Bisarylureas Based on 1H-Pyrazolo[3,4-d]pyrimidine Scaffold as Novel Pan-RAF Inhibitors with Potent Anti-Proliferative Activities: Structure-Based Design, Synthesis, Biological Evaluation and Molecular Modelling Studies

RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy. With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold 1a. Most of the synthesized compounds showed good to excellent inhibitory activities against BRAFV600E kinase, possessed moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3 and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine kidney, MDCK). The most promising compound, 1v, exhibited potent inhibitory activity against not only BRAFV600E (half maximal inhibitory concentration, IC50 = 23.6 nM) but also wild-type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative activities against A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound 1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression of MEK (mitogen-activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, the promise of compound 1v was further confirmed by molecular dynamics simulation and binding free energy calculations.


Introduction
The mitogen-activated protein kinase (MAPK) cascades consist of Ras/RAF(Ras activating factor)/MEK(mitogen-activated protein kinase kinase)/ERK(extracellular receptor kinase) signal transduction and play an important role in the regulation of cellular activities, including proliferation, differentiation, and survival [1,2]. There are three RAF isoforms, ARAF, BRAF (v-raf murine sarcoma viral oncogene homolog B1), and CRAF (RAF-1), which play central roles in this cascade [3,4]. Among them, BRAF isoform is more easily activated by Ras and has higher basal kinase activity than the The 1H-pyrazolo [3,4-d]pyrimidine nucleus is an important drug-like scaffold present in many pharmacologically active compounds, and the antitumor activity was initially reported many years ago [20]. Having similar structure of the purines, 1H-pyrazolo [3,4-d]pyrimidine, a template of tyrosine kinase hinge-binding, was extensively chemically modified to improve the biological activity and kinase selectivity. To find potent RAF inhibitors, encouraged by the success of Sorafenib, we designed Sorafenib analogue 1a by replacing the pyridine ring of Sorafenib with 1H-pyrazolo [3,4-d]pyrimidine moiety ( Figure 2). To the best of our knowledge, although the inhibitory activities of 1a and its analogues targeting VEGFR-2 (vascular endothelial growth factor receptor 2) and FLT3 (fms-related tyrosine kinase 3) have been reported [21], the RAF inhibitory activities of these derivatives are being reported for the first time by our group. Based on the promising activities as VEGFR-2 and FLT3 inhibitors with potent in vitro and in vivo activity against acute myeloid leukemia, we would like to examine the RAF inhibitory activities further. Model of 1a in the active site of BRAF V600E (Protein Data Bank (PDB) code: 1UWJ), which was reported to be in the DFG-out inactive conformation, was constructed by Surflex-Dock according to our previous protocol [22]. As shown in Figure 3, the model suggested that 1a overlapped well with the co-crystal ligand sorafenib in the active site of BRAF V600E . The 1H-pyrazolo [3,4-d]pyrimidine ring in 1a occupied the ATP-binding pocket, and formed hydrogen bonds with the hinge region of BRAF V600E . The diaryl urea portion extended into the large hydrophobic back pocket created by the rearrangement of the DFG motif, and the urea CO and NH moiety formed a hydrogen bond with the backbone-NH of Asp 594 and side-chain carboxylate of Glu 501 in the DFG motif, respectively. These suggested that the diaryl urea contributes significantly to the inhibitor´s affinity. Considering the importance of the diaryl urea for the potency of kinase, we decided to maintain the diaryl urea portion in our inhibitors and tried to focus on the replacement of the terminal phenyl to investigate their effects on kinase inhibitory activities in this paper. In order to illustrate the effect of the N1 hydrogen on the inhibitory activity, N1-methylated compounds were also designed. Herein, we would like to report the synthesis and biological evaluation of these series compounds as novel pan-BRAF inhibitors. The molecular modelling study was also discussed in this paper. carboxylate of Glu 501 in the DFG motif, respectively. These suggested that the diaryl urea contributes significantly to the inhibitor´s affinity. Considering the importance of the diaryl urea for the potency of kinase, we decided to maintain the diaryl urea portion in our inhibitors and tried to focus on the replacement of the terminal phenyl to investigate their effects on kinase inhibitory activities in this paper. In order to illustrate the effect of the N1 hydrogen on the inhibitory activity, N1-methylated compounds were also designed. Herein, we would like to report the synthesis and biological evaluation of these series compounds as novel pan-BRAF inhibitors. The molecular modelling study was also discussed in this paper.

