Patent ID: 12258331

DESCRIPTION OF THE EMBODIMENTS

The present invention will be further described in detail in combination with the following embodiments, but the implementation of the invention is not limited thereto.

Embodiment 1: Anti-Tumor Compound (±)-5b

Anti-tumor compound (±)-5b, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound (±)-5b

Preparation of intermediate 2: An aqueous solution (32 mL water) of sodium carbonate (66 mmol) were added to a solution of 5-fluoro-2-chloropyrimidine (33 mmol) in toluene (32 mL) and bis(triphenyl phosphine) palladium chloride (0.33 mmol), then added to a solution of acetyl phenylboronic acid (32 mmol) in ethanol (65 mL) dropwise. The reaction was stirred at 80° C. under the protection of nitrogen for 18 hours, cooled to room temperature, and filtrated. The filtrates were added into ethyl acetate and water, the organic phase was separated, dried over anhydrous sodium sulfate, and then the solvent was removed in vacuum. The crude product was separated by silica gel column chromatography (ethyl acetate:petroleum ether, 8:1) to get a light yellow solid, i.e., the intermediate 2. Yield: 61%.1H NMR (400 MHz, CDCl3) δ 8.98 (t, J=1.6 Hz,1H), 8.70 (s, 2H), 8.59 (s,1H), 8.09 (s,1H), 7.60 (s,1H), 2.71 (s, 3H).

Preparation of intermediate 3b: At 0° C., sodium borohydride (15 mmol) was added to a suspension of intermediate 2 (10 mmol) in absolute ethyl alcohol/tetrahydrofuran batchwise. After stirring at 0° C. for 1 hour, the ice bath is removed. After stirring at room temperature for 1 hour, water was slowly added into the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate for three times. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and rotary evaporated to get a crude product. The crude product was purified by column chromatography to obtain a white solid, i.e., the intermediate 3b (ethyl acetate:petroleum=4:1), yield: 83%.1H NMR (500 MHz, CDCl3) δ 8.65 (s, 2H), 8.38 (s,1H), 8.28 (d, J=7.5 Hz,1H), 7.52 (d, J=7.5 Hz,1H), 7.47 (t, J=7.6 Hz,1H), 5.01 (dd, J=6.0, 3.0 Hz,1H), 1.56 (d, J=6.4 Hz, 3H).

Synthesis of intermediate 4b: The intermediate 3b (5 mmol) was added batchwise into sulfoxide chloride (25 mmol) with stirring, and reacted at 30° C. for 2 hours. Excessive sulfoxide chloride was removed under reduced pressure. The residues were added into toluene and stirred. Toluene was removed under reduced pressure to get a chlorinated intermediate. The chlorinated intermediate and potassium carbonate (7.5 mmol) were added into a solution of 3-(6-oxo-1,6-dihydropyridazin-3-yl)-benzonitrile (6 mmol) in N-methyl pyrrolidinone, stirred at 80° C. to react for 20 hours. The reaction was quenched with water, extracted with ethyl acetate for 3 times. The organic phase was washed with saturated saline, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The crude product was separated by silica gel column chromatography (dichloromethane:methanol=100:1) to get a yellow solid, i.e., the intermediate 4b, yield: 68%.1H NMR (400 MHz, CDCl3) δ 8.70 (s, 3H), 8.33 (d, J=7.8 Hz,1H), 8.19 (s,1H), 7.98 (d, J=8.0 Hz,1H), 7.70 (d, J=7.7 Hz,1H), 7.66-7.52 (m, 3H), 7.46 (t, J=7.7 Hz,1H), 7.04 (d, J=9.7 Hz,1H), 6.56 (q, J=7.0 Hz,1H), 1.93 (d, J=7.1 Hz, 3H).

Synthesis of target (±)-5b: At 0° C., sodium hydride (60%, 1.5 mmol) was added batchwise into a solution of (1-methylpiperidin-4-yl)methanol (1.2 mmol) in N,N-dimethyl formamide, and stirred for 15 minutes. The intermediate 4b was then added into the reaction solution slowly at 0° C., warmed to room temperature slowly and stirred for 1 hour. The reaction was quenched with water, and extracted with ethyl acetate. The combined organic layer was dried, and concentrated. The crude product was purified by silica-gel column chromatography (dichloromethane:methanol=20:1) to obtain light yellow oil, i.e., the target (±)-5b, yield: 69%.1H NMR (400 MHz, CDCl3) δ 8.67 (s,1H), 8.49 (s, 2H), 8.28 (d, J=7.8 Hz,1H), 8.20 (s,1H), 7.97 (d, J=8.0 Hz,1H), 7.68 (d, J=7.7 Hz,1H), 7.61-7.50 (m, 3H), 7.42 (t, J=7.7 Hz,1H), 7.02 (d, J=9.7 Hz,1H), 6.54 (q, J=7.0 Hz,1H), 3.96 (d, J=5.9 Hz, 2H), 2.94 (d, J=11.5 Hz, 2H), 2.31 (s, 3H), 1.99 (d, J=10.2 Hz, 2H), 1.91 (d, J=7.0 Hz, 3H), 1.85 (m, 3H), 0.1.50 (dd, J=18.4, 8.6 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 159.22, 157.76, 151.21, 144.04, 141.88, 140.66, 137.72, 136.40, 132.46, 130.29, 129.82, 129.79, 129.74, 129.69, 128.79, 128.71, 127.30, 126.98, 118.70, 113.38, 71.78, 56.23, 54.19, 43.99, 33.64, 26.06, 20.12. HRMS (ESI) m/z calculated C30H31N6O2[M+H]+, 507.2508; found, 507.2519. HPLC purity: 95.2%, retention time: 16.68 min.

