrimidine-H),6.45(d,J=16.7Hz,1H,CH=),2.07(s,6H,Ph-CH 3 ×2). ESI-MS: m / z 286.3 (M+H) + ,C 15 H 12 C1N 3 O(285.07).

[0031] Example 2: Preparation of (E)-3-(4-((2-(Benzo[d]thiazol-5-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3a)

[0032]

[0033] (E)-3-(4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (0.52 g, 3.01 mmol), benzo[d]thiazol-5-amine (0.45 g, 3.01 mmol), palladium acetate (0.034 g, 0.15 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.086 g, 0.15 mmol) and cesium carbonate (1.46 g, 4.51 mmol) were dissolved in 20 mL of 1,4-dioxane, and reacted at 90°C for 8 hours under nitrogen protection. The reaction was monitored by TLC. The mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure. The target compound 3a was further purified by silica gel chromatography using methanol / dichloromethane as the eluent. It was obtained as a brown solid with a yield of 47.4%. 1 HNMR (600 MHz, DMSO-d 6)δ9.81(s,1H,NH),9.24(s,1H,thienyl-H),8.44(d,J=5.6Hz,1H,pyrimidyl-H),8.30(s,1H,Ph-H),7.82(d,J=8.5Hz,1H,Ph-H),7.64(d,J=16 .7Hz,1H,CH=),7.57(d,J=8.8Hz,1H,Ph-H),7.52(s,2H,Ph-H),6.54(d,J=5.6Hz,1H,pyrimidyl-H),6.44(d,J=16.7Hz,1H,CH=),2.12(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.78,160.70,160.27,156.56,154.23,152.49,150.67,139.72,134.04,131.58,12 9.85,128.87,125.87,121.98,120.15,118.47,112.51,98.68,96.71,17.53.ESI-MS:m / z 400.08(M+H) + ,C 22 H 17 N 5 OS(399.12).

[0034] Example 3: Preparation of (E)-3-(3,5-dimethyl-4-((2-(2-methylbenzo[d]thiazol-5-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3b)

[0035]

[0036] The preparation method is the same as that of Example 2, except that the raw material is replaced with 2-methylbenzo[d]thiazol-5-amine. Yellow solid, yield: 45.4%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.77(s,1H,NH),8.42(d,J=5.6Hz,1H,pyrimidyl-H),8.04(s,1H,Ph-H),7.68(s,1H,Ph-H),7.65(d,J=16.7Hz,1H,CH= ),7.52(s,2H,Ph-H),7.41(s,1H,Ph-H),6.55(d,J=7.2Hz,1H,pyrimidyl-H),6.44(d,J=16.7Hz,1H,CH=),2.75(s,3H,CH 3),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.78,167.31,160.72,160.23,154.13,152.09,150.91,139.11,131.92,131.49,12 8.90,128.17,121.45,119.47,117.46,111.71,97.95,96.51,20.25,16.56.ESI-MS:m / z 414.28(M+H) + ,C 23 H 19 N 5 OS(413.13).

[0037] Example 4: (E)-3-(4-((2-(Benzo[d]thiazol-6-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3c)

[0038]

[0039] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzo[d]thiazol-6-amine. Yellow solid, yield: 50.3%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.99(s,1H,NH),9.19(s,1H,thienyl-H),8.46(d,J=5.6Hz,1H,pyrimidyl-H),8.05(s,1H,Ph-H),7.82(d,J=8.9Hz,1H,Ph-H),7.72(d,J=16 .7Hz,1H,CH=),7.59(s,2H,Ph-H),7.43(d,J=9.0Hz,1H,Ph-H),6.65(d,J=5.6Hz,1H,pyrimidyl-H),6.51(d,J=16.7Hz,1H,CH=),2.12(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.76,160.93,160.02,153.75,151.96,150.66,148.67,148.14,138.61,134.72 ,131.81,129.76,128.76,122.97,118.96,109.60,98.17,96.96,16.56.ESI-MS:m / z 400.04(M+H)+ ,C 22 H 17 N 5 OS(399.12).

[0040] Example 5: (E)-3-(4-((2-(1,3-dihydroisobenzofuran-5-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile

[0041]

[0042] The preparation method is the same as that of Example 2, except that the raw material is replaced with 1,3-dihydroisobenzofuran-5-amine. Brown solid, yield: 48.3%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.73(s,1H,NH),8.39(d,J=5.6Hz,1H,pyrimidyl-H),7.68(d,J=16.7Hz,1H,CH=),7.54(s,2H,Ph-H),7.31(s,1H,Ph-H),7. 14(s,1H,Ph-H),7.01(d,J=8.1Hz,1H,Ph-H),6.56(d,J=5.6Hz,1H,pyrimidyl-H),6.49(d,J=16.7Hz,1H,CH=),4.86(s,2H,OCH 2 ),4.63(s,2H,OCH 2 ),2.09(s,6H). 13 C NMR (100 MHz, DMSO-d 6 )δ168.71,160.81,160.17,152.39,150.52,140.03,139.70,131.85,131.71,129.63,1 28.63,121.19,119.38,118.24,110.54,97.64,96.78,73.35,72.78,16.65.ESI-MS:m / z 385.03(M+H) + ,C 23 H 20 N 4 O 2 (384.16).

[0043] Example 6: (E)-3-(3,5-dimethyl-4-((2-((3-oxoisoindolin-5-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3e)

[0044]

[0045] The preparation method is the same as that of Example 2, except that the raw material is replaced with 6-aminoisoindol-1-one. Gray solid, yield 44.2%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.78(s,1H,NH),8.47(s,1H,amide-H),8.42(d,J=5.7Hz,1H,pyrimidyl-H),7.91(s,1H,Ph-H),7.67(d,J=7.3Hz,1H,Ph-H),7.63(d,J =16.7Hz,1H,CH=),7.51(s,2H,Ph-H),7.21(s,1H,Ph-H),6.52(d,J=5.7Hz,1H,pyrimidyl-H),6.44(d,J=16.7Hz,1H,CH=),4.25(s,2H,CH 2 ),2.12(d,J=15.1Hz,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ170.50,168.73,160.67,160.19,152.01,150.57,140.42,137.26,133.37,131.75,13 1.66,128.83,123.68,122.36,119.45,113.01,98.24,96.77,44.89,16.48.ESI-MS:m / z 396.07(MH) - ,C 23 H 19 N 5 O 2 (397.15).

[0046] Example 7: (E)-3-(3,5-dimethyl-4-(2-(2-methyl-1,3-diketoisoindolin-5-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3f)

[0047]

[0048] The preparation method is the same as that of Example 2, except that the raw material is replaced with 5-amino-2-methylisoindole-1,3-dione. Yellow solid, yield 50.7%. 1 H NMR (600 MHz, DMSO-d 6)δ10.25(s,1H,NH),8.51(d,J=5.7Hz,1H,pyrimidyl-H),8.07(s,1H,Ph-H),7.81(d,J=8.1Hz,1H,Ph-H),7.64(d,J=16.7Hz,1H,CH =),7.53(s,2H,Ph-H),7.50(d,J=8.3Hz,1H,Ph-H),6.68(d,J=5.6Hz,1H,pyrimidyl-H),6.45(d,J=16.7Hz,1H,CH=),2.98(s,3H,CH 3 ),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.83,168.28,168.16,160.81,159.67,151.92,150.49,146.25,133.68,131.90,131.5 9,128.88,123.94,123.88,122.39,119.33,112.77,99.70,96.84,24.05,16.52.ESI-MS:m / z 424.12(MH) - ,C 24 H 19 N 5 O 3 (425.15).

