Structure-based design and synthesisof 5-benzyl-2-phenylpyrimidin-4(3H)-one derivatives as novel MEK1 inhibitors 
Can Li, Hongyue Li, Jing Sun, Dandan Xi, Chao Wang, Lei Liang, Fengrong Xu, Yan Niu*, Ping Xu*
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
Abstract: Previous studies have shown that Ras/Raf/MEK/ERK signaling pathway is up-regulated in almost all cancer cells. Blocking of this pathway by MEK inhibition is an efficient therapeutic approach of cancer. In the present study, we described the discovery of 5-benzyl-2-phenylpyrimidin-4(3H)-one as a novel skeleton of allosteric MEK inhibitor. All acquired target compounds exhibited modest potency to inhibit MEK1 in Raf-MEK cascading assay, and docking studies revealed that the binding mode of the most potent compound (SJ3) was very similar to that of the well known diarylamine-based inhibitor (PD0325901). The results provided valuable guidance for further optimizations on this novel scaffold to achieve druggable molecules. 
Keywords: 5-Benzyl-2-phenylpyrimidin-4(3H)-one; MEK1 inhibitor; Docking  
CLC number:R 916                Document code: A                 Article ID: 10031057(2018)1068610
1. Introduction
The mitogen activated protein kinase (MAPK) signaling pathways are involved in many cellular processes in eukaryotic cells, such as proliferation, differentiation, apoptosis and so on[1]. The Ras/Raf/MEK/ERK signaling pathway is a member of MAPK pathway family, which plays a central role in mediating the extracellular stimuli, such as growth factors and cytokines, to cellular responses[2]. As one of the most often deregulated pathways in tumor cells, Ras/Raf/MEK/ERK signaling pathway has been linked to important oncological indications. Since MEK is the only known downstream kinase of Ras and Raf in the signal transduction and catalyzes the phosphorylation of ERK1 and ERK2 as its only known substrates, it has become a highly attractive chemotherapeutic target against cancer[3,4].
Up to date, many of the small molecule MEK1/2 inhibitors have been reported, and a majority of them bear a diarylamine scaffold. Several of them with good drug-like properties, in addition to high potency and selectivity, have been evaluated in the clinical practice (e.g. PD0325901[5]). Presently, only two of the reported MEK1/2 inhibitors have been approved by FDA, namely Trametinib[6] (in 2013) and Cobimetinib[7](in 2015), for the treatment of advanced melanoma. Besides, Selumetinib has been granted Orphan Drug Designation for the treatment of differentiated thyroid cancer[8] (Fig. 1).Since the structure-activity relationships of diarylamine inhibitors have been extensively studied and the diversity of available MEK1 inhibitors remains limited, the design and discovery of MEK1 inhibitors with novel scaffolds have become of great interest of medicinal chemists. We have focused for long on the discovery of MEK inhibitors with novel scaffolds[911]. In the present study, we report the identification of a serials of 5-benzyl-2-phenylpyrimidin-4(3H)-ones as moderate MEK1 inhibitors which could provide clues for the design of novel allosteric MEK1 inhibitors for clinic use.  
Figure 1. The structures of PD0325901, Trametinib, Cobimetinib and Selumetinib. 
2. Results and discussion
2.1. Molecular design
Inspired by the previously reported inhibitor TAK-733(IC50 = 3.2 nM)[12] and 3-benzyl-1,3-benzoxazine-2,4-dione derivative 9m (IC50 = 60.0 nM)[10] (Fig. 2), we designed a novel scaffold by incorporation of benzyl substitution onto pyrimidin-4(3H)-one ring with the assistance of molecular docking. This 5-benzylpyrimidin-4(3H)-one was further optimized to 5-benzyl-2-phenylpyrimidin-4(3H)-one by adding a phenyl ring in order to fully occupy the allosteric pocket of MEK1 in adjacent to the ATP-binding site (Fig. 3A). As observed from the binding mode, the carbonyl oxygen of pyrimidin-4(3H)-one formed tight hydrogen bonds (HBs) with Val211 and Ser212, and the 2-substituted phenyl ring overlapped well with one of the aryl rings within PD0325901 (Fig. 3B). Moreover, the 5-benzyl ring oriented properly in the hydrophobic site formed by Arg189, Asp190, Gly210, Leu215 and Ile216. To increase affinity, diverse flexible substitutions could be designed at position 6 of the pyrimidin-4(3H)-one ring to fit into the subpocket formed by Met219, Asp208 and Lys97. Therefore, both polar and unpolar side chains were designed in our work, and nine compounds (Fig. 3C) were synthesized and evaluated in vitro for MEK1 inhibitory potency to validate the feasibility of 5-benzyl-2-phenylpyrimidin-4(3H)-one as novel scaffold of MEK inhibitor.