Compound (±)-5a was prepared with a similar method.

Embodiment 2: Anti-Tumor Compound 6a

Anti-tumor compound 6a, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound 6a

Preparation of intermediate 3a: Into an aqueous solution (32 mL) of sodium carbonate (66 mmol) were added a solution of 5-fluoro-2-chloropyrimidine (33 mmol) in toluene (32 mL) and bis(triphenyl phosphine) palladium dichloride (0.33 mmol), then added a solution of hydroxymethyl phenyl boronic acid (32 mmol) in ethanol (65 mL). The reaction was stirred at 80° C. under the protection of nitrogen for 18 hours, cooled to room temperature, and filtrated. Into the filtrate were added ethyl acetate and water. The organic phase was separated, and dried over anhydrous sodium sulfate. The crude product was separated by silica gel column chromatography (ethyl acetate:petroleum ether, 8:1) to get a light yellow solid, i.e., the intermediate 3a. Yield: 61%.1H NMR (400 MHz, CDCl3) δ 8.66 (d, J=1.8 Hz,1H), 8.37 (d, J=4.9 Hz,1H), 8.31 (s,1H), 7.54-7.46 (m, 3H), 4.80 (s,1H), 1.99 (s,1H).

Preparation of intermediate 4a: The intermediate 3a (5 mmol) was added into sulfoxide chloride (25 mol) batchwise with stirring, and reacted at 25° C. for 2 hours. Excessive sulfoxide chloride was removed under reduced pressure. The residues were added into toluene. Toluene was removed under reduced pressure to get a chloride as the crude product. The crude product and potassium carbonate (2.75 mol) were added into a solution of 3-(6-oxo-1,6-dihydropyridazin-3-yl)-benzonitrile in N,N-dimethyl formamide, and stirred at 80° C. to react for 20 hours. The reaction was quenched with water, extracted with ethyl acetate for 3 times. The organic phase was washed with saturated saline, dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The crude product was separated by silica gel column chromatography (dichloromethane:methanol: 100:1) to get a yellow solid, i.e., the intermediate 4a, yield: 68%.1H NMR (500 MHz, CDCl3) δ 8.68 (s, 2H), 8.60 (s,1H), 8.34 (d, J=7.8 Hz,1H), 8.16 (s,1H), 7.99 (d, J=8.0 Hz,1H), 7.70 (d, J=7.7 Hz,1H), 7.65 (d, J=9.7 Hz,1H), 7.62-7.55 (m, 2H), 7.48 (t, J=7.7 Hz,1H), 7.09 (s,1H), 5.51 (s, 2H).

Synthesis of target 6a: N,N-dimethylamino chloropropane hydrochloride (2 mmol) and thiourea (2 mmol) are dissolved in absolute ethyl alcohol (50 mL), refluxed for 7 hours under the protection of nitrogen, cooled to room temperature, and rotary evaporated under reduced pressure to remove the solvent, thus obtaining isothiourea analogue, which was directly used in the following step without further purification. The intermediate 4a (1 mmol), isothiourea analogue (2 mmol) and sodium hydroxide solid (4 mmol) were placed in a single-port flask, with the nitrogen being replaced, into which were then added N,N-dimethyl formamide/water=5:1, stirred at room temperature for 15 minutes, then stirred at 60° C. for 3 hours. Upon the completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with water and brine, rotary evaporated and concentrated. The crude product was purified by column chromatography (CH2Cl2:CH3OH=20:1) to obtain a yellow oil, i.e., the target 6a, yield: 56%.1H NMR (400 MHz, CDCl3) δ 8.77 (s, 2H), 8.62 (s,1H), 8.36 (d, J=7.8 Hz,1H), 8.16 (s,1H), 8.00 (d, J=8.1 Hz,1H), 7.69 (s,1H), 7.65 (d, J=9.7 Hz,1H), 7.58 (dd, J=14.7, 7.0 Hz, 2H), 7.47 (t, J=7.7 Hz,1H), 7.08 (d, J=9.7 Hz,1H), 5.51 (s, 2H), 3.03 (t, J=7.3 Hz, 2H), 2.41 (t, J=7.0 Hz, 2H), 2.22 (s, 6H), 1.83 (dt, J=14.1, 7.1 Hz, 4H).13C NMR (101 MHz, CDCl3) δ 161.75, 159.45, 157.63, 142.21, 137.56, 136.25, 135.96, 132.58, 131.26, 130.85, 130.33, 129.93, 129.82, 129.71, 129.42, 129.11, 128.73, 127.81, 118.44, 113.39, 57.96, 55.49, 45.42, 31.75, 27.16. HRMS (ESI) m/z C27H26N6OS [M+H]+, calculated 483.1962; found 483.1947. HPLC purity: 96.8%, retention time: 9.65 min.

Compounds 6b-6h, 7a-7j were prepared with a similar method.