[0049] Example 8: (E)-3-(4-((2-(Benzo[d][1,3]dioxolan-5-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3 g)

[0050]

[0051] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzo[d][1,3]dioxolane-5-amine. Brown solid, yield 52.8%. 1 H NMR (600 MHz, DMSO-d 6)δ9.47(s,1H,NH),8.34(d,J=5.6Hz,1H,pyrimidyl-H),7.62(d,J=16.7Hz,1H,CH=),7.49(s,2H,Ph-H),7.04(s,1H,Ph-H ),6.82(s,1H,Ph-H),6.60(s,1H,Ph-H),6.46(d,J=5.6Hz,1H,pyrimidyl-H),6.42(d,J=16.7Hz,1H,CH=),5.88(s,2H,CH 2 ),2.08(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.67,160.67,160.16,152.19,150.62,147.33,142.04,135.08,131.82,131.65 ,128.70,119.39,111.66,107.94,101.43,101.07,97.41,96.60,16.53.ESI-MS:m / z 387.05(M+H) + ,C 22 H 18 N 4 O 3 (386.14).

[0052] Example 9: (E)-3-(3,5-dimethyl-4-(2-(2-methylbenzo[d]oxazol-5-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3h)

[0053]

[0054] The preparation method is the same as that of Example 2, except that the raw material is replaced with 2-methylbenzo[d]oxazol-5-amine. Yellow solid, yield 47.7%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.66(s,1H,NH),8.39(d,J=5.5Hz,1H,pyrimidyl-H),7.72(s,1H,Ph-H),7.64(d,J=16.7Hz,1H,CH=),7.51(s ,2H,Ph-H),7.31(s,2H,Ph-H),6.51(d,J=5.7Hz,1H,pyrimidyl-H),6.44(d,J=16.7Hz,1H,CH=),2.55(s,3H,CH 3 ),2.10(s,6H,CH 3 ×2).13 C NMR (100 MHz, DMSO-d 6 )δ168.73,164.42,160.73,160.25,152.12,150.84,145.99,141.72,137.26,131.94,13 1.60,128.83,119.48,116.62,109.79,109.38,97.71,96.56,16.18,14.55.ESI-MS:m / z 398.12(M+H) + ,C 23 H 19 N 5 O 2 (387.15).

[0055] Example 10: (E)-3-(4-((2-(Benzofuran-5-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3i)

[0056]

[0057] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzofuran-5-amine. Brown solid, yield 50.7%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.64(s,1H,NH),8.38(d,J=5.7Hz,1H,pyrimidyl-H),7.89(d,J=2.2Hz,1H,furan-H),7.72(s,1H,furan-H),7.69(d,J=16.7Hz,1 H,CH=),7.57(s,2H,Ph-H),7.34–7.19(m,3H,Ph-H),6.53(d,J=4.9Hz,1H,pyrimidyl-H),6.49(d,J=16.7Hz,1H,CH=),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.71,160.74,160.35,152.39,150.53,150.18,146.57,136.08,131.83,129.70 ,128.72,127.42,119.38,117.17,111.06,107.14,97.39,96.79,16.18.ESI-MS:m / z 383.11(M+H) + ,C 23 H 18 N4 O 2 (382.14).

[0058] Example 11: (E)-3-(4-((2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3j)

[0059]

[0060] The preparation method is the same as that of Example 2, except that the raw material is replaced with 2,2-difluorobenzo[d][1,3]dioxolane-5-amine. Red solid, yield 45.2%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.88(s,1H,NH),8.42(d,J=5.7Hz,1H,pyrimidyl-H),7.62(d,J=16.7Hz,1H,CH=),7.53(s,2H,Ph-H),7.33(s,1H,Ph-H),7. 13(d,J=8.7Hz,1H,Ph-H),7.04(s,1H,Ph-H),6.61(d,J=5.6Hz,1H,pyrimidyl-H),6.43(d,J=16.7Hz,1H,CH=),2.08(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.70,160.95,159.79,152.15,150.48,142.97,137.53,137.32,132.02,131.66, 128.70,119.29,114.04,109.96,101.08,98.20,96.63,15.95.ESI-MS:m / z421.07(MH) - ,C 22 H 16 F 2 N 4 O 3 (422.12).

[0061] Example 12: (E)-3-(4-((2-([1,2,4]triazolo[4,3-a]pyridin-6-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3k).

[0062]

[0063] The preparation method is the same as that of Example 2, except that the raw material is replaced with [1,2,4]triazolo[4,3-a]pyridin-6-amine. White solid, yield 51.8%. 1 H NMR (400 MHz, DMSO-d 6 )δ9.99(s,1H,NH),9.11(s,1H,triazole-H),8.50(d,J=5.6Hz,1H,pyrimidyl-H),8.30(s,1H,Ph-H),7.68(s,2H,Ph-H),7.6 4(d,J=16.6Hz,1H,CH=),7.53(s,2H,Ph-H),6.64(d,J=5.7Hz,1H,pyrimidyl-H),6.43(d,J=16.6Hz,1H,CH=),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.89,160.80,159.78,153.71,151.84,150.67,146.17,132.13,131.46,12 9.90,128.96,125.85,119.41,117.06,115.76,98.55,96.80,16.53.ESI-MS:m / z 384.20(M+H) + ,C 21 H 17 N 7 O(383.15).

[0064] Example 13: (E)-3-(4-((2-(Benzothiophene-5-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (31)

[0065]

[0066] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzothiophene-5-amine. White solid, yield 51.8%. 1 H NMR (400 MHz, DMSO-d 6)δ9.99(s,1H,NH),9.11(s,1H,triazole-H),8.50(d,J=5.6Hz,1H,pyrimidyl-H),8.30(s,1H,Ph-H),7.68(s,2H,Ph-H),7.6 4(d,J=16.6Hz,1H,CH=),7.53(s,2H,Ph-H),6.64(d,J=5.7Hz,1H,pyrimidyl-H),6.43(d,J=16.6Hz,1H,CH=),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.89,160.80,159.78,153.71,151.84,150.67,146.17,132.13,131.46,12 9.90,128.96,125.85,119.41,117.06,115.76,98.55,96.80,16.53.ESI-MS:m / z 384.20(M+H) + ,C 21 H 17 N 7 O(383.15).

[0067] Example 14: Benzo[b]thiophene-5-amine (E)-3-(3,5-dimethyl-4-((2-(2-methyl-1-oxoisoindolin-5-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3m)

[0068]

[0069] The preparation method is the same as that of Example 2, except that the raw material is replaced with 5-amino-2-methylisoindolin-1-one. White solid, yield 42.9%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.07(s,1H,NH),8.46(d,J=5.5Hz,1H,pyrimidyl-H),7.72(d,J=16.7Hz,1H,CH=),7.58(s,2H,Ph-H),7.53(s,1H,Ph-H),7.38(d ,J=8.3Hz,1H,Ph-H),7.30(d,J=8.4Hz,1H,Ph-H),6.67(d,J=5.6Hz,1H,pyrimidyl-H),6.55(d,J=16.7Hz,1H,CH=),3.97(s,2H,-CH 2 ),3.00(s,3H,-N-CH3 ),2.10(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.73,167.69,160.98,159.90,152.41,150.61,143.59,143.12,131.86,131.80,128.7 8,125.57,123.14,119.30,118.54,111.64,98.47,96.95,51.79,30.33,16.54.ESI-MS:m / z 412.04(M+H) + ,C 24 H 21 N 5 O 2 (411.17).

[0070] Example 15: (E)-6-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzo[d]thiazole-2-carbonitrile (3n)

[0071]

[0072] The preparation method is the same as that of Example 2, except that the raw material is replaced with 6-aminobenzo[d]thiazole-2-carbonitrile. White solid, yield 42.9%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.34(s,1H,NH),8.51(d,J=5.6Hz,1H,pyrimidyl-H),8.18(s,1H,Ph-H),8.01(d,J=9.0Hz,1H,Ph-H),7.78(d,J=16.7Hz,1 H,CH=),7.61(s,1H,Ph-H),7.59(s,2H,Ph-H),6.74(d,J=5.6Hz,1H,pyrimidyl-H),6.54(d,J=16.8Hz,1H,CH=),2.11(s,6H,CH 3 ×2). 13 C NMR (150 MHz, DMSO-d 6)δ168.76,161.02,159.70,152.29,150.60,146.57,141.69,137.22,133.03,132.04,13 1.88,128.76,124.82,121.20,119.28,114.00,108.33,99.08,97.14,16.63.ESI-MS:m / z 425.09(M+H) + ,C 23 H 16 N 6 OS(424.11).