Figure 2. The design of 5-benzylpyrimidin-4(3H)-one from TAK-733 and 9m. 
Figure 3. Predicted binding mode of 5-benzyl-2-phenylpyrimidin-4(3H)-one (yellow stick), in comparison with PD0325901 (green stick) within the allosteric site of MEK1 protein (3EQG). (A) The orientations of 5-benzyl-2-phenylpyrimidin-4(3H)-one and PD0325901 in MEK1 allosteric pocket in adjacent to ATP (pink stick) binding site; (B) The docked pose of 5-benzyl-2-phenylpyrimidin-4(3H)-one in comparison with PD0325901 with hydrogen bonds depicted in dashed lines; (C) General structure of designed 5-benzyl-2-phenylpyrimidin-4(3H)-ones. 
2.2. Chemistry
Substituted β-ketone ester was treated with diverse benzyl bromides in the presence of NaH to give intermediates 13[9], which were cyclized with benzamidinesto afford targeted compounds SJ1-4[13]. To obtain SJ5, the -NO2 within SJ4 was firstly reduced to -NH2, followed by condensation with methylsufonyl chloride to give the desired sulfamide SJ5. The6-hydroxypyrimidin-4(3H)-one intermediate 5 was acquired from cyclization of 4-fluro-benzamidine with diethylbenzylmalonate[13], and the -OH was further acylated by acylchloride or sulfonyl chloride to afford SJ6-8[14]. Diethyl 2-benzyl-3-oxosuccinate 6[15] was prepared from sodium diethyloxalacetate and benzyl bromide, and cyclization of 6 with benzamidine under reflux in ethanol readily gave SJ9[16] in good yield. Scheme 1 outlines the structures and synthetic route of the targeted compound SJ1-9.
Scheme 1. The synthetic route of the designed 5-benzyl-2-phenylpyrimidin-4(3H)-ones. Reagents and conditions: (a) NaH, THF, N2, reflux; (b) substituted benzamidines, EtOH, reflux; (c) 5% Pd/C, H2, MeOH; (d) pyridine, methyl sulfonyl chloride, r.t.; (e) NaOEt, EtOH, reflux; (f) R1COCl or R1SO2Cl, K2CO3, DMF; (g) THF, reflux; (h) 4-fluorobenzamidine hydrochloride, KOH, EtOH, reflux.
2.3. In vitro evaluation on MEK1 inhibition
According to our previous observation, allosteric inhibitor inhibits MEK1 by stabilizing the kinase in an inactivated conformation so as to prevent the activation of MEK by Raf. Therefore, we evaluated the potencies of the acquire compounds in Braf-MEK1 cascading assay at a concentration of 10 μM. The inhibitory ratios (%) were listed in Table 1.
Table 1. Structures, in vitro enzymatic inhibitory activities and docking scores of SJ1-9.
aValuesare expressed as the mean±SD determined in three independent experiments; bFitness of GOLDScore; cThe inhibition was tested at a concentration of 10 nM.   
Table 1 shows that all compounds exhibited very moderate potencies against Raf-MEK1 cascading. The best activity was observed from SJ3 with n-propyl substitution as R1, and replacement of the alkyl side chain into more polar ones as in SJ6-9 did not seem to improve binding. Electron-withdraw substitutions of R2 (SJ3-5) also barely contributed to binding, suggesting that electron-withdraw substitutions should be avoided on R2 in further optimizations on this scaffold.