Chemistry
The target compounds were prepared according to the synthetic routes outlined in Scheme 1, which was distinct from that described previously [22]. All target compounds were prepared from the key intermediate 4-chloro-1H-pyrazolo [3,4-d]pyrimidine (2). The preparation of 2 from allopurinol by chlorination was evaluated and determined to be unsuitable for large scale preparation due to poor stability of the product with respect to polymerization. We therefore synthesized compound 2 by cycloaddition reaction of 4,6-dichloropyrimidine-5-carbaldehyde and hydrazine monohydrate [23]. In order to avoid the side reaction of 1-NH of compound 2, we designed the pyran-protected compound 3. Compound 3 was synthesized from compound 2 and then nucleophilic substitution with 4-aminophenol in the present of Cs2CO3 gave the phenoxylated derivatives 4. Condensation of carboxylate of Glu 501 in the DFG motif, respectively. These suggested that the diaryl urea contributes significantly to the inhibitor´s affinity. Considering the importance of the diaryl urea for the potency of kinase, we decided to maintain the diaryl urea portion in our inhibitors and tried to focus on the replacement of the terminal phenyl to investigate their effects on kinase inhibitory activities in this paper. In order to illustrate the effect of the N1 hydrogen on the inhibitory activity, N1-methylated compounds were also designed. Herein, we would like to report the synthesis and biological evaluation of these series compounds as novel pan-BRAF inhibitors. The molecular modelling study was also discussed in this paper.

Chemistry
The target compounds were prepared according to the synthetic routes outlined in Scheme 1, which was distinct from that described previously [22]. All target compounds were prepared from the key intermediate 4-chloro-1H-pyrazolo [3,4-d]pyrimidine (2). The preparation of 2 from allopurinol by chlorination was evaluated and determined to be unsuitable for large scale preparation due to poor stability of the product with respect to polymerization. We therefore synthesized compound 2 by cycloaddition reaction of 4,6-dichloropyrimidine-5-carbaldehyde and hydrazine monohydrate [23]. In order to avoid the side reaction of 1-NH of compound 2, we designed the pyran-protected compound 3. Compound 3 was synthesized from compound 2 and then nucleophilic substitution with 4-aminophenol in the present of Cs2CO3 gave the phenoxylated derivatives 4. Condensation of

Chemistry
The target compounds were prepared according to the synthetic routes outlined in Scheme 1, which was distinct from that described previously [22]. All target compounds were prepared from the key intermediate 4-chloro-1H-pyrazolo [3,4-d]pyrimidine (2). The preparation of 2 from allopurinol by chlorination was evaluated and determined to be unsuitable for large scale preparation due to poor stability of the product with respect to polymerization. We therefore synthesized compound 2 by cycloaddition reaction of 4,6-dichloropyrimidine-5-carbaldehyde and hydrazine monohydrate [23].
In order to avoid the side reaction of 1-NH of compound 2, we designed the pyran-protected compound 3. Compound 3 was synthesized from compound 2 and then nucleophilic substitution with 4-aminophenol in the present of Cs 2 CO 3 gave the phenoxylated derivatives 4. Condensation of 4 with the corresponding phenyl isocyanate in CH 2 Cl 2 provided the urea 5 in moderate yields. Finally, compound 5 was deprotected using 4 M HCl in dioxane to produce the target compounds 1a-1t in high yields. N1-methylated compounds 1u and 1v were also prepared from intermediate 2. Compound 2 was methylated using iodomethane in the presence of sodium hydride to obtain compound 6, with subsequent nucleophilic substitution with 4-aminophenol in the presence of Cs 2 CO 3 to give the phenoxylated derivatives 7 in good yield. Finally, condensation with the corresponding phenyl isocyanate provided the target compounds 1u and 1v in high yields. with the corresponding phenyl isocyanate in CH2Cl2 provided the urea 5 in moderate yields. Finally, compound 5 was deprotected using 4 M HCl in dioxane to produce the target compounds 1a-1t in high yields. N1-methylated compounds 1u and 1v were also prepared from intermediate 2.
Compound 2 was methylated using iodomethane in the presence of sodium hydride to obtain compound 6, with subsequent nucleophilic substitution with 4-aminophenol in the presence of Cs2CO3 to give the phenoxylated derivatives 7 in good yield. Finally, condensation with the corresponding phenyl isocyanate provided the target compounds 1u and 1v in high yields.