Embodiment 3: Anti-Tumor Compound (R)-5b

Anti-tumor compound (R)-5b, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound (R)-5b

Preparation of intermediate 8b: At 0° C., sodium hydride (60%, 1.5 mmol) was added into a solution of (1-methylpiperidin-4-yl)methanol (1.2 mmol) in N,N-dimethyl formamide (10 mL) batchwise, and stirred for 15 minutes. The intermediate 2 was then slowly added into the mixture at 0° C. The reaction mixture was warmed to room temperature slowly and stirred for 1 hour. The reaction solution was washed with water, and extracted with ethyl acetate. The combined organic layer was dried, rotary evaporated to obtain a crude product. The crude product was purified by silica-gel column chromatography (dichloromethane:methanol=20:1) to obtain light yellow oil, i.e., the intermediate 8b, yield: 73%.1H NMR (400 MHz, CDCl3) δ 8.93 (t, J=1.5 Hz,1H), 8.56 (dt, J=7.8, 1.3 Hz,1H), 8.47 (s, 2H), 8.04 (dt, J=7.7, 1.3 Hz,1H), 7.57 (t, J=7.8 Hz,1H), 3.97 (d, J=5.8 Hz, 2H), 2.93 (d, J=11.0 Hz, 2H), 2.70 (s, 3H), 2.31 (s, 3H), 2.06-1.91 (m, 4H), 1.88 (d, J=2.6 Hz,1H), 1.48 (qd, J=13.6, 13.1, 3.6 Hz, 2H).

Preparation of intermediate (S)-9b: Dichloro(p-cymene)ruthenium (II) dimer (0.01 mmol) and a chiral ligand (S,S)-CsDPEN (0.02 mmol) were placed in a 50 mL single-port flask, into which was added pure water (5 mL), and stirred at 40° C. for 4 hours under the protection of nitrogen. After then, the intermediate 8b (1 mmol, dissolved in 8 mL dichloromethane) and sodium formate (5 mmol, dissolved in 3 mL pure water) were promptly added, and stirred at room temperature for 10 hours. Upon the completion of the reaction, 10 mL water was added into the reaction system. The water phase was extracted with dichloromethane. The combined organic layer was dried and concentrated. The crude product was purified by silica-gel column chromatography (dichloromethane:methanol=20:1) to obtain light yellow oil, i.e., the intermediate (S)-9b, yield: 70%.1H NMR (400 MHz, CDCl3) δ 8.44 (s, 2H), 8.34 (s,1H), 8.24 (d, J=7.1 Hz,1H), 7.53-7.42 (m, 2H), 4.99 (q, J=6.4 Hz,1H), 4.12 (t, J=6.3 Hz, 2H), 2.45 (t, J=7.2 Hz, 2H), 2.25 (s, 6H), 2.02-1.94 (m, 2H), 1.55 (d, J=6.4 Hz, 3H). 98.0% ee, Daicel AD column (0.46×25 cm), n-hexane/isopropanol=75/25, 0.5 mL/min, λ=254 nm, tR=15.79 (R), tS=18.85 (S).

Synthesis of the target (R)-5b: At 0° C., diisopropyl azodicarboxylate (0.600 mmol, dissolved in 1 mL N,N-dimethyl formamide) was slowly added into a solution of 3-(6-oxo-1,6-dihydropyridazin-3-yl)-benzonitrile (0.40 mmol), the intermediate (S)-9b (0.40 mmol) and triphenyl phosphine (0.60 mmol) in tetrahydrofuran (4 mL), warmed to room temperature slowly under the protection of nitrogen, stirred at room temperature for 18 hours, and rotary evaporated under reduced pressure to remove the solvent. The crude product was purified by silica-gel column chromatography (dichloromethane/methanol=20:1) to obtain a colorless oil, i.e., the target (R)-5b, yield: 57%.1H NMR (400 MHz, CDCl3) δ 8.67 (s,1H), 8.49 (s, 2H), 8.28 (d, J=7.8 Hz,1H), 8.20 (s,1H), 7.97 (d, J=8.0 Hz,1H), 7.68 (d, J=7.7 Hz,1H), 7.61-7.50 (m, 3H), 7.42 (t, J=7.7 Hz,1H), 7.02 (d, J=9.7 Hz,1H), 6.54 (q, J=7.0 Hz,1H), 3.96 (d, J=5.9 Hz, 2H), 2.94 (d, J=11.5 Hz, 2H), 2.31 (s, 3H), 1.99 (d, J=10.2 Hz, 2H), 1.91 (d, J=7.0 Hz, 3H), 1.85 (m, 3H), 1.50 (dd, J=18.4, 8.6 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 159.22, 157.76, 151.21, 144.04, 141.88, 140.66, 137.72, 136.40, 132.46, 130.29, 129.82, 129.79, 129.74, 129.69, 128.79, 128.71, 127.30, 126.98, 118.70, 113.38, 71.78, 56.23, 54.19, 43.99, 33.64, 26.06, 20.12. HRMS (ESI) m/z calculated C30H31N6O2[M+H]+, 507.2508; found, 507.2519. HPLC purity: 95.8%, retention time: 16.71 min.

Compounds (S)-5b, (R)-5a and (S)-5a were prepared with a similar method.