2.4. Docking studies
Docking result (Fig. 4) suggested that SJ3 could fit properly into the allosteric pocket of MEK1, and the docking scores for the targeted compounds (Table 1) were very similar, suggesting that the deviations on the scaffold remained to be explored in the future. Compared with the 5-benzyl-2-phenylpyrimidin-4(3H)-one core, the 6-propyl chain of SJ3 was observed overlappedwell with the amide side chain of PD0325901, and to improve potency, the length of this chain could beprolonged to 78 atoms. 3′- or 4′-substitutions on the 2-phenyl ring of SJ3 could be considered to improve potency due to the space around it to accommodate substitutions with proper size and electron distribution. The above observations provided plenty of information to guide further optimizations on the 5-benzyl-2-phenylpyrimidin-4(3H)-one scaffold. 
Figure 4. Binding modes of SJ3 (magentas stick) and PD0325901 (green stick) in the allosteric site of MEK1 (PDB code: 3EQG). (A) The docked pose of SJ3 in comparison with the crystallized pose of PD0325901 with hydrogen bonds depicted in dashed lines; (B) The orientations of SJ3 and PD0325901 in the allosteric pocket.  
3. Conclusions
In summary, we designed and synthesized a serials of 5-benzyl-2-phenylpyrimidin-4(3H)-ones and identified them in vitro as moderate MEK1 inhibitors through Raf-MEK cascading assay. The results presented a novel scaffold for the MEK inhibitor design. Docking study also provided plenty of structural information, which could be used as guidance for optimizations on the 5-benzyl-2-phenylpyrimidin-4(3H)-one to achieve more potent allosteric MEK inhibitors.
4. Experimental section
4.1. Molecular docking
To explore the binding mode of compounds with MEK1, GOLD 3.0 software (Cambridge Crystallographic Data Center, Chembridge, UK) was used for docking following the previously established method by our group[9]. The 3D structure of MEK1 was downloaded from Protein Data Bank (ID: 3EQG). Before docking, the protein and ligand structures (SJ1-9) were prepared in Discovery Studio 2.5 (Accelrys Software Inc, San Diego, CA) by adding all hydrogens and deleting waters from protein as well as minimizing ligand conformational energy with default settings. The native ligand of 3EQG (PD0325901) was used to define the binding site[17] and the radius of the active site was set as 10 Å. The default genetic algorithm was used to preset parameters, and GOLD Score function was used in docking. The top-scored 10 poses for each ligand were further analyzed. This docking protocol was validated by comparison of the docked and the active conformation of PD0325901 with an RMSD value of 0.89 Å. The binding modes were visually inspected by PyMol 0.99 (DeLanoScentific LLC Inc., San Carlos, CA).
4.2. Chemistry
The procedures of synthesis of all chemical products are presented below. The tetrahydrofuran (THF) used was dried by distillation. All the reagents werecommercially available and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) performed on silica gel GF254purchased from Qingdao Haiyang Chemical Co. (Qingdao,China). Nuclear magnetic resonance (NMR) spectroscopy was performed on Brukeravance III 400 spectrometers. Mass spectra were obtained with electrospray ionization (ESI) produced by Waters ACQ-SQDLC-MS spectrometer. Melting points (mp) were uncorrected and measured by an XT4A apparatus. 