Kinase Inhibitory Activity
The enzyme activities are summarized in Table 1. Based on the results of molecular docking, we focused on optimization of the terminal phenyl ring to improve the kinase inhibitory activities. The effects of ortho-, meta-, and para-substitution and disubstitution on the terminal phenyl were investigated. Several trends can be observed from Table 1. The results displayed that the 4-chloro-3-trifluomethylphenyl group is important for potency against BRAF V600E , which is consistent with the molecular modelling that the terminal phenyl group in 1a projects into a large hydrophobic pocket created by the rearrangement of the DFG motif. As we expected, compound 1e, bearing no substitution on the terminal phenyl, displayed no inhibitory activity even at 1000 nM, which clearly indicated the importance of the hydrophobic interaction between the inhibitor and the hydrophobic amino acid residue of the back pocket. A small group of ortho-substitution such as fluoro is tolerated, whereas large groups are not tolerated. For example, compounds 1f and 1r, which bear 2-Cl and 2-NO2, are less potent than the parent compound 1b. A variety of substituents at the meta-position, including both

Kinase Inhibitory Activity
The enzyme activities are summarized in Table 1. Based on the results of molecular docking, we focused on optimization of the terminal phenyl ring to improve the kinase inhibitory activities. The effects of ortho-, meta-, and para-substitution and disubstitution on the terminal phenyl were investigated. Several trends can be observed from Table 1. The results displayed that the 4-chloro-3-trifluomethylphenyl group is important for potency against BRAF V600E , which is consistent with the molecular modelling that the terminal phenyl group in 1a projects into a large hydrophobic pocket created by the rearrangement of the DFG motif. As we expected, compound 1e, bearing no substitution on the terminal phenyl, displayed no inhibitory activity even at 1000 nM, which clearly indicated the importance of the hydrophobic interaction between the inhibitor and the hydrophobic amino acid residue of the back pocket. A small group of ortho-substitution such as fluoro is tolerated, whereas large groups are not tolerated. For example, compounds 1f and 1r, which bear 2-Cl and 2-NO 2 , are less potent than the parent compound 1b. A variety of substituents at the meta-position, including both electron-donating and electron-withdrawing groups are tolerated, which suggested that the electronic interaction of the substituent had little effect on the kinase inhibitory activities. For example, introduction of electron-donating groups methyl and ethyl in compounds 1b and 1l increase the activities against BRAF V600E . Substitution with electron-withdrawing groups such as Cl and Br on the phenyl ring also leads to enhancement of kinase inhibitory activities (1g and 1s). Substitution at the para-position is also tolerated. For example, compound 1c with 4-chloro substitution is more potent than compound 1g. In addition, di-substitution with two electron-withdrawing groups such as F, Cl and CF 3 on the phenyl ring leads to dramatic improvement of kinase inhibitory activities (1a, 1c, 1i, 1j and 1n). electron-donating and electron-withdrawing groups are tolerated, which suggested that the electronic interaction of the substituent had little effect on the kinase inhibitory activities. For example, introduction of electron-donating groups methyl and ethyl in compounds 1b and 1l increase the activities against BRAF V600E . Substitution with electron-withdrawing groups such as Cl and Br on the phenyl ring also leads to enhancement of kinase inhibitory activities (1g and 1s). Substitution at the para-position is also tolerated. For example, compound 1c with 4-chloro substitution is more potent than compound 1g. In addition, di-substitution with two electron-withdrawing groups such as F, Cl and CF3 on the phenyl ring leads to dramatic improvement of kinase inhibitory activities (1a, 1c, 1i, 1j and 1n). The modelling suggested that 1a formed hydrogen bonds with the hinge region of BRAF V600E and elaboration at the interaction at NH position might not be tolerated. Surprisingly, the N-methylation compounds 1u and 1v exhibited higher inhibitory activities against BRAF V600E than the unsubstituted counterparts 1g and 1a, implying that the methyl substitution at the N1 position of 1H-pyrazolo [3,4- The modelling suggested that 1a formed hydrogen bonds with the hinge region of BRAF V600E and elaboration at the interaction at NH position might not be tolerated. Surprisingly, the N-methylation compounds 1u and 1v exhibited higher inhibitory activities against BRAF V600E than the unsubstituted counterparts 1g and 1a, implying that the methyl substitution at the N1 position of 1H-pyrazolo [3,4-d]pyrimidine was beneficial for the inhibitory activity of BRAF V600E . The favorable hydrophobic interaction imposed by the N1-methyl might be responsible for the improvement in potency.
Subsequently, we further evaluated the inhibitory activities of compounds 1l, 1u and 1v, which exhibited excellent inhibitory activities at 1000 nM and 100 nM ( Table 2). As shown in Table 2, 1l, 1u and 1v exhibited stronger inhibitory activities against BRAF V600E than the positive control Sorafenib.