Embodiment 4: Anti-Tumor Compound (R)-6f

Anti-tumor compound (R)-6f, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound (R)-6f

Preparation of intermediate (S)-3b: Dichloro(p-cymene)ruthenium (II) dimer (0.05 mmol) and a chiral ligand (S,S)-CsDPEN (0.10 mmol) were placed in a 100 mL single-port flask, into which was added pure water (25 mL), and stirred at 40° C. under the protection of nitrogen for 4 hours. After then, the intermediate 2 (5 mmol, dissolved in 40 mL dichloromethane) and sodium formate (25 mmol, dissolved in 15 mL pure water) were promptly added, and stirred at room temperature for 10 hours. Upon the completion of the reaction, 30 mL water was added into the reaction system. The water phase was extracted with dichloromethane. The combined organic layer was dried and concentrated. The crude product was purified by silica-gel column chromatography (petroleum ether:ethyl acetate=2:1) to obtain a white solid, i.e., the intermediate (S)-3b, yield: 71%.1H NMR (500 MHz, CDCl3) δ 8.65 (s, 2H), 8.38 (s,1H), 8.28 (d, J=7.5 Hz,1H), 7.52 (d, J=7.5 Hz,1H), 7.47 (t, J=7.6 Hz,1H), 5.01 (dd, J=6.0, 3.0 Hz,1H), 1.56 (d, J=6.4 Hz, 3H). [α]D20=−32.5 (c=0.1 g/100 mL, CH3OH). 97.1% ee. Daicel OD column (0.46×25 cm), n-hexane/isopropanol=85/15, 0.5 mL/min, λ=254 nm, tR=13.98 (R), tS=14.95 (S).

Preparation of intermediate (S)-11a: N,N-dimethylamino chloropropane hydrochloride (1 mmol) and thiourea (1 mmol) in absolute ethyl alcohol (50 mL) were refluxed for 7 hours under the protection of nitrogen, cooled to room temperature, rotary evaporated under reduced pressure to remove the solvent, thus obtaining an isothiourea analogue, which was directly used in the following step without further purification. The intermediate (S)-3b (1 mmol), the isothiourea analogue (2 mmol) and sodium hydroxide (4 mmol) were placed in a single-port flask, the air in which was replaced with nitrogen, and then into which was added N,N-dimethyl formamide/water=5:1, stirred at room temperature for 15 minutes, and then stirred at 60° C. for 3 hours. Upon the completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with water and brine, rotary evaporated and concentrated. The crude product was purified by column chromatography (dichloromethane:methanol=20:1) to obtain a colorless oil, i.e., the intermediate (S)-11a, yield: 56%.1H NMR (400 MHz, CDCl3) δ 8.75 (s, 2H), 8.40 (q, J=1.2, 0.7 Hz,1H), 8.32 (dt, J=7.6, 1.6 Hz,1H), 7.58-7.44 (m, 2H), 5.01 (q, J=6.4 Hz,1H), 3.08-2.97 (m, 2H), 2.39 (t, J=7.0 Hz, 2H), 2.21 (s, 6H), 1.85-1.78 (m, 2H), 1.56 (d, J=6.5 Hz, 3H).

Synthesis of target (R)-6f: At 0° C., diisopropyl azodicarboxylate (0.60 mmol, dissolved in 1 mL N,N-dimethyl formamide) was slowly added into a solution of 3-(6-oxo-1,6-dihydropyridazin-3-yl)-benzonitrile (0.40 mmol), the compound (S)-11a (0.40 mmol) and triphenyl phosphine (0.60 mmol) in tetrahydrofuran (4 mL), warmed to room temperature slowly under the protection of nitrogen, and stirred at room temperature for 18 hours. The solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica-gel column chromatography (dichloromethane/methanol=20:1) to obtain light yellow oil, i.e., the target (R)-6f, yield: 56%.1H NMR (500 MHz, CDCl3) δ 8.81 (s, 2H), 8.76 (s,1H), 8.36 (d, J=7.9 Hz,1H), 8.23 (s,1H), 7.97 (d, J=8.0 Hz,1H), 7.70 (d, J=7.8 Hz,1H), 7.59 (dt, J=12.6, 8.8 Hz, 3H), 7.46 (t, J=7.7 Hz,1H), 7.04 (d, J=9.7 Hz,1H), 6.55 (d, J=7.0 Hz,1H), 3.07 (t, J=7.1 Hz, 2H), 2.73-2.62 (m, 2H), 2.43 (s, 6H), 1.98 (dd, J=14.4, 7.2 Hz, 2H), 1.92 (d, J=7.1 Hz, 3H).13C NMR (126 MHz, CDCl3) δ 162.05, 159.23, 157.94, 141.86, 140.79, 137.37, 136.30, 132.46, 130.49, 130.31, 129.83, 129.79, 129.72, 128.89, 128.75, 127.75, 127.40, 118.64, 113.33, 57.40, 56.24, 44.56, 31.58, 26.04, 20.06. HRMS (ESI) m/z C28H28N6OS [M+H]+, calculated 497.2118; found 497.2111. HPLC purity: 99.0%, retention time: 17.24 min. 90.7% ee. Daicel OD column (0.46×25 cm), n-hexane/ethanol=80/20, 0.5 mL/min, λ=254 nm, tR=29.94 (R), tS=31.88 (S).

Compounds (S)-6f, (R)-7j and (S)-7j were prepared with a similar method.

Embodiment 5: Anti-Tumor Compound (R)-14

Anti-tumor compound (R)-14, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound (R)-14

Preparation of intermediate 12: 1,5,7 triazabicyclo[4.4.0]dec-5-ene (TBD, 0.5 mmol) was added into a solution of the intermediate 8b (5 mmol) in deuterated chloroform (30 mL), stirred at room temperature for 12 hours, and rotary evaporated to get a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=50:1) to obtain an intermediate 12.1H NMR (400 MHz, CDCl3) δ 8.93 (t, J=1.5 Hz,1H), 8.56 (dt, J=7.8, 1.3 Hz,1H), 8.47 (s, 2H), 8.04 (dt, J=7.7, 1.3 Hz,1H), 7.57 (t, J=7.8 Hz,1H), 3.97 (d, J=5.8 Hz, 2H), 2.93 (d, J=11.0 Hz, 2H), 2.70 (s, 3H), 2.06-1.91 (m, 4H), 1.88 (d, J=2.6 Hz,1H), 1.48 (qd, J=13.6, 13.1, 3.6 Hz, 2H).

