Potent Pan-Raf and Receptor Tyrosine Kinase Inhibitors Based on a

Article pubs.acs.org/jcim

Potent Pan-Raf and Receptor Tyrosine Kinase Inhibitors Based on a Cyclopropyl Formamide Fragment Overcome Resistance Yanmin Zhang,†,∥ Lu Wang,†,∥ Qing Zhang,‡ Gaoyuan Zhu,† Zhimin Zhang,† Xiang Zhou,† Yadong Chen,† Tao Lu,*,†,§ and Weifang Tang*,† †

School of Basic Science, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu 211198, China School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu 211198, China § State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China ‡

S Supporting Information *

ABSTRACT: While selective BRafV600E inhibitors have been proven effective clinically, acquired resistance rapidly develops through reactivation of the mitogen-activated protein kinase (MAPK) pathway. Simultaneous targeting of multiple nodes in the pathway offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Replacement pyridine group of Y-1 by a cyclopropyl formamide group afforded I-01 as a novel multitargeted kinase inhibitor template. I-01 displayed enzyme potency against Pan-Raf and receptor tyrosine kinases (RTKs). Based on the binding mode of I-01, analogues I-02−I-18 were designed and synthesized. The most promising compound I-16 potently inhibits all subtypes of Rafs with IC50 values of 3.49 (BRafV600E), 8.86 (ARaf), 5.78 (BRafWT), and 1.65 nM (CRaf), respectively. I-16 not only exhibit comparable antiproliferative activities with positive control compounds against HepG2, SW579, MV4-11, and COLO205 cell lines, but also suppress the proliferation of melanoma SK-MEL-2 harboring overexpressed BRafWT with IC50 values of 0.93 μM. The Western blot results for the ERK inhibition in human melanoma SK-MEL-2 cell lines show that I-16 inhibits the proliferation of SK-MEL-2 cell lines without paradoxical activation of ERK, which support the hypothesis that the inhibition of Pan-Raf and RTKs might be a tractable strategy to overcome the resistance of melanoma induced by the therapy with the current selective BRafV600E inhibitors. despite impressive initial responses in BRafV600E tumors, most patients developed acquired resistance (the resistance caused by vemurafenib) to vemurafenib after six months, and about 20% of patients have intrinsic resistance and do not respond to the drug despite the presence of BRafV600E.10 In BRafWT tumors, vemurafenib even activates the MAPK pathway, which thus enhances tumor growth in some xenograft models.11 Resistance is generally mediated by pathway reactivation, and multiple mechanisms have been described. As the repair mechanism of Path B1 in Figure 2, the addition of DFG-in BRafV600E inhibitors at a certain concentration range will bind to BRafV600E and inhibit the kinase, but they lack inhibitory activity against heterodimers of BRafWT and CRaf, which can induce the phosphorylation of MEK/ERK leading to acquired resistance. Raf homodimer of CRafs can directly phosphorylate MEK despite the absence of BRaf and ARaf as shown in Path B2.12 In Path B3, ARaf acts as a scaffold to stabilize BRaf-CRaf heterodimers.13 When there is upstream signaling from RTKs (receptor tyrosine kinases) amplication, Rafs form dimers

1. INTRODUCTION The upregulation or activation of the Ras-Raf-Mek-Erk (mitogen-activated protein kinase MAPK) signaling pathway always leads to abnormal cell proliferation and differentiation.1 As a downstream effector of Ras in the MAPK signaling pathway, Raf plays a crucial role in oncotherapy. BRaf demonstrates more potent biochemical activity and mutation rate compared with the other two members of Raf isoforms ARaf and CRaf.2 Over 60% of melanoma patients or 5% of colorectal carcinoma patients are associated with mutated BRaf (BRafV600E, Val600-Glu600, V600E).3 According to the binding modes of DFG (a conserved amino acid sequence of D594, F595, and G596), which derived from crystallographic analysis, Raf kinase inhibitors are categorized into two types: DFG-out and DFG-in series.4 The DFG-out inhibitors, such as sorafenib5 and LY3009120,6 engage the protein in DFG-out conformation and inhibit all the subtypes of Raf proteins. The DFG-in inhibitors, such as vemurafenib,7 bind to the ATP binding site of the kinase with a DFG-in conformation and inhibit BRafV600E with high selectivity (Figure 1). Vemurafenib increases overall survival in patients with BRafV600E melanoma, thus validating BRafV600E as therapeutic targets in this disease.7−9 However, © XXXX American Chemical Society

