The chemical reagents and solvents were purchased from commercial sources and were used without further purification.1H NMR and 13C NMR spectra were obtained using a Bruker DRX spectrometer at 400 and 100 MHz, respectively. Chemical shifts were reported in parts per million (ppm). Multiplicity of1H NMR signals was reported as singlet (s), doublet (d), triplet (t), quartert (q), and multiplet (m). ESI-MS data were recorded on an API 4000 spectrometer. Melting points were determined using open capillary on an uncorrected electrothermal melting point apparatus.
N-(2-Chloropyrimidin-4-yl)-N,2,3-trimethyl-2H-indazol-6-amine(1)and4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)benz-oic acid (10) were synthesized as reported methods[12].
4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)phen-ol (2).To a solution of 1 (0.50 g,1.74 mmol) and 4-aminophenol (0.23 g, 2.09 mmol) inisopropanol (30 mL) was added 2 drops of conc HCl, and the mixture was heated to reflux with stirring for 4 h. The mixture was cooled to room temperature and the resulting precipitate was collected viafiltration and washed with ethyl acetate, affording compound 2, white solid(0.43 g, 70%). ESI-MS m/z: 360.14 [M+H]+.
N2-(4-aminophenyl)-N4-(2,3-dimethyl-2H-indazol-6-yl)-N4-methylpyrimidine-2,4-diamine(3). To a solution of 1 (0.50 g,1.74 mmol) and benzene-1,4-diamine (0.23 g, 2.09 mmol) inisopropanol (30 mL) was added 2 drops of conc HCl, and the mixture was heated to reflux with stirring for 4 h. The mixture was cooled to room temperature and the resulting precipitate was collected viafiltration and washed with ethyl acetate, affording compound 3, white solid (0.41 g, 65%). ESI-MS m/z: 360.05 [M+H]+.
Methyl8-((4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)ami-no)phenyl)amino)-8-oxooctanoate(4).To a solution of 3 (0.46g, 1.29 mmol) in DMF (10 mL) in ice bath, was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU, 0.50 g, 1.54 mmol), followed by Et3N (0.16 g,1.54 mmol). 30 min later, suberic acid monomethyl ester (0.29 g, 1.54 mmol) was added. 12 h later, the solution was diluted by water and extracted with ethyl acetate. The combined organic extract was washed with saturated NaHCO3and brine, dried over Na2SO4overnight, and the solvent was evaporated under vacuum. The crude product was purified by silica gelcolumn chromatography (MeOH/CH2Cl2, 1/50 to 1/20) to affordcompound4, white solid (0.40 g, 58% yield). ESI-MS m/z: 530.14 [M+H]+.
N1-(4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)phe-nyl)-N8-hydroxyoctanediamide (5).KOH (28.55g, 509 mmol) and NH2OH·HCl (23.84 g, 343 mmol) were dissolved in 70 mL and 120 mL of MeOH to get solution A and solutionB, respectively. Then solution A was added dropwise to solution B. After filtering the precipitated KCl, a NH2OK solution was obtained. Compound 4(0.25 g, 0.47 mmol) was dissolved in 30 mL of NH2OK solution and stirred for 2 h. After the reaction was complete, it was evaporated under vacuum. The residue was acidified by addition of 1 M HCl to pH 5-6.The resulting precipitate was collected by filtration and dried to affordcompound5, white solid (0.11 g, 43% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.68 (s, 1H), 9.04 (s, 1H), 8.65 (s, 1H), 7.82 (d, J = 5.9 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 1.7 Hz, 1H), 7.37 (d, J = 8.9 Hz, 2H), 6.88 (dd, J = 8.8, 1.8 Hz, 1H), 5.76 (d, J = 6.0 Hz, 1H), 4.06 (s, 3H), 3.46 (s, 3H), 2.63 (s, 3H), 2.25 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.64 – 1.40 (m, 4H), 1.34 – 1.21 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 171.25, 169.62, 162.89, 158.01, 153.25, 147.39, 142.05, 135.81, 134.08, 132.71, 122.30, 120.07, 119.97, 119.91, 114.48, 96.64, 38.61, 37.86, 36.76, 32.75, 28.89, 25.60, 25.52, 9.89. HRMS (AP-ESI) m/z calcd for C28H35N8O3 [M+H]+531.2832, found 531.2882.