Preparation of intermediate (S)-13a: Dichloro(p-cymene)ruthenium (II) dimer (0.01 mmol) and a chiral ligand (S,S)-CsDPEN (0.05 mmol) were placed in a 50 mL single-port flask, into which was added heavy water (3 mL), and stirred at 40° C. under the protection of nitrogen for 4 hours. After then, the intermediate 12 (1 mmol, dissolved in 8 mL deuterated chloroform) and deuterated sodium formate (5 mmol, dissolved in 3 mL heavy water) were added promptly, and stirred at room temperature for 10 hours. Upon the completion of the reaction, 10 mL water was added into the reaction system. The water phase was extracted with dichloromethane. The combined organic layer was dried and concentrated. The crude product was recrystallized with ethyl acetate to obtain a white solid, i.e., the intermediate (S)-13a, yield: 70%.1H NMR (400 MHz, CDCl3) δ 8.44 (s, 2H), 8.34 (dd, J=1.9, 1.4 Hz,1H), 8.27-8.23 (m,1H), 7.50-7.44 (m, 2H), 3.92 (d, J=5.9 Hz, 2H), 2.91 (d, J=11.4 Hz, 2H), 2.29 (s, 3H), 2.00-1.93 (m, 2H), 1.87-1.81 (m, 3H), 1.47-1.35 (m, 2H).

Synthesis of target (R)-14: At 0° C., diisopropyl azodicarboxylate (0.60 mmol, dissolved in 1 mL N,N-dimethyl formamide) was slowly added into a solution of 3-(6-oxo-1,6-dihydropyridazin-3-yl)-benzonitrile (0.40 mmol), the compound (S)-13a (0.40 mmol) and triphenyl phosphine (0.60 mmol) in tetrahydrofuran (4 mL), warmed to room temperature slowly under the protection of nitrogen, and stirred at room temperature for 18 hours. The solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica-gel column chromatography (dichloromethane/methanol=20:1) to obtain light yellow oil, i.e., the target (R)-14, yield: 56%.1H NMR (400 MHz, Chloroform-d) δ 8.67 (t, J=1.6 Hz,1H), 8.50 (s, 2H), 8.28 (dt, J=7.8, 1.4 Hz,1H), 8.21 (t, J=1.5 Hz,1H), 7.98 (dt, J=8.0, 1.2 Hz,1H), 7.69 (m,1H), 7.60-7.50 (m, 3H), 7.43 (t, J=7.7 Hz,1H), 7.02 (d, J=9.7 Hz,1H), 3.96 (d, J=6.0 Hz, 2H), 2.94 (d, J=11.5 Hz, 2H), 2.31 (s, 3H), 2.05-1.94 (m, 2H), 1.90-1.83 (m, 3H), 1.48 (qd, J=12.9, 3.5 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 159.28, 157.28, 151.67, 143.93, 141.84, 140.56, 137.85, 136.34, 132.45, 130.29, 129.85, 129.75, 129.65, 129.35, 128.78, 128.70, 127.23, 126.95, 118.61, 113.37, 73.27, 55.24, 46.33, 35.19, 29.71, 28.80, 19.92. HRMS (ESI) m/z C30H27D4N6O2[M+H]+, calculated 511.2724; found 511.2714. HPLC purity: 99.0%, retention time: 15.03 min. 97.1% ee. Daicel AD column (0.46×25 cm), n-hexane/ethanol=80/20, 0.5 mL/min, λ=254 nm, tR=40.66 (R), tS=64.22 (S).

Compound (S)-14 was prepared with a similar method.

Embodiment 6: Anti-Tumor Compound (R)-18

Anti-tumor compound (R)-18, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound (R)-18

Preparation of intermediate 15: 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD, 0.5 mmol) was added into a solution of the intermediate 2 (5 mmol) in deuterated chloroform (30 mL), and stirred at room temperature for 12 hours. The organic layer was washed with water, dried over a saturated sodium chloride solution and anhydrous sodium sulfate, and rotary evaporated to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=50:1) to obtain the desired compound 15.1H NMR (400 MHz, CDCl3) δ 8.98 (t, J=1.6 Hz,1H), 8.70 (s, 2H), 8.60 (dt, J=7.8, 1.5 Hz,1H), 8.10 (dt, J=7.7, 1.5 Hz,1H), 7.60 (t, J=7.8 Hz,1H).

Preparation of intermediate (S)-16: Dichloro(p-cymene)ruthenium (II) dimer (0.05 mmol) and a chiral ligand (S,S)-CsDPEN (0.10 mmol) were placed in a 100 mL single-port flask, into which was added pure water (25 mL), and stirred at 40° C. under the protection of nitrogen for 4 hours. After then, the intermediate 15 (5 mmol, dissolved in 40 mL dichloromethane) and sodium formate (25 mmol, dissolved in 15 mL pure water) were added promptly, and stirred at room temperature for 10 hours. Upon the completion of the reaction, 30 mL water was added into the reaction system. The water phase was extracted with dichloromethane. The combined organic layer was dried and concentrated. The crude product was purified by silica-gel column chromatography (petroleum ether:ethyl acetate=2:1) to obtain a white solid, i.e., the intermediate (S)-16, yield: 71%.1H NMR (400 MHz, CDCl3) δ 8.73 (s, 2H), 8.40 (t, J=1.8 Hz,1H), 8.30 (dt, J=7.6, 1.6 Hz,1H), 7.55-7.43 (m, 2H), 4.98 (s,1H).