In Vitro Anti-Proliferative Activity
The cell growth inhibitory activities of newly synthesized compounds were evaluated against four human cancer cell lines, including wild-type BRAF cell lines (PC-3 and A549), and BRAF V600E cell lines (A375, HT-29) using standard MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay in vitro, with Sorafenib as the positive control (Table 3). Additionally, cytotoxic assays were conducted using normal cell line MDCK (Madin-Darby canine kidney). As depicted in Table 3, most compounds showed moderate to good anti-proliferative activities, while compounds 1e and 1r proved to be ineffective against all four cell lines (half maximal inhibitory concentration, IC 50 > 80 µM). Some of the synthesized compounds exhibited similar anti-proliferative activities compared with Sorafenib against two or more cell lines. Remarkably, compounds 1c, 1f, 1h, 1i, 1o and 1s demonstrated much higher anti-proliferative activities than the positive control Sorafenib against the PC-3 cell line. In particular, compounds 1f and 1i represented a 12-fold improvement in activity compared to Sorafenib with the IC 50 value of 1.12 µM. Meanwhile, compound 1f exhibited a 2-fold improvement compared to Sorafenib in inhibiting A549 cell line proliferation. Compound 1n displayed stronger anti-proliferative activities than Sorafenib against the A375 cell line. Unfortunately, the results indicated that most of the compounds were ineffective (IC 50 > 80 µM) on the HT-29 cell line. In addition, the results demonstrated that compounds with two electron-withdrawing substituents on the terminal phenyl ring (1a, 1c, 1i, and 1n) also presented promising anti-proliferative activities. It is worth noting that most compounds indicated no inhibitory activity against MDCK cell lines (IC 50 > 80 µM), which suggested that the compounds exhibit good selectivity towards cancer cells rather normal cells.

Kinase Inhibitory Profiles of Compound 1v
Compound 1v exhibited higher inhibitory activity against BRAF V600E than positive control Sorafenib with IC 50 values of 23.6 nM, effective cellular anti-proliferative potencies against four tumor cell lines (A375, HT-29, PC-3, A549) which were comparable to the efficacy of Sorafenib, as well as better selection to normal cells compared with Sorafenib. Therefore, we selected compound 1v for further study. The kinase selectivity profile of compound 1v was assessed over 19 different protein kinases (Table 4). The results exhibited that 1v had potent inhibitory activity against not only BRAF V600E (IC 50 = 23.6 nM) but also wild-type BRAF (IC 50 = 51.5 nM) and C-RAF (IC 50 = 8.5 nM), and almost no significant inhibitory activity against 13 other tested protein kinases even at concentration of 1000 nM. The results revealed that compound 1v had a very good selectivity profile and was a pan-RAF kinases inhibitor.

Flow Cytometric Analysis of Cell-Cycle Arrest
To study the effect of the target compounds on cell cycle progression, a flow-activated cell sorting analysis was performed to examine the cell cycle progression upon treatment with the representative compound 1v which exhibited most promising kinase inhibitory and anti-proliferative activities. As shown in Figure 4 and Table 5, cell cycle assay of the A375 cell line following treatment with 1v showed a dose-dependent cell cycle arrest in the 'G0/G1' phase and a decrease in 'G2/M' phase of cell cycle in the treated group compared to the control.  Propidium iodide-Annexin V.

Signaling Inhibition in A375 and HT-29 Cell Lines
To determine the potency of 1v as a RAF inhibitor, we preliminary investigated the in vitro MAPK signal suppression effect of 1v in A375 and HT-29 cell lines. The effect of compound 1v on the expression of phosphor-MEK protein (RAF's downstream target protein) in A375 and HT-29 cell lines was assessed by western blot analysis. As shown in Figure 5, 1v suppressed phosphor-MEK levels in a concentration-dependent manner in two cancer cell lines, reflecting the inhibition of the downstream molecules in MAPK signal transduction. Notably, the inhibitory potency of compound 1v on MEK phosphorylation was not so conspicuous at IC50 concentration, which may be related to the complicated mechanism of tumor cell growth and MEK phosphorylation. On one hand, the anti-proliferative IC50 values were determined by cell survival or proliferation, which is regulated by several different factors [24]. On the other hand, MEK phosphorylation is regulated by not only by RAF but also other