4.2.1. 5-Benzyl-2-(4-fluorophenyl)-6-methylpyrimidin-4(3H)-one (SJ1)
4-Fluro-benzamidine (1 mmol, 0.17 g) was dissolved in dry THF (6 mL) at 0 °C and stirred for 10 min, and then, NaH (2 mmol, 0.06 g) was added, followed by a continuous stirring foran additional 30 min beforecompound 1[9] (1.2 mmol, 0.26 g) in dry THF (1 mL) was added. The mixture was refluxed for 24 h. Subsequently, solvent was removed by evaporation under vacuum. The residue was added with 20 mL water, and the mixturewas fully extracted with ethyl acetate (EA). The organic layer was washed with saturated NaCl solution and dried over anhydrous sodium sulfate. After EA was removed, the crude product of SJ1 was acquired as white solid. The product was further recrystallized with EA to give SJ1 as white powder (0.11 g), 37% yield, mp: 233235 °C. 1H NMR (400 MHz, DMSO-d6)δ: 2.30 (s, 3H, CH3), 3.85 (s, 2H, CH2), 7.17 (t, J 6.8 Hz, 1H, Ph), 7.237.29 (m, 4H, Ph), 7.35 (t, J 8.8 Hz, 2H, Ph), 8.158.19 (dd, J1 7.0 Hz, J2 8.4 Hz, 2H, Ph), 12.71 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 22.15, 30.71, 115.94, 116.16 (d, JC-F 22 Hz, 2C), 126.37, 128.55 (4C), 128.79 (3C), 130.56 (d, JC-F 9 Hz, 2C), 130.65, 140.17, 164.45 (d, JC-F 248 Hz, 1C). MS (ESI) m/z 293.24 [M-H].
4.2.2. 5-Benzyl-2-(4-chlorophenyl)-6-methylpyrimidin-4(3H)-one (SJ2)
The procedure was the same as that of SJ1 except that 4-chloro-benzamidine was used as starting material. SJ2 was obtained as white powder, 0.09 g, 30% yield, mp: 234236 °C. 1H NMR (400 MHz, DMSO-d6) δ: 12.72 (brs, 1H, NH), 8.13 (d, J 8.4 Hz, 2H, Ph), 7.58 (d, J 8.4 Hz, 2H, Ph), 7.237.29 (m, 4H, Ph), 7.17 (t, J 6.8 Hz, 1H, Ph), 3.86 (s, 2H, CH2), 2.30 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ: 22.08, 30.74, 126.38 (2C), 128.55 (4C), 128.80 (3C), 129.12(2C), 129.85 (2C), 136.65, 140.10 (2C). MS (ESI) m/z 311.0872 [M+H]+.
4.2.3. 5-(4-Fluorobenzyl)-2-(4-fluorophenyl)-6-propylpyrimidin-4(3H)-one (SJ3)
4-Flubenzamid hydrochloride (3 mmol, 0.52 g) was dissolved in EtOH (20 mL), and then NaOEt/EtOH (7.5 mmol, 2.85 mL) solution was added, followed by a continuous stirring foran additional 10 min at r.t. before compound 2[9] (3.59 mmol, 0.95 g) in EtOH was added. The mixture was refluxed for 13 h under N2 protection. Then solvent was removed by evaporation under vacuum. The residue was added with 20 mL EA. The crude product was then purified by silica gel chromatography using DCM and MeOH (200:1, v/v) to give SJ3 as white powder (0.01 g), 3% yield, mp: 245247 °C. 1H NMR (400 MHz, DMSO-d6) δ: 12.75 (s, 1H, NH), 8.18 (dd, J1 8.6 Hz, J2 5.5 Hz, 2H, Ph), 7.36 (t, J 8.8 Hz, 2H, Ph), 7.26 (dd, J1 8.5 Hz, J2 5.7 Hz, 2H, Ph), 7.09 (t, J 8.9 Hz, 2H, Ph), 3.85 (s, 2H, CH2Ph), 2.602.53 (m, 2H, CH2), 1.51.61 (m, 2H, CH2), 0.87 (t, J 7.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)δ: 14.23, 21.41, 29.49, 36.28, 115.43 (d, JC-F 22 Hz, 2C), 115.53, 116.09 (d, JC-F 22 Hz, 2C), 130.19, 130.27, 130.23 (d, JC-F 8 Hz, 2C), 130.62 (d, JC-F 9 Hz, 2C), 136.79 (2C), 159.88, 161.08 (d, JC-F 240 Hz, 1C), 164.48 (d, JC-F 248 Hz, 1C). MS (ESI) m/z 339.26 [M-H].