[0073] Example 16: (E)-3-(4-((2-(Benzo[d]isoxazol-5-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3o)

[0074]

[0075] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzo[d]isoxazole-5-amine. Brown solid, yield 48.7%. 1 HNMR (400 MHz, DMSO-d 6 )δ10.60(s,1H,NH),9.59(s,1H,-CH=N-),8.37(d,J=5.6Hz,1H,pyrimidyl-H),7.65(s,1H,Ph-H),7.58(d,J=16.7Hz,1H,CH=),7.50(s,2 H,Ph-H),7.42(s,1H,Ph-H),6.73(d,J=8.5Hz,1H,Ph-H),6.52(d,J=5.5Hz,1H,pyrimidyl-H),6.40(d,J=16.7Hz,1H,CH=),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.75,160.73,160.05,155.34,152.02,150.56,132.79,131.77,131.54,128.7 9,126.20,122.10,119.44,117.36,116.61,98.56,97.93,96.74,16.53.ESI-MS:m / z 384.03(M+H) + ,C 22 H 17 N 5 O 2 (383.14).

[0076] Example 17: (E)-3-(4-((2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3p)

[0077]

[0078] The preparation method is the same as that of Example 2, except that the raw material is replaced with 2,3-dihydrobenzo[b][1,4]dioxin-6-amine. Brown solid, yield 49.7%. 1 H NMR (400 MHz, DMSO-d 6 )δ10.60(s,1H,NH),9.59(s,1H,-CH=N-),8.37(d,J=5.6Hz,1H,pyrimidyl-H),7.65(s,1H,Ph-H),7.58(d,J=16.7Hz,1H,CH=),7.50(s,2 H,Ph-H),7.42(s,1H,Ph-H),6.73(d,J=8.5Hz,1H,Ph-H),6.52(d,J=5.5Hz,1H,pyrimidyl-H),6.40(d,J=16.7Hz,1H,CH=),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.75,160.73,160.05,155.34,152.02,150.56,132.79,131.77,131.54,128.7 9,126.20,122.10,119.44,117.36,116.61,98.56,97.93,96.74,16.53.ESI-MS:m / z 384.03(M+H) + ,C 22 H 17 N 5 O 2 (383.14).

[0079] Example 18: (E)-3-(4-((2-(Benzo[d]isothiazol-6-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3q)

[0080]

[0081] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzo[d]isothiazol-6-amine. Brown solid, yield 52.6%. 1HNMR (600 MHz, DMSO-d 6 )δ9.98(s,1H,NH),8.53(s,1H,isothiazole-H),8.46(d,J=5.6Hz,1H,pyrimidyl-H),8.18(s,1H,Ph-H),7.94(d,J=8.8Hz,1H,Ph-H),7.72(d,J= 16.7Hz,1H,CH=),7.61(s,2H,Ph-H),7.56(d,J=9.1Hz,1H,Ph-H),6.63(d,J=5.6Hz,1H,pyrimidyl-H),6.53(d,J=16.7Hz,1H,CH=),2.13(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.77,160.86,160.20,155.39,152.33,150.39,144.72,138.34,136.97,131.91 ,131.82,128.77,121.85,120.34,119.35,111.37,98.22,97.01,15.95.ESI-MS:m / z 400.03(M+H) + ,C 22 H 17 N 5 OS(399.12).

[0082] Example 19: (E)-3-(4-((2-(Benzo[c][1,2,5]thiadiazol-5-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3r)

[0083]

[0084] The preparation method is the same as that of Example 2, except that the raw material is replaced with benzo[c][1,2,5]thiadiazole-5-amine. Brown solid, yield 50.4%. 1 H NMR (600 MHz, DMSO-d 6)δ10.16(s,1H,NH),8.53(d,J=5.6Hz,1H,pyrimidyl-H),8.16(s,1H,Ph-H),7.85(d,J=9.4Hz,1H,Ph-H),7.74(d,J=7.4Hz,1H,Ph- H),7.67(d,J=16.7Hz,1H,CH=),7.55(s,2H,Ph-H),6.69(d,J=5.6Hz,1H,pyrimidyl-H),6.45(d,J=16.7Hz,1H,CH=),2.12(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.82,160.83,159.95,156.00,152.00,151.7,150.80,141.78,132.14,131 .50,128.97,126.79,120.90,119.44,105.02,99.28,96.70,16.55.ESI-MS:m / z 399.10(MH) - ,C 21 H 16 N 6 OS(400.11).

[0085] Example 20: (E)-3-(4-((2-(imidazo[1,2-a]pyridin-6-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3s)

[0086]

[0087] The preparation method is the same as that of Example 2, except that the raw material is replaced with imidazo[1,2-a]pyridin-6-amine. White solid, yield 49.7%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.78(s,1H,NH),8.46(d,J=5.7Hz,1H,pyrimidyl-H),7.71(d,J=16.7Hz,1H,CH=),7.61(s,2H,Ph-H),7.44(s,2H,imidazole -H),7.41(s,1H,Ph-H),7.23(d,J=9.7Hz,2H),6.63(d,J=5.5Hz,1H,pyrimidyl-H),6.52(d,J=16.7Hz,1H,CH=),2.12(s,6H,CH 3 ×2). 13C NMR (100 MHz, DMSO-d 6 )δ168.81,160.88,159.92,152.20,150.35,142.04,133.19,131.93,131.89,129.76,128. 80,127.99,121.31,119.34,116.95,113.82,97.86,97.12,16.18.ESI-MS:m / z383.21(M+H) + ,C 22 H 18 N 6 O(382.15).

[0088] Example 21: (E)-3-(4-((2-(imidazo[1,2-a]pyridin-6-ylamino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (3s)

[0089]

[0090] The preparation method is the same as that of Example 2, except that the raw material is replaced with imidazo[1,2-a]pyridin-6-amine. White solid, yield 49.7%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.78(s,1H,NH),8.46(d,J=5.7Hz,1H,pyrimidyl-H),7.71(d,J=16.7Hz,1H,CH=),7.61(s,2H,Ph-H),7.44(s,2H,imidazole -H),7.41(s,1H,Ph-H),7.23(d,J=9.7Hz,2H),6.63(d,J=5.5Hz,1H,pyrimidyl-H),6.52(d,J=16.7Hz,1H,CH=),2.12(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.81,160.88,159.92,152.20,150.35,142.04,133.19,131.93,131.89,129.76,128. 80,127.99,121.31,119.34,116.95,113.82,97.86,97.12,16.18.ESI-MS:m / z383.21(M+H) + ,C 22 H 18 N 6 O(382.15).

[0091] Example 22: (E)-3-(3,5-dimethyl-4-((2-((1-methyl-1H-indazol-5-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3t)

[0092]

[0093] The preparation method is the same as that of Example 2, except that the raw material is replaced with 1-methyl-1H-indazol-5-amine. Pink solid, yield 48.2%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.66(s,1H,NH),8.39(d,J=5.6Hz,1H,pyrimidyl-H),7.73(d,J=16.6Hz,2H,,CH=;imidazole-H),7.59(s,2H,Ph-H),7.54(s,1H,P h-H),7.38(d,J=8.7Hz,1H,Ph-H),7.33(s,1H,Ph-H),6.54(d,J=2.2Hz,1H,pyrimidyl-H),6.52(d,J=9.0Hz,1H,CH=),3.96(s,3H,CH 3 ),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.72,160.73,160.37,152.46,150.58,136.31,133.82,132.06,131.84,131.72,12 8.73,123.86,120.89,119.39,109.67,108.19,97.27,96.77,35.73,16.57.ESI-MS:m / z 397.14(M+H) + ,C 23 H 20 N 6 O(396.17).