Received: December 31, 2016 Published: May 9, 2017 A

DOI: 10.1021/acs.jcim.6b00795 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Article

Journal of Chemical Information and Modeling

Figure 1. Binding mode of BRafV600E (A) DFG-out conformation for PDB 1UWJ (crystallized with sorafenib) and (B) DFG-in conformation for PDB 3OG7 (crystallized with vemurafenib).

development of Pan-Raf/RTKs MTKIs to overcome the resistance caused by vemurafenib.

2. MATERIALS AND METHODS 2.1. Computational Methods. 2.1.1. Binding Site Detection. Two programs including FTMap and SiteMap in the Schrödinger suite23 were used to detect the active binding site of BRaf. As a particularly useful and accurate in predicting the binding site of proteins, FTMap globally samples small organic molecules as probes on the surface of a target protein by a library of 16 small organic probe molecules, finds favorable positions, clusters conformations, and ranks the consensus sites (CSs) (also called “hot spots”) on the basis of average energy and the number of probe clusters they included.24 It was performed through its online server (http://ftmap.bu.edu). SiteMap24 incorporated in the Schrodinger software is a fast, accurate, and practical binding site identification methods. It was carried out to detect the binding site of BRaf crystal structures and predict the druggability of those sites. 2.1.2. Molecular Docking. The crystal structures of BRaf (PDB code 1UWJ, 3IDP, and 4XV2) were downloaded from Protein Data Bank (PDB) and prepared by the Protein Preparation Wizard in the Schrödinger suite.23 The small molecules were initially minimized by the LigPrep module in Schrödinger. The Glide module (extra precision [XP] mode) was selected for molecular docking due to its excellent performance through a self-docking analysis,25 and the 10 best poses of each ligand were minimized by a post docking program with the best pose saved for further analysis. 2.1.3. Molecular Dynamics Simulations. The protein complexes after molecular docking were used for MD simulation. The MD simulations were carried out using GROMACS26 with AMBER03 force field27 and TIP3P water model.28 The proteins were simulated in a water filled dodecahedron box with the distance at least 12 Å between the complex and the box edges. The charges of system were neutralized by adding counterions, either Na+ or Cl− by the genion tool. Then, the system was relaxed through an energy minization process with steepest descent algorithm first and then with conjugate gradient algorithm. Afterward, an NVT simulation was performed with temperature going from 100 to 300 K and followed by an NPT simulation to equilibrate the pressure. Finally, a production MD was performed for 20 ns at 300 K. Bond lengths were constrained using LINCS algorithm.29 The last 5 ns of each production simulation were extracted at every 10 ps interval for calculating the molecular mechanics/Poisson−Boltzmann surface area binding energy (MM-PBSA) using the AMBER12 package.30 Average

Figure 2. Acquired resistance is induced by vemurafenib through paths A and B.