4.2.4. 2-(4-Fluorophenyl)-5-(3-nitrobenzyl)-6-propylpyrimidin-4(3H)-one (SJ4)
The procedure was the same as that of SJ3 except that compound 3[9] was used as starting material. SJ4 was obtained as yellow powder, 0.56 g, 41% yield, mp: 256258 °C.1H NMR (400 MHz, DMSO-d6) δ: 0.87 (t, J 7.3 Hz, 3H, CH3), 1.581.63 (m, 2H, CH2), 2.632.54 (m, 2H, CH2), 4.01 (s, 2H, CH2Ph), 7.37 (t, J 8.8 Hz, 2H, Ph), 7.51 (d, J 8.6 Hz, 2H, Ph), 8.17 (t, J 8.4 Hz, 4H, Ph), 12.82 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6) δ: 14.18, 21.47, 30.05, 36.30, 116.11 (d, JC-F 22 Hz, 2C), 121.55 (2C), 123.09 (3C), 130.27 (2C), 130.70 (d,JC-F 9 Hz, 2C), 135.44 (2C), 143.08, 148.25, 164.54 (d, JC-F 248 Hz, 2C). MS (ESI) m/z 366.25 [M-H].
4.2.5. N-(3-((2-(4-Fluorophenyl)-6-oxo-4-propyl-1,6-dihydropyrimidin-5-yl)methyl)phenyl)methanesulfo-namide (SJ5)
SJ4 (1.187 mmol, 0.4367 g) was dissolved in MeOH, which was added with 5% Pd/C (0.044 g). The mixture was reacted in a H2 atmosphere for 11 h before filtered by celite. The solvent was then removed by evaporationunder vacuum, and the residue (compound 4, 1.16 mmol, 0.39 g) was dissolved in DCM (10 mL), to which pyridine (1.27 mmol, 0.1 mL) and DCM (1 mL) were added, followed by a continuous stirring for 36 h. Then 20 mL water was added, and the mixture was fully extracted with EA. The organic layer was washed with saturated NaCl (40 mL) solution and dried over anhydrous sodium sulfate. The crude product was then purified by silica gel chromatography using DCM and MeOH (100:1, v/v) to give SJ5 as white powder, 0.08 g, 18% yield, mp: 220221°C. 1H NMR (400 MHz, DMSO-d6) δ: 0.88 (t, J 7.1 Hz, 3H, CH3), 1.61 (m, 2H, CH2), 2.56 (t, J 7.2 Hz, 2H, CH2), 2.94 (s, 3H, SO2 CH3), 3.84 (s, 2H, CH2Ph), 6.97 (d, J 7.6 Hz, 1H, Ph), 7.03 (d, J 7.9 Hz, 1H, Ph), 7.08 (s, 1H, Ph), 7.22 (t, J 7.5 Hz, 1H, Ph),7.36(t, J8.1 Hz, 2H, Ph), 8.19 (s, 2H, Ph), 9.62 (s, 1H, NHSO2), 12.73 (br s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6) δ: 14.28, 21.38 (2C), 30.30, 36.28, 116.11 (d, JC-F 22 Hz,2C), 117.62, 119.85 (2C), 124.06 (2C), 129.62 (2C), 130.61 (d, JC-F 9 Hz, 2C), 138.86 (2C), 141.84 (2C), 164.49 (d, JC-F 248 Hz, 1C). MS (ESI) m/z 414.29 [M-H].