Signaling Inhibition in A375 and HT-29 Cell Lines
To determine the potency of 1v as a RAF inhibitor, we preliminary investigated the in vitro MAPK signal suppression effect of 1v in A375 and HT-29 cell lines. The effect of compound 1v on the expression of phosphor-MEK protein (RAF's downstream target protein) in A375 and HT-29 cell lines was assessed by western blot analysis. As shown in Figure 5, 1v suppressed phosphor-MEK levels in a concentration-dependent manner in two cancer cell lines, reflecting the inhibition of the downstream molecules in MAPK signal transduction. Notably, the inhibitory potency of compound 1v on MEK phosphorylation was not so conspicuous at IC 50 concentration, which may be related to the complicated mechanism of tumor cell growth and MEK phosphorylation. On one hand, the anti-proliferative IC 50 values were determined by cell survival or proliferation, which is regulated by several different factors [24]. On the other hand, MEK phosphorylation is regulated by not only by RAF but also other kinases such as Mos (an upstream activator of mitogen-activated protein kinase) and MEKK-1 (mitogen activated protein kinase kinase kinase 1) [25,26]. When compound 1v was applied at its anti-proliferative IC 50 concentration, the expression of MEK phosphorylation via RAF regulation might have been just partly inhibited, and other kinases could still activate MEK phosphorylation, which resulted in the unobvious inhibitory effects of MEK phosphorylation. However, when the concentration reached twice the IC 50 , MEK phosphorylation was obviously inhibited, implying that the compound 1v can effectively inhibit the Ras/RAF/MEK/ERK pathway at higher concentrations. kinases such as Mos (an upstream activator of mitogen-activated protein kinase) and MEKK-1 (mitogen activated protein kinase kinase kinase 1) [25,26]. When compound 1v was applied at its anti-proliferative IC50 concentration, the expression of MEK phosphorylation via RAF regulation might have been just partly inhibited, and other kinases could still activate MEK phosphorylation, which resulted in the unobvious inhibitory effects of MEK phosphorylation. However, when the concentration reached twice the IC50, MEK phosphorylation was obviously inhibited, implying that the compound 1v can effectively inhibit the Ras/RAF/MEK/ERK pathway at higher concentrations.

Molecular Modelling
In order to analyze in depth the interaction of synthesized compounds with BRAF, we performed a molecular dynamics (MD) study on 1a and N1-methylated compound 1v in complex with BRAF V600E . Firstly, compound 1v was also successfully docked into the active site of BRAF V600E (PDB code: 1UWJ) using Surflex-Dock. MD simulations were then carried out on inhibitors 1a and 1v in complex with BRAF V600E in explicit aqueous solution for 50 ns. The stability of the system under simulation was evaluated by the root-mean-square deviation (RMSD) of the backbone atoms relating to the starting structures ( Figure 6). As can be seen in the plot, all systems were stable during the 50 ns MD simulation. We compared the representative snapshots of each complex taken from the last 10 ns MD trajectories with the initial structure ( Figure 7). As illustrated in Figure 7, the binding modes of 1a and 1v from MD simulated results were nearly the same as the docked structures. The 1H-pyrazolo [3,4-d]pyrimidine moiety inserted into the deep cleft of ATP-binding site, and the diaryl urea portion extended into the large hydrophobic back pocket created by the rearrangement of the DFG motif.
The hydrogen bond plays an important role in inhibitor binding to kinase. The major interactions were the important H-bonding interactions between inhibitors 1a and 1v and with Glu 501, Asp594 and Cys532 ( Table 6). The CO and NH motifs of the urea group of compounds 1a and 1v formed hydrogen bond interactions with the backbone-NH of Asp594 and side-chain carboxylate of Glu 501 in the DFG motif with very high hydrogen-bond occupancies, respectively. The N7 of 1H-pyrazolo [3,4-d]pyrimidine of compounds 1a and 1v formed moderate hydrogen bond interactions with Cys532 in the hinge region of BRAF V600E . The 1H-pyrazolo [3,4-d]pyrimidine N1H of compound 1a established a weak hydrogen bond interaction with backbone of Cys 532.