Methyl 6-((4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amin-o)phenyl)amino)hexanoate (6).To a solution of 3 (0.40g, 1.11 mmol) in DMF (10 mL), was added potassium carbonate (K2CO3, 0.18 g, 1.33 mmol), followed by methyl 6-bromohexanoate (0.28 g,1.33 mmol). The reaction mixture was stirred at 70°C. 12 h later, the solution was diluted by water and extracted with ethyl acetate. The combined organic extract was washed with saturated NaHCO3 and brine, dried over Na2SO4 overnight, and the solvent was evaporated under vacuum. The crude product was purified by silica gelcolumn chromatography (MeOH/CH2Cl2, 1/50 to 1/20) to afford compound 6, white solid (0.21 g, 39% yield). ESI-MS m/z: 488.25 [M+H]+.
Methyl 2-(4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amin-o)phenoxy)acetate (8a). To a solution of 2 (0.48g, 1.33 mmol) in DMF (10 mL), was added caesium carbonate (Cs2CO3, 0.52 g, 1.60 mmol), followed by methyl bromoacetate (0.24 g,1.60 mmol). The reaction mixture was stirred at 80°C. 6 h later, the solution was diluted by water and extracted with ethyl acetate. The combined organic extract was washed with saturated NaHCO3 and brine, dried over Na2SO4 overnight, and the solvent was evaporated under vacuum. The crude product was purified by silica gelcolumn chromatography (MeOH/CH2Cl2, 1/50 to 1/20) to affordcompound8a, white solid (0.42 g, 73% yield). ESI-MS m/z: 433.23 [M+H]+.
Compounds 8b-8e were prepared from compound 2 in a similarmanner as described for compound 8a, respectively.
Methyl 4-(4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amin-o)phenoxy)butanoate (8b). White solid. 70% yield. ESI-MS m/z: 461.21 [M+H]+.
Methyl 5-(4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amin-o)phenoxy)pentanoate (8c). White solid. 67% yield. ESI-MS m/z: 475.19 [M+H]+.
Methyl 6-(4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amin-o)phenoxy)hexanoate (8d). White solid. 65% yield. ESI-MS m/z: 489.32 [M+H]+.
Methyl 7-(4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amin-o)phenoxy)heptanoate (8e). White solid. 75% yield. ESI-MS m/z: 503.31 [M+H]+.
Compounds 7, 9a-9e were respectively prepared from compound 6, 8a-8e in a similarmanner as described for compound 5.
6-((4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2yl)amino)pheny-l)amino)-N-hydroxyhexanamide (7).White solid. 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.80 (s, 1H), 8.64 (s, 1H), 7.74 (t, J = 7.7 Hz, 2H), 7.43 (s, 1H), 7.36 (d, J = 8.7 Hz, 2H), 6.90 – 6.83 (m, 1H), 6.45 (d, J = 8.6 Hz, 2H), 5.69 (d, J = 6.1 Hz, 1H), 4.06 (s, 3H), 3.44 (s, 3H), 2.94 (t, J = 6.8 Hz, 2H), 2.62 (s, 3H), 1.96 (t, J = 7.4 Hz, 2H), 1.53 (p, J = 7.2 Hz, 4H), 1.39 – 1.28 (m, 2H). HRMS (AP-ESI) m/z calcd for C26H33N8O2 [M+H]+ 489.2726, found 489.2749.
2-(4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)pheno-xy)-N-hydroxyacetamide (9a). white solid (0.16 g, 50% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.07 (s, 1H), 8.93 (s, 1H), 7.80 (d, J = 6.1 Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.44 (s, 1H), 6.88 (dd, J = 8.8, 1.5 Hz, 1H), 6.81 (d, J = 8.9 Hz, 2H), 5.76 (d, J = 6.0 Hz, 1H), 4.39 (s, 2H), 4.06 (s, 3H), 3.46 (s, 3H), 2.63 (s, 3H). 13C NMR (101 MHz, Methanol-d4) δ 166.56, 163.04, 158.33, 153.41, 152.56, 147.46, 142.86, 133.94, 133.43, 121.64, 121.54, 119.95, 119.81, 114.53, 113.54, 96.08, 66.48, 37.41, 36.21, 8.28. HRMS (AP-ESI) m/z calcd for C22H24N7O3 [M+H]+ 434.1941, found 434.1922.