Preparation of intermediate (S)-17: N,N-dimethylamino chloropropane hydrochloride (1 mmol) and thiourea (1 mmol) in absolute ethyl alcohol (50 mL) were refluxed under the protection of nitrogen for 7 hours, and cooled to room temperature. The solvent was removed by rotary evaporation under reduced pressure to obtain an isothiourea analogue, which was directly used in the following step without further purification. The intermediate (S)-16 (1 mmol), the isothiourea analogue (2 mmol) and sodium hydroxide (4 mmol) were placed in a single-port flask, the air in which was replaced with nitrogen, and then into which was added N,N-dimethyl formamide/water=5:1, stirred at room temperature for 15 minutes, and then stirred at 60° C. for 3 hours. Upon the completion of the reaction, the reaction solution was diluted with ethyl acetate, washed with water and brine, rotary evaporated and concentrated. The crude product was purified by column chromatography (dichloromethane:methanol=20:1) to obtain colorless oil, i.e., the intermediate (S)-17, yield: 56%.1H NMR (400 MHz, CDCl3) δ 8.75 (s, 2H), 8.40 (q, J=1.2, 0.7 Hz,1H), 8.32 (dt, J=7.6, 1.6 Hz,1H), 7.58-7.44 (m, 2H), 5.01 (s,1H), 3.08-2.97 (m, 2H), 2.39 (t, J=7.0 Hz, 2H), 2.21 (s, 6H), 1.85-1.78 (m, 2H).

Synthesis of target (R)-18: At 0° C., diisopropyl azodicarboxylate (0.60 mmol, dissolved in 1 mL N,N-dimethyl formamide) was slowly added into a solution of 3-(6-oxo-1,6-dihydropyridazin-3-yl)-benzonitrile (0.40 mmol), the compound (S)-17 (0.40 mmol) and triphenyl phosphine (0.60 mmol) in tetrahydrofuran (4 mL), warmed to room temperature slowly under the protection of nitrogen, and stirred at room temperature for 18 hours. The solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica-gel column chromatography (dichloromethane/methanol=20:1) to obtain light yellow oil, i.e., the target (R)-18, yield: 61%.1H NMR (400 MHz, CDCl3) δ 8.78 (s, 2H), 8.71 (s,1H), 8.35 (d, J=7.7 Hz,1H), 8.18 (s,1H), 7.99 (d, J=7.7 Hz,1H), 7.69 (d, J=7.9 Hz,1H), 7.58 (dt, J=11.2, 8.8 Hz, 3H), 7.45 (t, J=7.7 Hz,1H), 7.03 (d, J=9.7 Hz,1H), 6.53 (s,1H), 3.03 (t, J=7.2 Hz, 2H), 2.41 (t, J=7.1 Hz, 2H), 2.22 (s, 6H), 1.88-1.77 (m, 2H).13C NMR (101 MHz, CDCl3) δ 161.89, 159.26, 157.73, 141.82, 140.78, 137.46, 136.33, 132.46, 130.34, 130.19, 129.83, 129.78, 129.65, 128.88, 128.68, 127.70, 127.39, 118.55, 113.41, 57.89, 56.19, 45.27, 31.75, 27.01. HPLC purity: 96.8%, retention time: 16.83 min HRMS (ESI) m/z C28H25D3N6OS [M+H]+, calculated 500.2306; found 500.2293. 92.6% ee. Daicel OD column (0.46×25 cm), n-hexane/ethanol=80/20, 0.5 mL/min, λ=254 nm, tR=28.96 (R), tS=31.90 (S).

Compound (S)-18 was prepared with a similar method.

Embodiment 7: Anti-Tumor Compound 19

Anti-tumor compound 19, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound 19

The preparation of the intermediate 12 is the same as that in Embodiment 5; Except for using sodium formate as the reducing agent in the preparation of the intermediate (S)-13b, other processes are the same as in the process of Embodiment 5.

1H NMR (500 MHz, CDCl3) δ 8.65 (s,1H), 8.48 (s, 2H), 8.26 (d, J=7.6 Hz,1H), 8.18 (s,1H), 7.96 (d, J=8.0 Hz,1H), 7.67 (d, J=7.6 Hz,1H), 7.60-7.50 (m, 3H), 7.41 (t, J=7.7 Hz,1H), 7.00 (d, J=9.6 Hz,1H), 6.52 (s,1H), 3.94 (d, J=5.8 Hz, 2H), 2.90 (d, J=11.3 Hz, 2H), 2.28 (s, 3H), 1.97 (t, J=11.5 Hz, 2H), 1.85 (d, J=12.9 Hz, 3H), 1.46 (td, J=12.8, 6.6 Hz, 2H).13C NMR (126 MHz, CDCl3) δ 159.26, 157.28, 151.68, 143.92, 141.79, 140.63, 137.87, 136.35, 132.43, 130.29, 129.84, 129.74, 129.64, 129.36, 128.76, 128.66, 127.21, 126.94, 118.59, 113.38, 73.32, 56.19, 55.30, 46.43, 35.24, 28.91.