shown in Path B.12 These dimers induce MEK/ERK phosphorylation and increased MAPK pathway output leading to acquired resistance. This would not happen with Pan-Raf inhibitors of similar activities against all subtypes of Raf kinases.14 It is also demonstrated that the feedback activation in BRafWT or CRaf bearing cancers can be significantly suppressed by DFG-out Pan-Raf inhibitors.15 Almost all major human cancers seem to harbor not a single but several concomitant dysregulations of signaling pathways.16 Crosstalk and compensatory mechanism exists in different pathways.17,18 RTKs, upstream kinases of MAPK and PI3K/ Akt/mTOR pathway, mediate resistance in melanoma suggesting potential upfront therapeutic strategies to prevent resistance (Path A in Figure 2).19 A compensatory mechanism of acquired resistance primarily involves Ras activation in response to upstream RTK signals such as EPHA2,17,18 FGFR,20 and PDGFRb.21 Drugs, which inhibit the pathway of upstream and downstream multiple targets, can achieve collaborative treatment to overcome the resistance.22 There is rarely a report on the treatment of acquired resistant melanoma cancers or intrinsic resistant colorectal cancers by using a Pan-Raf/RTK MTKI (multitargeted kinase inhibitor). Herein, we would like to describe the discovery of a series of inhibitors based on a cyclopropyl formamide fragment as novel MTKIs. These compounds potently inhibit the kinase activities of Pan-Raf and RTKs with low IC50 values. Furthermore, the compounds also display potent inhibition on the proliferation of a panel of vemurafenib-resistant cancer cells harboring overexpressed BRafV600E, representing new leads for further B


Article

Journal of Chemical Information and Modeling Scheme 1. Synthetic Route of I-01 to I-17a

a Reagents and conditions: (a) diethyl malonate, NaH, THF, 0 °C, 0.5 h, then reflux, 2.5 h, 86.2%; (b) EtONa, EtOH, reflux, 2.5 h, 92.4%; (c) 1,2dibromoethane, NaOH, DMF, rt, 5 h, 90.2%; iodomethane, NaOH, DMF, rt, 5 h, 94.3%; 1,4-dibromobutane, NaOH, DMF, rt, 5 h, 90.2%; (d) A1− A11, Al(CH3)3, toluene, nitrogen atmosphere, 80 °C, 5 h, 70.2−89.2%; (e) 1 N HCl, CH3OH, rt, 2 h, 72.4−85.6%.

The value of rHB = 3.5 Å corresponds to the first minimum of the radial distribution function (RDF) of simple point charge (SPC) water. 2.2. Synthesis of Compounds. The preparation of the target purine derivatives I-01−I-17 are shown in Scheme 1. The reaction of commercially available 6-chloro-9-(tetrahydro2H-pyran-2-yl)-9H-purine with diethyl malonate in the presence of sodium hydride gave diethyl 2-(9-(tetrahydro-2Hpyran-2-yl)-9H-purin-6-yl) malonate I-a in 86.2%. The prepared I-a was allowed to react with sodium ethoxide to afford the desired I-b in 92.4%. Subsequent alkylation of I-b with 1,2-dibromoethane, iodomethane or 1,4-dibromobutane in the presence of sodium hydroxide afforded the corresponding alkylated derivatives I-c-1 to I-c-3 in 90.2%, 93.4%, or 90.2%, respectively. Amide-forming reactions of I-c-1 to I-c-3 with the corresponding anilines A1−A11 were carried out in the

of the energy for the 1000 snapshots with each one for 50 ps was saved for further analysis.31 The radius of gyration (Rg), root-mean squared deviation (RMSD), and the number of hydrogen bond formed was analyzed by g_gyrate, r_rms, and g_hond tool, respectively, in the GROMACS package.32 In addition, the program gmx hbond analyzes the hydrogen bonds (H-bonds) between all possible donors D and acceptors A. To determine if an H-bond exists, a geometrical criterion (r ≤ rHB = 3.5 Å, α ≤ rHB = 30 °C) is used, see below:

C


Article

Journal of Chemical Information and Modeling Scheme 2. Synthesis of Intermediates A1−A8a

a Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, rt, 2 h; (b) 4-chloro-3 (trifluoromethyl) aniline, 4-methyl-3-nitroaniline or 3-nitroaniline, Et3N, CH2Cl2, rt, 5 h; (c) CDI, CH2Cl2, rt, 34 h; (d) Fe, NH4Cl, 75% EtOH, reflux, 3 h.