4-(4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)ph-eno-xy)-N-hydroxybutanamide (9b). White solid. 55% yield.1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.99 (s, 1H), 8.69 (s, 1H), 7.80 (d, J = 6.0 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.9 Hz, 2H), 7.43 (d, J = 1.8 Hz, 1H), 6.87 (dd, J = 8.8, 1.8 Hz, 1H), 6.79 – 6.74 (m, 2H), 5.75 (d, J = 6.0 Hz, 1H), 4.06 (s, 3H), 3.89 (t, J = 6.3 Hz, 2H), 3.45 (s, 3H), 2.63 (s, 3H), 2.12 (t, J = 7.4 Hz, 2H), 1.91 (p, J = 6.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.17, 162.88, 159.52, 155.41, 153.55, 147.45, 142.41, 134.56, 132.61, 122.15, 120.86, 120.20, 119.94, 114.73, 114.39, 96.35, 67.44, 38.33, 37.84, 29.27, 25.42, 9.88. HRMS (AP-ESI) m/z calcd for C24H28N7O3 [M+H]+ 462.2254, found 462.2276.
5-(4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)ph-eno-xy)-N-hydroxypentanamide (9c). White solid. 50% yield.1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.11 (s, 1H), 8.67 (s, 1H), 7.79 (d, J = 6.2 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.60 – 7.55 (m, 2H), 7.44 (d, J = 1.7 Hz, 1H), 6.88 (dd, J = 8.8, 1.8 Hz, 1H), 6.81 – 6.75 (m, 2H), 5.76 (d, J = 6.1 Hz, 1H), 4.06 (s, 3H), 3.90 (t, J = 5.9 Hz, 2H), 3.46 (s, 3H), 2.63 (s, 3H), 2.01 (t, J = 6.7 Hz, 2H), 1.71 – 1.59 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 169.42, 162.85, 158.81, 154.27, 153.90, 147.41, 142.21, 134.03, 132.65, 122.23, 121.16, 120.08, 120.00, 114.73, 114.42, 96.42, 67.66, 38.45, 37.85, 32.41, 28.76, 22.30, 9.88.HRMS (AP-ESI) m/z calcd for C25H30N7O3 [M+H]+ 476.2410, found 476.2489.
6-(4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)ph-eno-xy)-N-hydroxyhexanamide (9d). White solid. 47% yield. 1H NMR (400 MHz, DMSO-d6)δ 10.35 (s, 1H), 8.93 (s, 1H), 8.67 (s, 1H), 7.80 (d, J = 6.0 Hz, 1H), 7.75 (dd, J = 8.7, 0.8 Hz, 1H), 7.60 (d, J = 9.1 Hz, 2H), 7.42 (dd, J = 1.8, 0.8 Hz, 1H), 6.87 (dd, J = 8.8, 1.8 Hz, 1H), 6.75 (d, J = 9.1 Hz, 2H), 5.73 (d, J = 5.9 Hz, 1H), 4.06 (s, 3H), 3.87 (t, J = 6.4 Hz, 2H), 3.45 (s, 3H), 2.63 (s, 3H), 1.97 (t, J = 7.3 Hz, 2H), 1.68 (p, J = 6.6 Hz, 2H), 1.55 (p, J = 7.4 Hz, 2H), 1.43 – 1.33 (m, 2H).HRMS (AP-ESI) m/z calcd for C26H32N7O3 [M+H]+ 490.2567, found 490.2517.