[0094] Example 23: (E)-3-(3,5-dimethyl-4-((2-(2-oxoindolin-6-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3u)

[0095]

[0096] The preparation method is the same as that of Example 2, except that the raw material is replaced with 6-aminoindol-2-one. Yellow solid, yield 51.4%. 1H NMR (600 MHz, DMSO-d 6 )δ10.21(s,1H,-CONH),9.53(s,1H,NH),8.36(d,J=5.7Hz,1H,pyrimidyl-H),7.63(d,J=16.7Hz,1H,CH=),7.51(s,2H,Ph-H),7.11(s,1H ,Ph-H),7.06(d,J=8.2Hz,1H,Ph-H),6.81(s,1H,Ph-H),6.46(d,J=5.5Hz,1H,pyrimidyl-H),6.43(d,J=16.7Hz,1H,CH=),3.34(s,2H,CH 2 ),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ177.25,168.68,160.56,160.27,152.11,150.45,144.16,140.06,131.75,129.68,12 8.72,124.30,119.37,119.00,111.76,101.13,97.77,96.82,35.72,16.18.ESI-MS:m / z 396.23(MH) - ,C 23 H 19 N 5 O 2 (397.15).

[0097] Example 24: (E)-3-(3,5-dimethyl-4-((2-(4-methyl-2-oxo-2H-benzopyran-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3v)

[0098]

[0099] The preparation method is the same as that of Example 2, except that the raw material is replaced with 7-amino-4-methyl-2H-chromen-2-one. Grey solid, yield 47.8%. 1 H NMR (600 MHz, DMSO-d 6)δ10.11(s,1H,NH),8.48(d,J=5.7Hz,1H,pyrimidyl-H),7.64(d,J=16.7Hz,1H,CH=),7.54(s,1H,Ph-H),7.53(s,2H,Ph-H),7.42(d,J=10.5 Hz,1H,Ph-H),7.32(d,J=7.8Hz,1H,Ph-H),6.67(d,J=5.6Hz,1H,pyrimidyl-H),6.43(d,J=16.7Hz,1H,CH=),6.17(s,1H,,CH),2.34(s,3H,CH 3 ),2.11(s,6H,CH 3 ×2).ESI-MS:m / z 425.07(M+H) + ,C 25 H 20 N 4 O 3 (424.15).

[0100] Example 25: (E)-3-(3,5-dimethyl-4-((2-(2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3w)

[0101]

[0102] The preparation method is the same as that of Example 2, except that the raw material is replaced with 7-amino-4-(trifluoromethyl)-2H-chromen-2-one. Brown solid, yield 46.2%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.35(s,1H,NH),8.52(d,J=5.6Hz,1H,pyrimidyl-H),7.64(d,J=16.8Hz,1H,CH=),7.59(s,1H,Ph-H),7.56(s,2H,Ph-H),7.37(d,J=9.3 Hz,1H,Ph-H),7.31(d,J=9.1Hz,1H,Ph-H),6.79(s,1H,CH),6.74(d,J=5.6Hz,1H,pyrimidyl-H),6.44(d,J=16.8Hz,1H,CH=),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.78,160.96,159.52,159.20,155.31,152.03,150.34,145.21,139.83(dd,J=32.1Hz),131.97,131.60,128.84,124.9 9,122.16(dd,J=275.5Hz),120.19,119.32,118.05,113.22(dd,J=5.9Hz),106.88,105.35,99.67,96.86,17.00.ESI-MS:m / z 477.07(MH)-,C 25 H 17 F 3 N 4 O 3 (478.13).

[0103] Example 26: (E)-3-(3,5-dimethyl-4-(2-((1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3x)

[0104]

[0105] The preparation method is the same as that of Example 2, except that the raw material is replaced with 7-amino-3,4-dihydroisoquinolin-1(2H)-one. White solid, yield 50.7%. 1 H NMR (400 MHz, DMSO-d 6 )δ9.64(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.94(s,1H,-CONH),7.85(s,1H,Ph-H),7.64(d,J=16.4Hz,1H,CH=),7.54(s,1H,Ph- H),7.51(s,2H,Ph-H),6.89(d,J=8.5Hz,1H,Ph-H),6.48(d,J=5.6Hz,1H,pyrimidyl-H),6.44(d,J=16.8Hz,1H,CH=),3.31(d,J=3.0Hz,2H,CH 2 ),2.77(t,J=6.6Hz,2H,CH 2 ),2.10(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.72,164.97,160.69,160.27,152.15,150.50,148.66,139.26,132.62,131.74,129.90,128 .73,127.58,122.38,119.37,118.30,97.93,96.77,39.8,27.61,15.95.ESI-MS:m / z412.06(M+H) + ,C 24 H 21 N 5 O 2 (411.17).

[0106] Example 27: (E)-3-(3,5-dimethyl-4-(2-((1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (3y)

[0107]

[0108] The preparation method is the same as that of Example 2, except that the raw material is replaced with 7-amino-3,4-dihydroisoquinolin-1(2H)-one. White solid, yield 50.7%. 1 H NMR (400 MHz, DMSO-d 6 )δ9.64(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.94(s,1H,-CONH),7.85(s,1H,Ph-H),7.64(d,J=16.4Hz,1H,CH=),7.54(s,1H,Ph- H),7.51(s,2H,Ph-H),6.89(d,J=8.5Hz,1H,Ph-H),6.48(d,J=5.6Hz,1H,pyrimidyl-H),6.44(d,J=16.8Hz,1H,CH=),3.31(d,J=3.0Hz,2H,CH 2 ),2.77(t,J=6.6Hz,2H,CH 2 ),2.10(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.72,164.97,160.69,160.27,152.15,150.50,148.66,139.26,132.62,131.74,129.90,128 .73,127.58,122.38,119.37,118.30,97.93,96.77,39.8,27.61,15.95.ESI-MS:m / z412.06(M+H) + ,C 24 H 21 N 5 O 2 (411.17).

[0109] Example 28: (E)-tert-butyl 7-((4-(4-(2-cyanovinyl)-2-methylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate (4)

[0110]

[0111] The preparation method is the same as that of Example 2, except that the raw material is replaced with tert-butyl 7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate. Brown solid, yield 44.3%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.58(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.62(d,J=16.7Hz,1H,CH=),7.51(s,2H,Ph-H),7.21(s ,2H,Ph-H),6.85(s,1H,Ph-H),6.54(d,J=5.6Hz,1H,pyrimidyl-H),6.41(d,J=16.4Hz,1H,CH=),4.08(s,2H,CH 2 ),3.48(t,J=5.9Hz,2H,CH 2 ),2.61(t,J=5.9Hz,2H,CH 2 ),2.11(s,6H,CH 3 ×2),1.46(s,9H,CH 3 ×3).ESI-MS:m / z 498.40(M+H) + ,C 29 H 31 N 5 O 3 (497.24).

[0112] Example 29: (E)-3-(3,5-dimethyl-4-(2-(1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (5)

[0113]

[0114] Dissolve 1.0 equivalent of compound 4 in 15 mL of dichloromethane, add 3.0 equivalents of trifluoroacetic acid, stir at room temperature for 2 h, and monitor the reaction completion by TLC. Adjust the pH to 9 with saturated sodium carbonate solution, extract with dichloromethane (3×10 mL), combine the organic phases, wash with saturated sodium chloride (3×20 mL), dry with anhydrous sodium sulfate, filter and concentrate the filtrate under reduced pressure. Recrystallize from ethyl acetate to obtain the crude product 5. White solid, yield 84.3%. ESI-MS: m / z 398.07 (M+H) + ,C 24 H 23 N 5 O(397.19).