Molecular Modelling
In order to analyze in depth the interaction of synthesized compounds with BRAF, we performed a molecular dynamics (MD) study on 1a and N1-methylated compound 1v in complex with BRAF V600E . Firstly, compound 1v was also successfully docked into the active site of BRAF V600E (PDB code: 1UWJ) using Surflex-Dock. MD simulations were then carried out on inhibitors 1a and 1v in complex with BRAF V600E in explicit aqueous solution for 50 ns. The stability of the system under simulation was evaluated by the root-mean-square deviation (RMSD) of the backbone atoms relating to the starting structures ( Figure 6). As can be seen in the plot, all systems were stable during the 50 ns MD simulation. We compared the representative snapshots of each complex taken from the last 10 ns MD trajectories with the initial structure ( Figure 7). As illustrated in Figure 7, the binding modes of 1a and 1v from MD simulated results were nearly the same as the docked structures. The 1H-pyrazolo [3,4-d]pyrimidine moiety inserted into the deep cleft of ATP-binding site, and the diaryl urea portion extended into the large hydrophobic back pocket created by the rearrangement of the DFG motif.  The hydrogen bond plays an important role in inhibitor binding to kinase. The major interactions were the important H-bonding interactions between inhibitors 1a and 1v and with Glu 501, Asp594 and Cys532 (Table 6). The CO and NH motifs of the urea group of compounds 1a and 1v formed hydrogen bond interactions with the backbone-NH of Asp594 and side-chain carboxylate of Glu 501 in the DFG motif with very high hydrogen-bond occupancies, respectively. The N7 of 1H-pyrazolo [3,4-d]pyrimidine of compounds 1a and 1v formed moderate hydrogen bond interactions with Cys532 in the hinge region of BRAF V600E . The 1H-pyrazolo [3,4-d]pyrimidine N1H of compound 1a established a weak hydrogen bond interaction with backbone of Cys 532.   The binding free energies were calculated by using MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) and MM-GBSA (molecular mechanics Generalized-Born surface area) programs in AMBER ( Table 7). As can been seen, MM-PBSA and MM-GBSA calculations verified that ligand 1v  The binding free energies were calculated by using MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) and MM-GBSA (molecular mechanics Generalized-Born surface area) programs in AMBER ( Table 7). As can been seen, MM-PBSA and MM-GBSA calculations verified that ligand 1v showed improved potency compared to that of 1a, which was in good agreement with the experimental data. According to the energy individual components of the binding free energies, the favorable contributors to ligand binding were van der Waals (vdW) terms, electrostatic and nonpolar salvation energies, whereas polar solvation and entropy terms oppose binding. The favorable electrostatic interactions were counteracted by the unfavorable electrostatics of desolvation upon binding. Consequently, the total electrostatic interaction contributions were unfavorable to binding in all systems.

Chemistry
Melting points were determined using an X-4 Melting-point Apparatus with Microscope (Yuhua Instrument Co., Ltd., Henan, China) and were uncorrected. 1 H-and 13 C-NMR spectra were recorded on a Bruker AVIII 400 MHz or Bruker AVIII 600 MHz and 100 MHz spectrometer (Bruker Co., Fällanden, Switzerland) with tetramethylsilane (TMS) as the internal standard, the values of the chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hz. Mass spectra (ESI-MS) were performed on Waters ZQ4000 (Waters Co., Milford, MA, USA). All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates at 254 nm under an ultraviolet (UV) light. Flash column chromatography separations were performed on normal phase silica gel (200-300 mesh, Merck Co., Elkton, VA, USA) or reverse phase silica gel by using Yamazen AI-580 flash chromatography (Yamazen Co., Osaka, Japan) with UV detection at 254 nm. All reagents were purchased from commercial vendors and were used without further purification. All oxygen-sensitive or moisture-sensitive reactions were run under nitrogen atmosphere. Yields were not optimized. (2). To the solution of 4,6-dichloropyrimidine-5-carbaldehyde (1.0 g, 0.006 mol) in methanol (20 mL) at −65 • C, triethylamine (0.97 mL,1.2 equivalents (eq.)) was added. A solution of hydrazine monohydrate (0.274 mL 1.0 eq.) in methanol (10 mL) was slowly dripped into above stirred solution by using a constant-pressure dropping funnel. The mixture was allowed to warm to room temperature and stirred for 2-3 h. The reaction mixture was concentrated in vacuo and crude product was diluted with water (20 mL), and extracted with EtOAc (60 mL × 3). The combined organic layer was washed with saturated solution of NaCl (60 mL × 3), dried over  (3). Compound 2 (1.88 g, 0.012 mol) was dissolved in EtOAc (50 mL) and heated to 50 • C. After 10 min pyridinium 4-toluenesulfonate (PPTs) (50 mg) were added, followed by the addition of 3,4-dihydro-2H-pyran. The resulting reaction mixture was at stirred 50 • C. After the reaction was complete according to the TLC detection, the mixture was cooled to room temperature, washed with water (60 mL × 1), and a saturated solution of NaCl (50 mL × 2), and dried over MgSO 4 . The ethyl acetate was removed and petroleum ether (60 mL × 2) was added. The mixture was heated and filtered through cotton. Removal of petroleum ether in vacuo gives compound 3 as light yellow colored solid. Yield: 76.5%. 1 (4). To the mixture of para-aminophenol (0.28 g) in dimethylformamide (DMF, 2 mL), Cs 2 CO 3 (1.2 eq.) was added under the protection of nitrogen. The reaction mixture was stirred at room temperature for 1.5-2 h, and then compound 3 was added slowly. After stirring overnight, the reaction mixture was diluted with EtOAc (50 mL) and washed with 1 M NaOH (60 mL × 1), water (80 mL × 1), 5% LiCl (50 mL × 2) and brine. The organic layer was dried over MgSO 4 and concentrated to give compound 4 as a brown oily solid. Yield: 79.5%. 1