HRMS (ESI) m/z C30H28D3N6O2[M+H]+, calculated 510.2691; found 510.2682. HPLC purity: 98.6.0%, retention time: 15.17 min 97.1% ee. Daicel AD column (0.46×25 cm), n-hexane/ethanol=80/20, 0.5 mL/min, λ=254 nm, tR=42.61 (R), tS=63.30 (S).

Embodiment 8: Anti-Tumor Compound 20

Anti-tumor compound 20, the structural formula of which is as shown below, and the synthesis steps of which are as below:

Structural Formula of Compound 20

In the preparation of the chiral alcohol intermediate (S)-13c, except for directly using the intermediate 8b as the reactant, and using dichloromethane and water as the reaction solvents, others are the same as in the process of Embodiment 5.

1H NMR (400 MHz, CDCl3) δ 8.67 (t, J=1.6 Hz,1H), 8.50 (s, 2H), 8.28 (d, J=7.8 Hz,1H), 8.20 (s,1H), 7.98 (d, J=8.0 Hz,1H), 7.69 (d, J=7.8 Hz,1H), 7.56 (m, 3H), 7.43 (t, J=7.7 Hz,1H), 7.02 (d, J=9.7 Hz,1H), 3.96 (d, J=5.9 Hz, 2H), 2.92 (d, J=11.4 Hz, 2H), 2.30 (s, 3H), 1.97 (m, 2H), 1.93-1.82 (m, 6H), 1.52-1.41 (m, 2H).13C NMR (101 MHz, CDCl3) δ 159.22, 157.30, 151.70, 143.94, 141.78, 140.58, 137.89, 136.38, 132.42, 130.29, 129.82, 129.73, 129.64, 129.35, 128.75, 128.64, 127.23, 126.94, 118.57, 113.41, 73.36, 55.32, 46.44, 35.27, 28.94, 19.91.

HRMS (ESI) m/z C30H29D1N6O2[M+H]+, calculated: 508.2566; found 508.2553. HPLC purity: 99.0%, retention time: 15.03 min. 97.1% ee. Daicel AD column (0.46×25 cm), n-hexane/ethanol=80/20, 0.5 mL/min, λ=254 nm, tR=40.66 (R), tS=64.22 (S).

Test Example: Anti-Tumor Activities of the Novel Pyrimidine Derivatives of the Present Invention

The novel pyrimidine derivatives of the invention were tested on their anti-tumor activities. Human highly metastatic hepatoma cells MHCC-97H were employed in the MTT process to evaluate the anti-tumor cell proliferation activities. The specific operation steps were as below: tumor cells were inoculated in a 96-well plate by a certain number of cells, at a cell density of 5×103-10×103/well. They were incubated in an incubator at 37° C. and a carbon dioxide concentration of 5% overnight, into which was then added the compound samples to be tested. After incubation for 72 hours, MTT was added to keep on for 4 hours, and dissolved with the addition of DMSO with shaking, and then detected with a microplate reader (570 nm).

Median inhibitory concentration IC50values of the novel pyrimidine derivatives of the invention on human highly metastatic hepatoma cells MHCC-97H are as shown in Table 1. It is indicated from the data in Table 1 that, the introduction of a chiral center in the molecule has great effects on the anti-tumor activities of the compound. The anti-tumor activities of the two enantiomers are significantly different. For example, the racemate (+)-5b (No. 4) inhibits the activity of human highly metastatic hepatoma cells MHCC-97H, the IC50of which is 0.0353 μM, while IC50of (R)-5b (No. 5) for inhibiting this tumor cell is 0.0037 μM, being superior to that of the racemate and Tepotinib (No. 14, its activity is 0.0134 μM). Accordingly, the inhibitory activity of(S)-5b (No. 6) is 0.1182 μM, much lower than that of the racemate and (R)-5b (No. 5). The activity of the compound (R)-5b for inhibiting tumor cells is 9.5 and 31.9 times that of (±)-5b and (S)-5b, respectively. As it can be seen that, the introduction of a chiral center at the position between pyridazinone and benzene ring plays an important role in enhancing the inhibition on the activity of human highly metastatic hepatoma cells MHCC-97H. There are similar structure-function relationships in other compounds. For example, in other three series, (±)-5a, (R)-5a and (S)-5a; (±)-6f, (R)-6f and (S)-6f; as well as (±)-7j, (R)-7j and (S)-7j, the compounds in (R)-configuration have better tumor cell inhibitory activities than the corresponding racemates (±) and compounds in (S)-configuration. It is demonstrated from the above results that, the spatial configuration of compounds in (R)-configuration plays an important role in inhibiting the activities of human highly metastatic hepatoma cells MHCC-97H.

The metabolic stability of a compound is a particularly important index of drug-like properties, and an important factor in determining whether a compound has the potential to become medicine. The new extracted SD rat liver microsomes were used to study the metabolic stabilities of the compounds (R)-5b and (R)-14. Firstly, a SD rat (200-300 g) was fasted for 12 hours, and then infused rapidly with normal saline. Liver was taken out from the rat, from which liver microsomes were extracted at 0-4° C., ready for use after calibrating the concentration of liver microsomes. At 37° C., liver microsomes were incubated in combination with a certain concentration of compounds and auxiliary reagents for a period of time. The reaction was then quenched with acetonitrile. High performance liquid chromatography was used to determine the contents of compounds (R)-5b and (R)-14. Prior to determining the metabolic stability of compounds in liver microsome of SD rats, the activity of liver microsome of SD rats was firstly tested by the same test method with the compound testosterone as the positive control compound; furthermore, Tepotinib was used as the control compound. Hydrogen atoms on the chiral central carbon and hydrogen atoms on the methyl of the compound (R)-5b were all substituted with its isotope deuterium to obtain a deuterated compound (R)-14. The compound (R)-14 has a better metabolic stability than (R)-5b while retaining good performance of (R)-5b on inhibiting the activity of tumor cells. As shown in Table 2 below, the metabolic half-life of (R)-5b in liver microsomes is 26.8 minutes, while the metabolic half-life of the compound (R)-14 is 35 minutes, both higher than the metabolic half-life of Tepotinib in liver microsomes (20.7 minutes), with improvements in stabilities. It has positive significance for improving the efficacy of anti-tumor compounds in vivo.