[0115] Example 30: (E)-7-(4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinoline-2(1H)-sulfonamide (6a)

[0116]

[0117] (E)-3-(3,5-dimethyl-4-(2-(1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile 5 (0.15 g, 0.37 mmol) was dissolved in 10 mL of dichloromethane, triethylamine (62 μL, 0.45 mmol) and aminosulfonyl chloride (0.043 g, 0.37 mmol) were slowly added under ice bath conditions, reacted at 0°C for 4 hours, and the reaction was completed after TLC monitoring. Water was added to quench the reaction (20 mL), extracted with dichloromethane (3×8 mL), the organic phases were combined, washed with saturated sodium chloride (3×15 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure, and recrystallized from ethyl acetate to obtain compound 6a. White solid, yield 84.8%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.53(s,1H,NH),8.37(d,J=5.6Hz,1H,pyrimidyl-H),7.61(d,J=16.7Hz,1H,CH=),7.51(s,2H,Ph-H),7.26(s,1H,Ph-H),7.18(s,1H,Ph-H),6.89(s,2H,NH 2),6.83(d,J=8.3Hz,1H,Ph-H),6.51(d,J=5.6Hz,1H,pyrimidyl-H),6.41(d,J=16.7Hz,1H,CH=),3.97(s,2H,CH 2 ),3.20(t,J=5.8Hz,2H,CH 2 ),2.76(t,J=5.9Hz,2H,CH 2 ),2.11(s,6H,CH 3 ×2). 13 CNMR (150MHz, DMSO-d 6 )δ168.75,160.66,160.28,152.22,150.39,138.65,132.70,131.78,131.72,128.87,128.68,126 .71,119.36,117.78,116.21,97.80,96.87,48.24,44.33,27.94,17.40.ESI-MS:m / z477.06(M+H) + ,C 24 H 24 N 6 O 3 S(476.16).

[0118] Example 31: (E)-3-(3,5-dimethyl-4-((2-(methylsulfonyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (6b)

[0119]

[0120] The preparation method is the same as that of Example 30, except that the raw material is replaced with methanesulfonyl chloride. White solid, yield 82.6%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.61(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.61(d,J=16.7Hz,1H,CH=),7.52(s,2H,Ph-H),7.25(s,1H,Ph-H),7. 16(s,1H,Ph-H),6.88(d,J=8.4Hz,1H,Ph-H),6.54(d,J=5.6Hz,1H,pyrimidyl-H),6.43(d,J=16.7Hz,1H,CH=),4.02(s,2H,CH 2 ),3.34(d,J=5.7Hz,2H,-CH 2),2.92(s,3H,methylsulfonyl-CH 3 ),2.76(t,J=5.9Hz,2H,-CH 2 ),2.10(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6 )δ168.75,160.69,160.24,152.22,150.48,138.80,132.35,131.75,129.62,129.13,128.66, 126.56,119.41,117.94,115.73,97.81,96.90,47.52,43.67,35.33,27.97,16.59.ESI-MS:m / z 476.06(M+H) + ,C 25 H 25 N 5 O 3 S(475.17).

[0121] Example 32: (E)-dimethyl(7-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)phosphonate (6c)

[0122]

[0123] The preparation method is the same as that of Example 30, except that the raw material is replaced with dimethyl chlorophosphate. White solid, yield 83.7%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.57(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.64(d,J=16.7Hz,1H,CH=),7.52(s,2H,Ph-H),7.16(s,1H,Ph-H),7. 07(s,1H,Ph-H),6.84(d,J=7.2Hz,1H,Ph-H),6.54(d,J=5.4Hz,1H,pyrimidyl-H),6.45(d,J=16.7Hz,1H,CH=),3.84(s,2H,-CH 2 ),3.58(s,3H,-OCH 3 ),3.56(s,3H,-OCH 3 ),3.22(m,2H,-CH 2 ),2.61(t,J=6.0Hz,2H,-CH2 ),2.10(s,6H,CH 3 ×2). 13 C NMR (150 MHz, DMSO-d 6 )δ168.76,160.68,160.26,152.26,150.47,138.55,131.81,131.73,129.60,129.37,128.66, 127.17,119.41,117.60,115.29,97.67,96.86,53.00,46.51,42.23,27.99,16.58.ESI-MS:m / z 506.42(M+H) + ,C 26 H 28 N 5 O 4 P(505.19).

[0124] Example 33: (E)-dimethyl(7-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)-yl)phosphonate (6d)

[0125]

[0126] The preparation method is the same as that of Example 30, except that the raw material is replaced with 2-methylsulfonylacetyl chloride. White solid, yield 72.7%. 1 H NMR (400 MHz, DMSO-d 6 )δ9.62(s,1H,NH),8.39(d,J=5.6Hz,1H,pyrimidyl-H),7.63(d,J=16.7Hz,1H,CH=),7.54(s,2H,Ph-H),7.25(s,1H,Ph-H),7. 12(s,1H,Ph-H),6.87(d,J=8.4Hz,1H,Ph-H),6.54(d,J=5.5Hz,1H,pyrimidyl-H),6.41(d,J=16.6Hz,1H,CH=),4.55(s,2H,CH 2 ),4.31(s,2H,CH 2 ),3.70(t,J=5.9Hz,2H,CH 2 ),3.15(s,3H,CH 3 ),2.77–2.62(m,2H,CH 2 ),2.11(s,6H,CH 3 ×2). 13C NMR (100 MHz, DMSO-d 6 )δ168.76,161.88,160.70,160.23,152.22,150.55,138.86,133.21,131.84,131.72,128.72,127.58,11 9.34,117.70,116.08,97.77,96.84,57.38,44.76,43.93,42.25,28.45,16.59.ESI-MS:m / z518.39(M+H) + ,C 27 H 27 N 5 O 4 S(517.18).

[0127] Example 34: (E)-3-(4-((2-(cyanomethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (6e)

[0128]

[0129] The preparation method is the same as that of Example 30, except that the raw material is replaced with bromoacetonitrile. White solid, yield 81.2%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.56(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.67(d,J=16.6Hz,1H,CH=),7.55(s,2H,Ph-H),7.15(s,1H,Ph-H),7. 07(s,1H,Ph-H),6.83(d,J=8.3Hz,1H,Ph-H),6.54(d,J=5.7Hz,1H,pyrimidyl-H),6.48(d,J=16.7Hz,1H,CH=),3.86(s,2H,CH 2 ),3.24(s,2H,-CH 2 -CN), 2.71(d, J=5.5Hz,2H,-CH 2 ),2.68(d,J=5.4Hz,2H,-CH 2 ),2.11(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.63,160.70,160.25,152.26,150.29,138.36,138.35,134.07,131.77,129.67,128.80,128. 67,126.22,117.62,116.34,115.85,97.59,96.99,54.80,49.08,43.36,27.85,17.00.ESI-MS:m / z 435.13(MH) - ,C 26 H 24 N 6 O(436.20).

[0130] Example 35: (E)-tert-butyl 6-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7)

[0131]

[0132] The preparation method is the same as that of Example 2, except that the raw material is replaced with tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate. Brown solid, yield 44.3%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.58(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidyl-H),7.62(d,J=16.7Hz,1H,CH=),7.51(s,2H,Ph-H),7.21(s ,2H,Ph-H),6.85(s,1H,Ph-H),6.54(d,J=5.6Hz,1H,pyrimidyl-H),6.41(d,J=16.4Hz,1H,CH=),4.08(s,2H,CH 2 ),3.48(t,J=5.9Hz,2H,CH 2 ),2.61(t,J=5.9Hz,2H,CH 2 ),2.11(s,6H,CH 3 ×2),1.46(s,9H,CH 3 ×3).ESI-MS:m / z 498.40(M+H) + ,C 29 H 31 N 5 O 3 (497.24).