Kinase Inhibitory Activity
The BRAF V600E inhibitory activity was evaluated by using the Z'-LYTE technology platform (Life Technologies, Carlsbad, CA, USA), and Sorafenib was employed as the positive control. The experiments were performed according to the instructions of the manufacturer, and single point concentration testing with two independent data points (n = 2). According to the anti-proliferative activities and percent inhibition values of compounds, we chose compounds 1l, 1u and 1v to evaluate kinase activity at ten concentration gradients from 1000 to 0.0508 nM (3-fold dilution). The IC 50 values were calculated from the inhibition curves from two separate experiments.

Cell proliferation Inhibition Assay
The anti-proliferative activities of compounds were evaluated against A375, HT-29, PC-3, A549 and MDCK cell lines by the standard MTT assay in vitro, with Sorafenib as the positive control. All cell lines were purchased from Cell Bank of China Science Academy (Shanghai, China). All chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Gibco (Gibco BRL, Grand Island, NY, USA). The PC-3 and A375 cell lines was maintained in Dulbecco's modified eagle (DEME) medium supplement with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The others were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplement with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Approximately 5 × 10 3 cells, suspended in medium, were plated into each well of a 96-well plate and grown at 37 • C in a humidified atmosphere with 5% CO 2 for 24 h. The following day, various concentrations of tested compounds were added to the culture medium and incubated for 48 h. Fresh MTT was added to each well at the terminal concentration of 5 mg/mL in phosphate buffered saline (PBS), and incubated with cells at 37 • C for 4 h.
The formazan crystals in each well were dissolved in 150 µL DMSO, and the absorbency at 570 nm was measured with an enzyme-linked immunosorbent assay plate reader. All of the compounds were tested three times in each of the cell lines.

Flow-Activating Cell Sorting Analysis
The effect of compound 1v on cell cycle phase distribution of human malignant melanoma A375 was assessed using flow cytometry. When the cells grew to about 70% confluence in 6-well microplates, they were treated with compound 1v at given concentrations (6.11 µmol/L, 12.21 µmol/L, 24.42 µmol/L). After 48 h, cells were harvested by trypsinization, washed with PBS, and fixed in 70% ice cold (4 • C) ethanol overnight. They were then washed with PBS, incubated with Ribonuclease (RNase) (50 mg/mL final concentration) at 37 • C for 30 min, stained with propidium iodide (50 mg/mL final concentration), and analyzed by flow cytometry (BD FACS Canto II, BD Biosciences, San Jose, CA, USA).

Western Blot Analysis
Total cells were harvested after treatment, washed twice with cold PBS, and lysed with RIPA lysis buffer (KeyGEN BioTECH, Nanjing, China) including 1% cocktail (Selleck Chemicals, Houston, TX, USA). Lysates were centrifuged at 12,000× g at 4 • C for 15 min, and the supernatant was collected. Total protein concentration was quantified using the bicinchoninic acid (BCA; Beyotime Institute of Biotechnology, Nanjing, China) protein assay kit (Sigma Chemical Co., St. Louis, MO, USA). The proteins were separated electrophoretically using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then the gel was transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were subsequently blocked with the use of 5% milk in tris-buffered saline Tween (TBST) for 1 h and incubated overnight with antibodies against p217/p221-MEK (Pmek, Cell Signaling Technology, Beverly, MA, USA), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Inc., Santa cruz, CA, USA) in TBST containing 5% defatted milk at 4 • C followed by incubation with horseradish peroxidase-linked secondary antibodies (Beyotime Institute of Biotechnology, Nanjing, China) at 4 • C for additional 1 h. The immunobands were detected with an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology, Nanjing, China).