7-(4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)ph-eno-xy)-N-hydroxyheptanamide (9e). White solid. 53% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.93 (s, 1H), 8.65 (s, 1H), 7.80 (d, J = 6.0 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 9.0 Hz, 2H), 7.44 – 7.41 (m, 1H), 6.87 (dd, J = 8.8, 1.7 Hz, 1H), 6.75 (d, J = 9.0 Hz, 2H), 5.73 (d, J = 5.9 Hz, 1H), 4.06 (s, 3H), 3.88 (t, J = 6.5 Hz, 2H), 3.45 (s, 3H), 2.63 (s, 3H), 1.99 – 1.91 (m, 2H), 1.67 (dt, J = 14.8, 6.8 Hz, 2H), 1.51 (dt, J = 14.8, 6.6 Hz, 2H), 1.39 (m, 2H), 1.34 – 1.27 (m, 2H). HRMS (AP-ESI) m/z calcd for C27H34N7O3 [M+H]+ 504.2723, found 504.2746.
4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)benzohy-drazide (11).To a solution of 10 (0.40 g, 1.03 mmol) in dichloromethane (10 mL) in ice bath, was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 0.40 g, 1.24 mmol), followed by Et3N (0.13 g,1.24 mmol). 30 min later, hydrazine hydrate (0.06 g, 1.24 mmol) was added. 12 h later, the solution was diluted by water and extracted with dichloromethane. The combined organic extract was washed with saturated NaHCO3 and brine, dried over Na2SO4 overnight, and the solvent was evaporated under vacuum. The crude product was purified by silica gelcolumn chromatography (MeOH/CH2Cl2, 1/50 to 1/20) to affordcompound11, white solid (0.22 g, 52% yield). ESI-MS m/z: 403.21 [M+H]+.
4-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-N'-prop-ylbenzohydrazide (12). Compound 11 (0.60 g, 1.49 mmol) and propionaldehyde (0.10 g, 1.79 mmol) were added to 15 mL anhydrous methanol, then p-toluenesulfonic acid (0.025 g, 0.15 mmol) was added at room temperature. 8h later, the reaction solution was filtered and concentrated. The obtained residue was dissolved in 15 mL anhydrous methanol, and NaBH3CN (0.14 g, 2.24mmol) was added. The pH value of the solution was adjusted to 5 by Conc. HCl/MeOH (v:v=1:1). 12h later, the pH of the solution was adjusted to 8 by saturated NaHCO3. The organic phase was decompressed and evaporated, and the residual was extracted with ethyl acetate. The combined organic extract was washed with saturated NaHCO3 and brine, dried over Na2SO4 overnight, and the solvent was evaporated under vacuum. The crude product was purified by silica gel column chromatography (MeOH/CH2Cl2, 1/100 to 1/45) to afford compound 12, white solid (0.21 g, 31% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 9.47 (s, 1H), 7.90 (d, J = 6.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 1.7 Hz, 1H), 6.90 (dd, J = 8.8, 1.7 Hz, 1H), 5.86 (d, J = 6.0 Hz, 1H), 5.10 (s, 1H) 4.07 (s, 3H), 3.50 (s, 3H), 2.74 (t, J = 7.1 Hz, 2H), 2.64 (s, 3H), 1.47 (q, J = 7.3 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 165.69, 162.90, 159.59, 156.18, 147.48, 144.41, 142.38, 132.62, 128.07, 125.25, 122.20, 120.20, 119.97, 117.84, 114.43, 97.38, 53.72, 38.44, 37.83, 21.32, 12.15, 9.88. HRMS (AP-ESI) m/z calcd for C24H29N8O1 [M+H]+445.2464, found 445.2478.
In vitro HDACs inhibition assay
In vitro HDACs inhibition assays were conducted according to the reported methods [12]. Briefly, 10 μL of enzyme solution (HDAC1, HDAC4, HDAC6 or HDAC11) was mixed with different concentrations of tested compound (50 μL). The mixture was incubated at 37℃ for 5 mins, followed by adding 40 μL fluorogenic substrate (Boc-Lys(acetyl)-AMC for HDAC1 and HDAC6, Boc-Lys(triflouroacetyl)-AMC for HDAC4, Ac-Leu-GlyLys(Ac)-AMC for HDAC11). After incubation at 37℃ for 30 mins, the mixture was quenched by adding100 μL of developer containing Trichostatin A (TSA) and trypsin. After another 20 min of incubation at 37 ºC, fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 390 and 460 nm, respectively. The inhibition ratios were calculated from the fluorescence intensity readings of tested wells relative to those of control wells, and the IC50 values were calculated using nonlinear regression with normalized dose-response fit using Prism GraphPad software.