Scheme 3. Synthesis of Intermediates A9−A11a

Reagents and conditions: (a) morphine, DMSO, 100 °C, 5 h, 94.2%; (b) NBS, DCE, reflux, 5 h, 86.4%; (c) morphine, NEt3, THF, reflux, 2 h; (d) 3-morpholinopropan-1-ol, NaH, DMF, ice bath, 0.5 h, then 100 °C, 5 h; (e) Fe, NH4Cl, 75% EtOH, reflux, 2 h; (f) 4-methyl-3-nitrobenzoyl chloride, Et3N, CH2Cl2, r.t., 5 h; (g) Fe, NH4Cl, 75% EtOH, reflux, 2 h.

a

fonyl chloride was converted to the desired nitroaniline in the presence of 3-nitroaniline. Subsequent treatment of the resulting nitroaniline with Fe/NH4Cl gave the corresponding anilines A1−A7 in 68.6−80.4%. Urea A8 was generated from condensation reaction using CDI (N,N-carbonyldiimidazole), while A1−A7 were obtained by condensation of benzoyl chloride with their corresponding anilines. The synthetic route of anilines A9−A11 was outlined in Scheme 3. The reaction of 1-fluoro-4-nitro-2-(trifluoromethyl) benzene with morphine through SNAr substitution reaction provided 1 in 94.2%. Hydrogenation of the nitro group of 1 gave the corresponding aniline. Subsequent treatment of the resulting aniline with intermediate 4-methyl-3-nitrobenzoyl chloride provided nitroaniline, which hydrogenated to afford

presence of trimethylaluminum (2 M solution of toluene) under the condition of nitrogen atmosphere to give I-d-01 to Id-17 in 70.2−89.2%. Finally, the THP (pyran) group was deprotected by using 1 N HCl in methanol to provide compounds I-01 to I-17 in 72.4−85.6%. A total of 11 important intermediates of amide analogs A1− A11 were synthesized according to Schemes 2 and 3 via a two/ five-step process. Intermediates of amide analogs A1−A8 were prepared by the method shown in Scheme 2. Substituted benzoyl chloride, prepared in the presence of benzoic acid with various R1 groups (R1 = CH3, F, Cl, H) and oxalyl chloride, was allowed to react with 4-chloro-3-(trifluoromethyl) aniline or 4-methyl-3-nitroaniline to afford the corresponding nitroaniline. BenzenesulD


Article

Journal of Chemical Information and Modeling Scheme 4. Synthetic Route of I-18a

a

Reagents and conditions: (a) diethyl carbonate, LDA, THF, rt, 12 h, 70.1%; (b) 1,2-dibromoethane, NaOH, DMF, rt, 1 h, 65.0%; (c) Zn(CN)2, Pd(PPh3)4, DMF, N2, 90 °C, 2 h, 88.0%; (d) NH3·H2O, H2O2, DMF, 3 h, rt, 3 h, 85.0%; (e) iodomethane, NaH, DMF, rt, 0.5 h, 93.0%; (f) A1, Al(CH3)3, toluene, 80 °C, 5 h, 60.0%.

10 uCi/uL) (P-ERK in Elmer, NEG302H001 MC) was added to initiate the reaction, the reactions were carried out at 25 °C for 120 min. The kinase activities were detected by filterbinding method. IC50 values and curve fits were obtained by Prism (GraphPad Software). 2.4. Antiproliferative Assay against Different Cell Lines. Cell planking: Vi-Cell XR cell counter was used to count living cells, which collected in exponential phases. After diluting to 3 × 103−1.5 × 104 cells/mL with the complete medium, 90 μL of cells suspension was added to each well of 96-well culture plates and cultured in DMEM (Dulbecco minimum essential medium) or McCoy’s 5a/10% fetal bovine serum (CrownBio Corporation) for 24 h, with 5% CO2 water saturated atmosphere at 37 °C. Compound dispensation: Each selected compounds dissolved in DMSO as the storage solution (10 mM). Then with medium diluted to 10 times solution respectively, each 2 holes (inhibition ratio) or 3 holes (IC50 value). The final drug concentration for 10 μM (inhibition ratio) or initial drug concentration for 10 μM. The compound dispensation, which volume was 10 μL, was further incubated at 37 °C for another 72 h in a humidified atmosphere with 5% CO2. Plate detection: 50 μL CTG solution, melt in advance and balance to room temperature, was added to each hole. According to the operating instructions of CTG, oscillators with microporous plate blend 2 min. At room temperature for 10 min, Envision2104 was used to measure the luminescence signal values. Data processing: inhibition ratio = 1 − Vsample/Vvehicle control × 100%. Vsample for drug treatment group, Vvehicle control for solvent control group. Using GraphPad Prism 5.0 software and nonlinear regression model S type dose draw survival rate curve and calculate the IC50 value. 2.5. P-ERK Cellular Assay in A375 Cells and SK-MEL-2 Cells. 2.5.1. Recovery, Culture, and Passage of Cells. A375 and SK-MEL-2 human melanoma cell line were both obtained from American Type Culture Collection (Manassas, VA). Removed melanoma cells cryopreserved tube from liquid nitrogen tank, shaking in 37 °C water bath to make it melt quickly. The centrifugal tube was added cell suspension in superclean bench, 5 mL EMEM (Eagle’s minimal essential medium) was added. Centrifugalized the mixture 5 min with