Molecular Modelling
Molecular docking was performed with Surflex-Dock program that is interfaced with Sybyl 7.3 [27]. The crystal structure of BRAF V600E kinase in complex with Sorafenib (PDB ID: 1UWJ [28]) was obtained from the PDB. All the water and ligands were removed and the random hydrogen atoms were added. The structures of the synthesized compounds were generated and minimized using tripos force fields. All the other default parameters were used. The highest-scored conformation based on the Surflex-Dock scoring functions, was selected as the final bioactive conformation.
The MD simulations were performed using AMBER 12 software package (San Francisco, CA, USA) [29,30] according to our previously published protocol [31]. The missing residues, which were not solved in the crystal structures, were modeled using the loop building routine in Modeler [32]. Geometry optimization and the electrostatic potential calculations for 1a and 1v were carried out at the HF/6-31G* level of the Gaussian03 suite [33]. The atomic partial charge was obtained by using the restrained electrostatic potential (RESP) fitting technique [34] implemented in AmberTools [35]. The force field parameters for 1a and 1v were generated by the general amber force field (GAFF) [36] using the Antechamber program. The AMBER 99SB force field [37] was used to simulate the protein structure and the ionization states of amino acid residues were set according to the standard protocol. The model was neutralized by adding suitable counterions and were solvated in a truncated octahedron box of the transferable interaction potential (TIP3P) [38] water molecules with a margin distance of 12 Å. The fully solvated and neutralized system was subjected to energy minimization. Following minimization, the system was gradually heated from 0 to 300 K in 50 ps using a Langevin thermostat with a coupling coefficient of 1.0/ps with a force constant 2.0 kcal·mol −1 ·Å −2 on the complex. Then, 50 ps of density equilibration with a force constant 2.0 kcal·mol −1 ·Å −2 on the complex was performed. Subsequently the systems were again equilibrated for 500 ps by releasing all the restraints. Finally, the 50-ns MD production was performed at 300 K with 1.0 atm pressure. During the MD simulations, the long-range Coulombic interactions were handled using the particle mesh Ewald (PME) method [39]. The cutoff distance for the long-range vdW energy term was set at 10.0 Å. Periodic boundary conditions were applied to avoid edge effects in all calculations. The SHAKE algorithm [40] was employed on all atoms covalently bond to hydrogen atoms, allowing for an integration time step of 2 fs. Coordinate trajectories were recorded every 10 ps throughout all equilibration and production runs. The binding free energies were calculated by using the MM-PBSA and MM-GBSA [41] procedure in AMBER12. An average of 1000 snapshots were extracted from the last 10 ns MD trajectory for the calculations. The calculations of entropic terms are time-consuming and have low prediction accuracy, thus the conformational entropy change was omitted.

Conclusions
We designed and synthesized pan-RAF bisarylurea inhibitors bearing 1H-pyrazolo [3,4d]pyrimidine scaffold targeting DFG-out conformation on the basis of structure-based drug design using docking of compound 1a into the active site of X-ray cocrystal structures of BRAF. Molecular docking indicated that these compounds bind to the ATP-binding site of DFG-out BRAF conformation, with the urea portion forming hydrogen bonds with Asp594 and Glu501, and the terminal-substituted phenyl projecting into the large hydrophobic pocket. By optimizing the substituents at both terminal phenyl ring and 1-NH, a series of compounds were obtained. In particular, the most promising compound, 1v, exhibited potent inhibitory activity against not only BRAF V600E (IC 50 = 23.6 nM) but also wild-type BRAF (IC 50 = 51.5 nM) and C-RAF (IC 50 = 8.5 nM), and effective cellular anti-proliferative potencies against A375, HT-29, PC-3 and A549 cell lines, as well as a very good selectivity profile. Furthermore, flow-activated cell sorting analysis revealed that compound 1v arrested the cell cycle of the A375 cell line in the G0/G1 phase with a concentration-dependent effect. Moreover, compound 1v showed significant suppression of MEK phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, molecular dynamics simulation and binding free energy calculations were conducted to analysis the interaction of synthesized compounds with BRAF, and the results suggested that ligand 1v showed improved potency compared to that of 1a.