In vitro VEGFRs inhibition assay
VEGFR1, VEGFR2 and VEGFR3 inhibitory activity was measured using Kinase-GloTM Luminescent Kinase Assay by HUAWEI PHARMA (Ji’nan, China). In brief, the tested compounds, kinases, substrate, and ATP were diluted in kinase buffer to the indicated concentrations, covered the assay plate, and incubated at room temperature for 40 min, then the Kinase-Glo reagent was added. Over an additional 15 min of incubation, the luminescence was recorded on a microplate reader (SpectraMax M5). The IC50values were calculated using nonlinear regression with normalized dose-response fit using Prism GraphPad software.
In vitro anti-proliferative assay
All cell lines were maintained in RPMI1640 medium containing 10% FBS at 37℃ in a 5% CO2 humidified incubator. Antiproliferation assay was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2h-tetrazoliumbromide) method. Briefly, cells were passaged the day before seeded into a 96-well plate, allowed to grow for 12 h, and then treated with different concentrations of compound for 48 h. A 0.5% MTT solution was added to each well. After incubation for another 4 h, formazan formed from MTT was extracted by adding 200 uL of DMSO. Absorbance was then determined using an ELISA reader at 570 nm.
HUVECs tube formation assay
HUVEC tuber formation assay was conducted according to the reported methods [12]. Briefly, matrigel (100 μL; BD biosciences, NJ) was added into test well of 96-well plates and then allowed to polymerize for 0.5 h at 37 °C. HUVECs were trypsinized and seeded at the density of 40000 per well in M199 (5% FBS) containing DMSO or test compounds for 6 h at 37 °C in a CO2 incubator. The morphological changes of the cells and tubes formation were observed under a phase-contrast microscope (OLYMPUS IX51) and photographed at a 200 magnification. Experiments were repeated at least two times.
Rat thoracic aorta ring (TARs) assay
TARs assay was conducted according to the reported methods [12]. Briefly, matrigel (100 μL; BD biosciences, NJ) was added into test well of 96-well plates and then allowed to polymerize for 0.5 h at 37 °C. Sprague–Dawley rats of 4 to 6-week-old were sacrificed and aortas were harvested. Each aorta was cut into 1-mm slices and embedded in additional 100 μL of matrigel in 96-well plates. After that, the rings were incubated for 30 min at 37 °C and 5% CO2. Aorta rings were treated with vehicle or tested compounds each day for 6 days and photographed on the 7th day at ×200 magnification. Experiments were repeated at least two times.
Western blot analysis
A549 or HUVEC cells were treated with compounds or DMSO for a specified period of time. Then the cells were washed twice with cold PBS and lysed in ice-cold RIPA buffer. Lysates were cleared by centrifugation. Protein concentrations were determined using the BCA assay. Equal amounts of cell extracts were then resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with ac-histone H4 antibody, ac-α-tubulin antibody, β-actin antibody, phosphorylated VEGFR2 antibody and total VEGFR-2 antibody, respectively. Blots were imaged using an enhanced chemiluminescence system.
In vitro liver microsomal stability assay
Mice liver microsomes containing tested compounds were incubated with NADPH at 37 °C. At the specific time points, acetonitrile was added to the samples to terminate the reaction, then the samples were subjected to vortex mixing for 5 min and stored in a freezer at -80 °C. Before analysis, the samples were centrifuged at 4000 rpm for 15 min. The remaining of tested compounds in the supernatants were analyzed by LC-MS/MS. The t1/2 values were calculated using the equation t1/2 = -0.693/k, where k is the slope found in the linear fit of the natural logarithm of the fraction remaining of tested compounds vs. incubation time.