[0133] Example 36: (E)-3-(3,5-dimethyl-4-(2-(1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (8)

[0134]

[0135] The preparation method is the same as that of Example 29, except that the raw material is replaced with (E)-6-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester 7. White solid, yield 82.3%. ESI-MS: m / z 398.23 (M+H) + ,C 24 H 23 N 5 O(397.19).

[0136] Example 37: (E)-3-(4-((2-(cyanomethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (6e)

[0137]

[0138] The preparation method is the same as that of Example 30, except that the raw material is replaced with (E)-3-(3,5-dimethyl-4-(2-(1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile 8. White solid, yield 82.3%. 1 HNMR (400 MHz, DMSO-d 6 )δ9.58(s,1H,NH),8.39(d,J=5.5Hz,1H,pyrimidyl-H),7.66(d,J=16.7Hz,1H,CH=),7.55(s,2H,Ph-H),7.22(s,1H,Ph-H),7.12(s,1H,Ph-H),6.87(s,2H,NH 2 ),6.84(s,1H,Ph-H),6.55(d,J=3.9Hz,1H,pyrimidyl-H),6.48(d,J=18.4Hz,1H,CH=),4.03(s,2H,piperidine-CH 2 ),3.19–3.11(m,2H,piperidine-CH 2 ),2.52(s,2H,piperidine-CH 2 ),2.10(s,6H,CH 3 ×2). 13C NMR (100 MHz, DMSO-d 6 )δ168.76,160.65,160.15,152.28,150.51,138.93,133.63,131.74,129.75,128.76,126. 75,125.67,119.34,117.87,117.28,97.69,96.70,47.73,43.15,29.25,14.59.ESI-MS:m / z 475.01(MH) - ,C 24 H 24 N 6 O 3 S(476.16).

[0139] Example 38: (E)-3-(4-((2-(cyanomethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (6e)

[0140]

[0141] The preparation method is the same as that of Example 37, except that the raw material is replaced by methanesulfonyl chloride. White solid, yield 81.5%. 1 HNMR (600 MHz, DMSO-d 6 )δ9.59(s,1H,NH),8.38(d,J=5.6Hz,1H,pyrimidyl-H),7.65(d,J=16.7Hz,1H,CH=),7.53(s,2H,Ph-H),7.22(s,1H,Ph-H),7.11(s, 1H,Ph-H),6.87(d,J=8.5Hz,1H,Ph-H),6.54(d,J=5.6Hz,1H,pyrimidyl-H),6.46(d,J=16.7Hz,1H,CH=),4.20(s,2H,piperidine-CH 2 ),3.31(d,J=5.9Hz,2H,piperidine-CH 2 ),2.90(s,3H,CH 3 ),2.51(s,2H,piperidine-CH 2 ),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.75,160.73,160.21,150.49,139.08,133.60,131.80,131.75,128.74,126.69,125. 32,119.30,118.10,117.41,97.75,96.76,46.07,43.13,33.87,29.30,14.60.ESI-MS:m / z 476.02(M+H) + ,C 25 H 25 N 5 O 3 S(475.17).

[0142] Example 39: (E)-dimethyl(6-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)-3,4-dihydroisoquinolin-2(1H)yl)phosphonate (9c)

[0143]

[0144] The preparation method is the same as that of Example 37, except that the raw material is replaced with dimethyl chlorophosphate. White solid, yield 75.2%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.54(s,1H,NH),8.37(d,J=5.6Hz,1H,pyrimidyl-H),7.65(d,J=16.7Hz,1H,CH=),7.53(s,2H,Ph-H),7.17(s,1H,Ph-H),7.04(s,1H,P h-H),6.81(d,J=8.3Hz,1H,Ph-H),6.53(d,J=5.5Hz,1H,pyrimidyl-H),6.46(d,J=16.7Hz,1H,CH=),4.07(d,J=6.3Hz,2H,piperidine-CH 2 ),3.55(s,3H,-OCH 3 ),3.53(s,3H,-OCH 3 ),3.20(d,J=9.0Hz,2H,piperidine-CH 2 ),2.35(s,2H,piperidine-CH 2 ),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.75,160.73,160.22,152.30,150.50,138.69,134.26,131.78,131.71,128.74,126.88, 126.23,119.30,118.52,117.23,97.61,96.69,52.97,45.70,42.10,29.38,16.55.ESI-MS:m / z 506.02(M+H) + ,C 26 H 28 N 5 O 4 P(505.19).

[0145] Example 40: (E)-3-(3,5-dimethyl-4-(2-(2-(methylsulfonyl)acetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (9d)

[0146]

[0147] The preparation method is the same as that of Example 37, except that the raw material is replaced with 2-methylsulfonylacetyl chloride. White solid, yield 81.6%. 1 H NMR(400MHz,Chloroform-d)δ9.59(s,1H,NH),8.38(d,J=5.7Hz,1H,pyrimidyl-H),7.65(d,J=16.8Hz,1H,CH=),7.53(d,J=10.3Hz,2H,Ph-H),7. 22(s,1H,Ph-H),7.12(s,1H,Ph-H),6.87(d,J=8.3Hz,1H,Ph-H),6.53(d,J=5.5Hz,1H,pyrimidyl-H),6.46(d,J=16.7Hz,1H,CH=),4.59(s,1H,CH 2 ×1 / 2),4.51(s,3H,CH 2 ; CH 2 ×1 / 2),3.62(m,2H,CH 2 ),3.12(s,3H,CH 3 ),2.58(s,1H,CH 2 ×1 / 2),2.37(s,1H,CH 2 ×1 / 2),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d 6)δ168.74,161.82,161.71,160.72,160.22,152.25,150.48,139.03,134.74,131.79,128.71,126.67, 126.51,119.36,118.01,117.40,97.74,96.85,57.44,47.26,42.25,30.26,28.87,16.00.ESI-MS:m / z 518.47(M+H) + ,C 27 H 27 N 5 O 4 S(517.18).

[0148] Example 41: (E)-3-(3,5-dimethyl-4-(2-(2-(methylsulfonyl)ethyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)amino)pyrimidin-4-yl)oxy)phenyl)acrylonitrile (9e)

[0149]

[0150] The preparation method is the same as that of Example 37, except that the raw material is replaced with dimethyl chlorophosphate. White solid, yield 75.2%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.51(s,1H,NH),8.36(d,J=5.6Hz,1H,pyrimidyl-H),7.64(d,J=16.7Hz,1H,CH=),7.52(s,2H,Ph-H),7.14(s,1H,Ph-H),7.03(s, 1H,Ph-H),6.73(d,J=8.3Hz,1H,Ph-H),6.51(d,J=5.6Hz,1H,pyrimidyl-H),6.45(d,J=16.7Hz,1H,CH=),3.47(s,2H,piperidine-CH 2 ),3.35(t,J=6.8Hz,2H,CH 2 ),3.00(s,3H,CH 3 ),2.84(t,J=6.8Hz,2H,CH 2 ),2.60(t,J=5.9Hz,2H,piperidine-CH 2 ),2.43(s,2H,piperidine-CH 2 ),2.09(s,6H,CH 3 ×2). 13 C NMR (100 MHz, DMSO-d6 )δ168.74,160.68,160.26,152.30,150.54,138.55,134.22,131.76,131.73,128.72,127.89,126.55,119.3 8,118.07,116.90,97.51,96.71,55.03,51.73,51.40,50.63,42.03,29.67,16.56.ESI-MS:m / z504.00(M+H) + ,C 27 H 29 N 5 O 3 S(503.20).

[0151] Example 42: Anti-HIV Activity Experiment (MT-4 Cell Model)

[0152] See ① Pauwels R, et al. J. Virol. Methods. 1988, 20, 309. ② Pannecouque C, et al. Nat Protocols 2008, 3, 427.

[0153] Explanation of terms

[0154] MT-4 cells: human acute lymphoblastic leukemia cells; MTT assay: MTT is 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide; trade name: Thiazole blue; DMSO: dimethyl sulfoxide.