4.2.6. 5-Benzyl-2-(4-fluorophenyl)-6-oxo-1,6-dihydro-pyrimidin-4-yl benzenesulfonate (SJ6)
Compound 5[13] (0.66 mmol, 0.2 g) was dissolved in toluene, to which trimethylamine (0.8 mmol, 0.11 mL) and benzene sulfochloride (0.8 mmol, 0.1 mL) were added, and the mixture was refluxed for 19 h before 2 N HCl (40 mL) was added. The mixture was fully extracted with DCM. The organic layer was dried over anhydrous sodium sulfate. After DCM was removed, the crude product was purified by silica gel chromatography using DCM and MeOH (100:1, v/v) to give SJ6 as white powder, 0.03 g, 12% yield, mp: 192194 °C. 1H NMR (400 MHz, DMSO-d6) δ: 3.76 (s, 2H, CH2), 7.207.36 (m, 7H, Ph), 7.72 (t, J 7.8 Hz, 2H, Ph), 7.827.88 (m, 3H, Ph), 8.028.04 (m, 2H, Ph), 13.20 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 28.69, 116.24 (d, JC-F 22 Hz, 2C), 126.73, 128.59 (3C), 128.72 (4C), 128.85 (3C), 130.12 (3C), 130.89 (2C), 135.28, 137.53, 139.06, 164.49 (d, JC-F 248 Hz, 1C). MS (ESI) m/z 435.17 [M-H].
4.2.7. 5-Benzyl-2-(4-fluorophenyl)-6-oxo-1,6-dihydro-pyrimidin-4-yl 4-methylbenzenesulfonate (SJ7)
Compound 5[13] (0.98 mmol, 0.29 g) was dissolved in DMF, to which K2CO3 (1.96 mmol, 0.27 g) and p-toluenesulfonylchloride (1.96 mmol, 0.37 g) were added, followed by a continuous stirring for 10 h at r.t. The residue was added with 20 mL water, and the mixture was fully extracted with EA. The organic layer was washed with saturated NaCl solution and dried over anhydrous sodium sulfate. After filtration, the crude product was then purified by silica gel chromatographyusing DCM and MeOH (100:1, v/v) to give SJ7 as white powder, 0.11 g, 26% yield, mp: 183185 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.46 (s, 3H, CH3), 3.74 (s, 2H, CH2), 7.157.32 (m, 5H, Ph), 7.36 (t, J 8.8 Hz, 2H, Ph), 7.51 (d, J 8.3 Hz, 2H, Ph), 7.90 (d, J 8.2 Hz, 4H, Ph), 13.21(s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 21.64, 28.69, 116.26 (d, JC-F 22 Hz, 2C), 126.72 (2C), 128.72 (6C), 128.84 (3C), 130.48 (3C), 130.95 (d,JC-F 10 Hz, 2C), 134.44, 139.09, 146.23, 164.49 (d, JC-F 248 Hz, 1C). MS (ESI) m/z 449.24 [M-H]. 
4.2.8. 5-Benzyl-2-(4-fluorophenyl)-6-oxo-1,6-dihydro-pyrimidin-4-yl 4-nitrobenzenesulfonate (SJ8)
The procedure was the same as that of SJ7 except that compound 5[13] was used as starting material.SJ8 was obtained as white powder, 0.07 g, 45% yield, mp: 195197 °C. 1H NMR (400 MHz, DMSO-d6) δ: 3.78(s, 2H, CH2), 7.197.36 (m, 7H, Ph), 7.757.84 (m, 2H, Ph), 8.31 (d, J 8.8 Hz, 2H, Ph), 8.48 (d, J 8.8 Hz, 2H, Ph), 13.30 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 28.67, 116.3 (d, JC-F 22 Hz, 2C), 125.30 (2C), 126.79, 128.71 (4C), 128.89 (3C), 128.82, 130.33(2C), 130.48, 130.96 (d, JC-F 10 Hz, 2C), 138.88, 142.63, 151.37, 164.49 (d, JC-F 248 Hz, 1C). MS (ESI) m/z 480.07 [M-H].