Studies on pharmacokinetic properties of the present novel pyrimidine derivatives in mice:

Shanghai Medicilon Bio-pharmaceutical Co., LTD was commissioned to research pharmacokinetic properties of the novel pyrimidine derivatives (R)-14, (R)-5b, and Tepotinib in mice. The specific processes are: An appropriate amount of the compounds (R)-14, (R)-5b and Tepotinib were accurately weighed and dissolved in a solution containing 2% Solutol and 98% 100 mM sodium acetate to obtain a clear dosing solution at a concentration of 2 mg/mL for intravenous administration. An appropriate amount of the compounds (R)-14, (R)-5b were accurately weighed and dissolved in 5% Solutol and 95% saline to obtain a clear dosing solution at a concentration of 5 mg/mL. An appropriate amount of Tepotinib was accurately weighed and dissolved in 5% Solutol, 5% DMSO and 90% saline, for oral administration and injection. Blood was collected from the test SD rats via jugular vein puncture, about 0.3 mL for per sample, and using heparin sodium as the anticoagulant. The time points for blood sampling were as below (including intravenous injection and oral administration): before administration, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h post administration. After collection, blood samples were placed on ice, and plasma was separated by centrifugation within 30 minutes (centrifugation conditions: 8000 rpm, 6 minutes, 2-8° C.). The collected plasma was stored at −80° C. before analysis. Plasma samples were all analyzed qualitatively and quantitatively by LC-MS/MS. The dosage and ways for administration were intravenous injection (IV, 2 mg/kg) and intragastric administration (PO, 5 mg/kg) respectively, with the pharmacokinetic data shown in Table 3. As shown in Table 3, the clearance rates (CL) of the compounds (R)-14, (R)-5b are 819.12 mL/h/kg and 932.87 mL/h/kg respectively, much less than that of Tepotinib (3554.03 mL/h/kg); the drug exposure value (AUC) of the compounds (R)-14 and (R)-5b for intravenous injection (IV) and intragastric administration (PO) are 2441.64 h. ng/mL (IV), 6309.37 h. ng/mL (PO) and 2143.91 h. ng/mL (IV), 4580.23 h. ng/mL (PO) respectively, greatly higher than those of the positive drug Tepotinib (562.74 h. ng/mL (IV) and 981.69 h. ng/mL (PO)). Furthermore, the half-life (T1/2) of the compounds (R)-14 and (R)-5b are 4.48 and 4.23 hours respectively, significantly superior that of Tepotinib (1.79 hours). This result is consistent with the results of previous experiments on the stability of rat liver microsomes, indicating that the compounds (R)-14, (R)-5b have better in vivo stability than Tepotinib. Finally, the bioavailabilities of the compounds (R)-14, (R)-5b are 103.73% and 85.97% respectively, higher than that of the positive drug Tepotinib (67.77%). Overall, it is demonstrated from the study on pharmacokinetic properties in mice that, compared with the positive drug Tepotinib, the novel pyrimidine derivatives (R)-14 and (R)-5b have lower clearance rates, higher drug exposure value, longer metabolic half-life in the body, and higher bioavailability. These results fully demonstrate that, due to the introduction of a chiral structure, the novel pyrimidine derivatives (R)-14 and (R)-5b do have great improvements in terms of anti-tumor cell activities and pharmacokinetic properties compared to the positive drug Tepotinib. It can be seen from further comparing the pharmacokinetic properties of the compounds (R)-14 and (R)-5b, due to introducing an isotope of hydrogen (deuterium) with more stable chemical properties at the key site, (R)-14 has better pharmacokinetic properties in mice, more meeting the requirements of being medicine.

TABLE 1The activity of the present novel pyrimidine derivatives on inhibiting human highlymetastatic hepatoma cell MHCC-97HHuman highlymetastatic hepatomacell MHCC-97HNo.Structural formula of compounds(μM)10.047620.014031.041540.035350.003760.118270.027280.013990.1890100.0270110.0117120.4150130.0035140.0037151.117160.0033170.0134

TABLE 2Test on the stability of rat liver microsomescompdK (min−1)T1/2(min)atestosteroneb0.30132.3(R)-5b0.025926.8(R)-140.019835.0Teponinib0.013420.7

TABLE 3Pharmacokinetic data of the compounds(R)-5b, (R)-14 and Tepotinib in micePK parameters(R)-14(R)-5bTepotinibIV 2 mg/kgCL (mL/h/kg)819.12932.873554.03Vz(mL/kg)5289.045687.119178.68AUC0-∞(h · ng/mL)2441.642143.91562.74T1/2(h)4.484.231.79PO 5 mg/kgCmax(ng/mL)613.84495.30211.23Tmax(h)421AUC0-∞(h · ng/mL)6309.374580.23981.69F %103.7385.9767.77IV = intravenous injection, PO = intragastric administration.