[0155] Test Principle

[0156] Since HIV-infected MT-4 cells will develop pathological changes within a certain period of time (5-7 days), a solution of the compound to be tested was added to the suspension of HIV-infected MT-4 cells. After a period of culture (5-7 days), the viability of MT-4 cells was determined by MTT assay to obtain the drug concentration that protects 50% of cells from cytopathic effects (EC 50 ) can be used to obtain the anti-HIV activity of the target compound. At the same time, the concentration of the target compound that causes 50% of cells not infected with HIV to become pathological (CC 50 ). Principle of MTT analysis: MTT is 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, which can bind to succinate dehydrogenase in living cells but not react with dead cells. MTT method is an enzyme analysis method that quickly reflects cell viability.

[0157] Test Materials and Methods

[0158] (1) HIV-1 wild-type strain III B , single mutants L100I, K103N, Y181C, Y188L, E138K and double mutants K103N / Y181C (RES056) and F227L / V106A, HIV-2 ROD strain: provided by the Institute of Microbiology and Immunology, Rega Institute, University of Leuven, Belgium.

[0159] (2) MT-4 cells: provided by the Institute of Microbiology and Immunology, Rega Institute, University of Leuven, Belgium.

[0160] (3) MTT: purchased from Sigma, USA.

[0161] (4) Sample treatment: Before use, the sample was dissolved in DMSO to an appropriate concentration and diluted 5-fold with double distilled water, with 5 dilutions each.

[0162] (5) Positive control drugs: doravirine (DOR), nevirapine (NVP), efavirenz (EFV), and etravirine (ETV).

[0163] (6) Test method: The sample was diluted and added to the suspension of HIV-infected MT-4 cells. After a period of time, the cell viability was determined by MTT colorimetry. The absorbance (A) value was recorded at 590 nm in an ELISA reader and the EC was calculated. 50 、CC 50 and SI.

[0164] (7) MTT staining method: After adding the sample and culturing for a period of time, add 20 μL of MTT solution to each well and continue culturing for several hours. Then discard the staining solution and add 150 μL of DMSO to each well. Mix thoroughly and record the absorbance at 590 nm in a microplate reader.

[0165] The specific operation is as follows: the compound is dissolved in DMSO or water and then diluted with phosphate buffer. 5 MT-4 cells were pre-incubated with 100 μL of compound solutions of different concentrations at 37°C for 1 h. Then, 100 μL of virus dilutions of appropriate concentrations were added to the mixture, and the cells were incubated at 37°C for 1 h. After washing three times, the cells were resuspended in culture matrix with or without compounds. The cells were then incubated at 5% CO 2The cells were incubated at 37°C for another 7 days in an environment, and the original culture medium was supplemented with culture medium containing or without the compound on the third day after infection. Each culture condition was repeated twice. The cytopathic effect of the virus was monitored every day using an inverted optical microscope. In general, the virus dilution used in this experiment often causes cytopathic effects on the fifth day after virus infection. The drug inhibitory concentration (EC) is the concentration at which the drug produces a 50% inhibitory effect on the virus-induced cytopathic effect while being non-toxic to the cells. 50 ) is indicated. It is worth emphasizing that when the compound is poorly water-soluble and needs DMSO to dissolve, the volume ratio of DMSO relative to water is generally less than 10% (the final concentration of DMSO in MT-4 cell culture medium is less than 2%). Because DMSO can affect the antiviral activity of the test compound, the antiviral activity comparison blank experiment containing the same concentration of DMSO solution should also be performed in parallel. In addition, the final DMSO concentration (1 / 1000) is far lower than the concentration required to affect HIV-1 replication in MT-4 cells.

[0166] The in vitro anti-HIV activity screening data of the target compounds were provided by the Institute of Microbiology and Immunology, Rega Institute, University of Leuven, Belgium. All activity data were measured in at least three independent and parallel experiments.

[0167] First, we tested the antiviral activity and cytotoxicity of all compounds against HIV-1 wild-type strains, RES056 mutant strains, and HIV-2 ROD strains. As shown in Table 1, the diaryl pyrimidine compounds containing fused heterocycles provided by the present invention showed good anti-HIV-1 wild-type activity, and their EC 50 The range is between 0.0018 and 0.12 μM. Among them, compound 3a (EC 50 =0.0019 μM), 3d (EC 50 =0.0019 μM), 3k (EC 50 =0.0019 μM), 3s (EC 50 =0.0020 μM) and 3u(EC 50 =0.0018 μM) was significantly more active than all control drugs (EC 50 =0.0032–0.13 μM); for HIV-1RES056 mutant, 3k (EC 50 =0.35μM) showed the highest antiviral activity, which was better than the marketed drug NVP (EC 50 >9.51μM, comparable to EFV (EC 50 =0.36 μM); for HIV-2 ROD strain, three compounds 3a, 3k, and 3r showed micromolar antiviral activity (EC 50=1.26–7.04 μM), significantly superior to all control drugs (which showed no antiviral activity at the tested concentrations).

[0168] Table 1 Anti-wild-type and RES056 mutant HIV-1 and HIV-2 activity and cytotoxicity of the compounds

[0169]

[0170]

[0171] Note: a EC 50 : The concentration of compound that protects 50% of HIV-infected MT-4 cells from cytopathic effects; b CC 50 : The concentration of the target compound that causes 50% of cells not infected with HIV to become diseased; c SI: Selection Index, CC 50 With EC 50 The ratio of d NA: The result cannot be calculated.

[0172] The compounds with excellent antiviral activity in the first round of testing were further tested for their activity against HIV-1 mutants L100I, K103N, Y181C, Y188L, E138K and F227L / V106A. As shown in Table 2, compound 3k (EC 50 =0.0019 μM) was particularly active against the K103N mutant, outperforming all positive control drugs (EC 50 =0.0030–5.22 μM); the activities for mutants L100I, Y181C, and E138K were 0.0087, 0.032, and 0.011 μM, respectively, which were better than or equivalent to the positive control drug. Therefore, this type of compound has great research and development value and can be used as a candidate drug for the preparation of anti-HIV.

[0173] Table 2 Antiviral activity of compounds against common clinical mutants of HIV-1

[0174]

[0175] Note: a EC 50 : The concentration of compound that protects 50% of HIV-1 infected MT-4 cells from cytopathic effects.

[0176] Example 43: Anti-CHIKV activity experiment (SJCRH30 cell model)

[0177] See ①Gupta DK, et al. Mar Drugs. 2014, 12(1), 115-27.

[0178] Test Principle

[0179] Alamar-Blue Cell Viability Assay Kit is a kit for detecting cell proliferation using the redox indicator resazurin. Resazurin is in an oxidized state, appears blue and has little fluorescence. When it is taken up by cells or bacteria and reduced by metabolic enzymes, it turns into a pink reduction product, resorufin. Resorufin can emit strong red fluorescence, and changes in cell vitality can be detected by fluorescence.

[0180] The plaque test is an experimental method used to measure the infectivity of a virus. A virus suspension of appropriate concentration is added to a monolayer of cells that has already formed to allow the virus to adsorb, and then covered with a layer of melted agar. After each virus replicates in the infected cell, it can produce a localized infection focus, the plaque. When living cells are stained with neutral red, unstained plaques can be seen. Each plaque is produced by the replication of an infectious virus body, called a plaque forming unit (PFU), that is, one plaque is equivalent to one virus body. The amount of infectious virus contained in the virus suspension is expressed in plaque forming units that can be formed per milliliter, that is, PFU / ml.