4.2.9. Ethyl 5-benzyl-2-(4-fluorophenyl)-6-oxo-1,6-dihydropyrimidine-4-carboxylate (SJ9)
4-Flubenzamid hydrochloride (7.2 mmol, 1.38 g) was dissolved in EtOH (20 mL), to which KOH (7.2 mmol, 0.41 g) was added, followed by a continuous stirring for 15 min at r.t. After the mixture was filtered, compound 6[15] (7.2 mmol, 2 g) was added, and the mixture was refluxed for 13 h before the solvent was removed by evaporation under vacuum. The crude product was then purified by silica gel chromatographyusing DCM to give SJ9 as white powder, 0.2 g, 8% yield, mp: 173174 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.23 (t, J 7.1 Hz, 3H, CH3), 3.89 (s, 2H, CH2Ph), 4.31 (q, J 7.1 Hz, 2H, CH2), 7.167.19 (m, 1H, Ph), 7.327.21 (m, 4H, Ph), 7.38 (t, J 8.9 Hz, 2H, Ph), 8.15 (s, 2H, Ph), 13.12 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 14.31, 30.91, 62.11, 116.22 (d, JC-F 22 Hz,2C), 126.56, 128.65 (5C), 128.88 (5C), 130.97 (d, JC-F 9 Hz, 2C), 139.35, 164.49 (d, JC-F 248 Hz, 1C). MS (ESI)m/z 351.13 [M-H].
4.3. Biological assays
Purified recombinant inactive GST-tagged MEK1 protein, in addition to activated GST-tagged BRaf kinase, was purchased from Carna Biosciences Inc., Japan. Anti-Phospho MEK1/2(Ser217/221)-cryptate (pAb) and anti-GST-XL665 (mAb) antibodies were obtained from Cisbio Bioassays. All the synthesized derivatives were tested for MEK inhibitory potency in BRaf/MEK1 cascade assay. Assays were conducted following the general protocol as described below[10].
The homogeneous time resolved fluorescence (HTRF) assay was performed on a Proxiplate-384 F plus solidwhite plate (Greiner) with 2 μL kinase (0.44 ng/μL BRaf), 2 μL substrate (30 nM inactive MEK1), 2 μL (100 μM) ATP and 4 μL test compound at a variety of concentrations,which were incubated at room temperature for 2 h. Then 10 μL of the detection buffer containing 200× anti-phosphoMEK1/2 (Ser217/221)-Cryptate and 26 nM mAbanti-GST-XL665 was added, and the mixture was incubated at room temperature for another 3 h. Finally, the energy transfer was detected according to an increasein the fluorescence emission of the tracer at 668 nm and a decrease in the fluorescence emission of europium at 620 nm with FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, USA). The following formula was used to calculate the inhibition rate:
Inhibition (%) = (Rn Rc)/(Rn Rb) × 100%
Where Rn is negative control, Rc is compound signal, and Rb is blank control. The curve-fitting software GraphPad Prism was used to generate the curves and determine IC50 values for individual compounds tested. Values were expressed as the mean±SD determined in two to three independent experiments, each based on three biological replicates. 
This work was supported byNational Natural Science Fund of China (Grant No. 21172012), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20120001110010) and Beijing Natural Science Foundation of China (Grant No. 7162110).
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李灿, 李宏月, 孙静, 席丹丹, 王超, 梁磊, 许凤荣, 牛彦*, 徐萍*
北京大学医学部 药学院 药物化学系, 北京 100191       
摘要: Ras/Raf/MEK/ERK通路在人类的多种肿瘤中均表现异常活化, 通过抑制MEK激酶阻断该通路是有效的癌症治疗策略。本文描述了5-苄基-2-苯基嘧啶-4(3H)-酮类作为新型MEK变构抑制剂骨架的设计与合成。所得目标化合物在Raf-MEK级联实验中表现出中等的MEK1抑制效力, 对接研究结果显示活性最好的化合物SJ3的结合模式与常见的二芳胺类抑制剂PD0325901的结合模式非常相似。这些结果为基于该新型母核结构的进一步设计和优化并最终获得成药性分子提供了有力的依据。 
关键词: 5-苄基-2-苯基嘧啶-4(3H)-; MEK1 抑制剂; 对接
Received: 2018-05-31, Revised: 2018-07-17, Accepted: 2018-09-10.
Foundation items: National Natural Science Fund of China (Grant No. 21172012), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20120001110010) and Beijing Natural Science Foundation of China (Grant No. 7162110).
*Corresponding author. Tel.: +86-010-82802632, Fax: +86-010-82801117, E-mail:;

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