[0181] Test Materials and Methods

[0182] (1) Cell and compound preparation: BHK21 (baby hamster kidney) cells and SJCRH30 rhabdomyosarcoma cells (ATCC CRL-2061) were cultured at 37°C and 5% CO 2 Culturing was carried out under the condition of 1% fetal calf serum (FCS) in RPMI-1640 medium. C6 / 36 mosquito cells isolated from embryonic tissue of Aedes albopictus were cultured in L-15 medium containing 10% FCS at 28°C. CHIKV-122508 (SGEHICHD 122508, provided by the Institute of Environmental Health, Singapore) was propagated in C6 / 36 cells and stored at -80°C before use. Compounds were dissolved in DMSO to a final stock concentration of 10 mM and stored at -80°C.

[0183] (2) Cell viability assay: SJCRH30 cells were seeded in a 96-well cell culture plate and incubated at 37°C and 5% CO 2 The compounds were diluted in 2% FCS and then incubated overnight at 37°C and 5% CO 2After the incubation period, the cells were washed once with phosphate-buffered saline (PBS) and 10 μL of The reagents were then incubated at 37°C and 5% CO 2 The cells were incubated for 4 h under the same conditions. A microplate reader (Tecan, The detected fluorescence was read at an excitation wavelength of 570 nm and an emission wavelength of 600 nm (Sigma, Switzerland). The measured values ​​of cells treated with the compound or 0.1% DMSO solvent (control) were normalized to those of untreated cells. Cytotoxic concentration (CC 50 ) was defined as the concentration of the compound that caused 50% of the cells to become cytopathic.

[0184] (3) Drug inhibition study: SJCRH30 cells were seeded into 96-well plates and cultured at 37°C and 5% CO 2 The cells were incubated overnight under the conditions of 10% PBS. The cells were then infected with CHIKV-122508 at 1 MOI (multiplicity of infection) for 1.5 hours. Subsequently, the cells were washed twice with PBS, the desired drug concentration was added to the cells and incubated for 24 hours. Subsequently, the supernatant of CHIKV infected cells was extracted for viral plaque assay. Three independent experiments were performed on each drug concentration group used.

[0185] (4) Virus plaque assay: BHK cells were seeded on 24-well plates and incubated at 37°C and 5% CO 2 The supernatant of CHIKV treatment was diluted 10-fold from 101 to 106 serially, and 100 μL of the diluted supernatant was added to BHK cells. Incubation was performed for 1.5 h with 15 min shaking intervals for virus binding. After incubation, the plates were washed twice with PBS and covered with 1% carboxymethylcellulose (CMC). The assay plates were incubated at 37 °C and 5% CO 2 The cells were incubated for 3 days under the same conditions. CMC was removed and the cells were stained and fixed with 10% paraformaldehyde-1% crystal violet (Sigma-Aldrich Chemical, St. Louis, MO, USA) solution to observe and count plaques. Virus titers were expressed as plaque forming units (PFU) per ml.

[0186] The in vitro anti-CHIKV activity screening data of the target compounds were provided by Yong Loo Lin School of Medicine, National University of Singapore. All activity data were measured in at least three independent and parallel experiments.

[0187] First, the cytotoxicity of the compounds was tested using the Alamar Blue Cell Viability Assay Kit, and the compounds with a cell viability of more than 80% at a concentration of 10 μM were further tested for their anti-CHIKV activity. As shown in Table 3, compounds 3b, 3g, 3j, 3k, 3q, 3r, 6a, and 9d showed good anti-CHIKV activity at a concentration of 10 μM. In particular, compounds 3q and 3r showed 3.4-log and 3.1-log inhibitory activity, respectively, compared to the negative control (0.1% DMSO). This is the first report that diaryl pyrimidine derivatives containing fused heterocycles have anti-CHIKV activity. Given that no specific drugs for CHIKV have been approved for marketing at this stage, this type of compound has great research and development value and can be used as a candidate drug for the preparation of anti-CHIKV.

[0188] Table 3 Activity of compounds in inhibiting CHIKV replication

[0189] Compounds <![CDATA[ a Inhibition at 10μM]]> 3b 1.61-log 3g 1.98-log 3j 1.99-log 3k 1.41-log 3q 3.4-log 3r 3.1-log 6a 1.74-log 9d 1.86-log

[0190] a Log inhibition of CHIKV by compounds at 10 μM concentration compared to 0.1% DMSO.

[0191] IV. Conclusion

[0192] The diaryl pyrimidine derivatives containing fused heterocycles provided by the present invention are a series of compounds with novel structures. As can be seen from Table 1, the activities of the five compounds 3a, 3d, 3k, 3s and 3u against HIV-1 wild strains are significantly better than all control drugs; for HIV-2 ROD strains, the antiviral activities of compounds 3a, 3k and 3r are significantly better than all control drugs. As can be seen from Table 2, the activity of compound 3k against the K103N mutant strain is significantly better than all positive control drugs. As can be seen from Table 3, compared with the negative control (0.1% DMSO), compounds 3q and 3r showed significant anti-CHIKV activity at a concentration of 10 μM. Therefore, this type of compound has a very great research and development value and can be used and developed as a candidate drug for preparing anti-HIV and anti-CHIKV.

Claims

1. A diaryl pyrimidine compound containing a fused heterocyclic ring or a pharmaceutically acceptable salt thereof, having a structure shown in the following general formula I: in, R1 is CH3, CN, p-C4H6-CN or CH=CHCN; R2 is H, CH3, CN, CF3, COCH3, NH2, NO2, F, Br, I, Cl, PO(CH3)2, PO(OCH3)2, SO2CH3, SOCH3, OCH3, SCH3, NHCH3, NHCOCH3 or NHSO2CH3; R3 is H, CH3, CN, CF3, COCH3, NH2, NO2, F, Br, I, Cl, PO(CH3)2, PO(OCH3)2, SO2CH3, SOCH3, OCH3, SCH3, NHCH3, NHCOCH3 or NHSO2CH3; X is 2. The diaryl pyrimidine compound containing a fused heterocycle according to claim 1, characterized in that The diaryl pyrimidine compound containing a fused heterocyclic ring is one of the following:

3. The diaryl pyrimidine compound containing a fused heterocycle according to claim 1, characterized in that The pharmaceutically acceptable salt of the compound is sodium salt, hydrochloride, sulfate, tartrate or citrate.

4. The diaryl pyrimidine compound containing a fused heterocycle as claimed in claim 2, characterized in that the step include: A method for preparing a diaryl pyrimidine derivative containing a fused heterocycle, comprising the following steps: first, using 2,4-dichloropyrimidine 1 as a raw material, reacting it with (E)-3,5-dimethyl-4-hydroxyphenyl acrylonitrile in an N,N-dimethylformamide solution to generate an intermediate 2; the intermediate 2 and the fused heterocycle aniline undergo a coupling reaction under the catalysis of palladium acetate to obtain a target compound 3(ax) or an intermediate 4 or 7; then, removing the Boc group from the intermediate 4 or 7 to obtain an intermediate 5 or 8, and the intermediate 5 or 8 reacts with different sulfonyl chlorides, phosphorus oxychlorides, acyl chlorides or alkyl halides to obtain a final product 6(ae) or 9(ae); Reagents and conditions: (i) (E)-3,5-dimethyl-4-hydroxybenzene acrylonitrile, N,N-dimethylformamide, potassium carbonate, 50°C; (ii) palladium acetate, 4,5-bis(diphenylphosphino-9,9-dimethylxanthene), cesium carbonate, nitrogen, 1,4-dioxane, 90°C; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) sulfonyl chloride, phosphorus oxychloride, acyl chloride or alkyl halide, triethylamine, 0°C.

5. Use of the diaryl pyrimidine compound containing a fused heterocycle as claimed in claim 1 in the preparation of anti-HIV drugs.

6. Use of the diaryl pyrimidine compound containing a fused heterocycle as claimed in claim 1 in the preparation of an anti-Chikungunya virus drug.

7. A pharmaceutical composition comprising the diaryl pyrimidine compound containing a fused heterocycle according to claim 1 and one or more pharmaceutically acceptable carriers or excipients.

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