the desired intermediate A9 in 85.4%. The desired A10 in 83.8% utilized 1-methyl-4-nitro-2-(trifluoromethyl) benzene as starting material. With NBS (N-bromosuccinimide), it was converted to 2 in 86.4%. 3 was prepared by 2 in the presence of NEt3 as alkali, THF as solvent. The last three steps of A10 were same as previous reaction (e, f and g) adopted for A9. The reaction of commercially available 1-fluoro-4-nitro-2-(trifluoromethyl) benzene with 3-morpholinopropan-1-ol in the presence of sodium hydride gave 4. The last three steps were same as previous reaction (e, f, and g) adopted for A9. Then, the desired intermediate A11 was afforded in 89.4%. The target pyridine derivative I-18 was prepared by the method as shown in Scheme 4. The reaction of commercially available 2-bromo-4-methylpyridine with diethyl carbonate in the presence of lithium diisopropylamide gave ethyl 2-(2bromopyridin-4-yl) acetate 5 in 70.1%. Subsequent alkylation of 5 with 1, 2-dibromoethane in the presence of sodium hydroxide afforded the alkylated derivative 6 in 65.0%. The prepared 6 was allowed to react with dicyanozinc in the presence of Pd(PPh3)4 under the condition of nitrogen atmosphere to afford the desired 7 in 88.0%. A solution of 7 and ammonium hydroxide was added hydrogen peroxide to achieve 8 in 85.0%. Subsequent methylation of 8 with iodomethane in the presence of sodium hydride afforded the methylated derivative 9 in 93.0%. Amide-forming reactions of 9 with the corresponding aniline A1 were carried out in the presence of Al(CH3)3 (2 M solution of toluene) under the condition of nitrogen atmosphere to give I-18 in 60.0%. 2.3. BRafV600E Inhibitory Assay. Activity of full length BRafV600E was determined using Hot-SpotSM kinase assay which was performed by Reaction Biology Corp. (Malvern PA). A 5 nM portion of human GST-tagged BRafV600E protein (AA416− 766) (Invitrogen, Cat no. PV3894) was mixed with 20 μM of the substrate His 6-Tagged Full-length Human MEK1 (K97R) (Reaction Biology Corp.) in reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) at room temperature, the compounds dissolved in 100% DMSO at indicated doses (starting at 30 μM with 3-fold dilution) was delivered into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 min at room temperature. After 10 uM 33P-γ-ATP (specific activity E


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Figure 3. Binding pockets of BRafV600E and the alignment of several potent BRafV600E inhibitors with sorafenib. For A−C, the purple colored compound is Sorafenib, the greened colored compounds are the cognate ligands for PDB 3IDP, the cognate ligands for PDB 4XV2, and compound Y-1, accordingly. The yellow dots are the sitemap results and the fragments in different colored lines are binding hot spots detected by FTMap. For D−F, the green colored compounds are always compound Y-1, the orange compounds are the lead fragment, compound I-01 and compound I-02, respectively.

Figure 4. Compounds based on cyclopropyl formamide fragment and four dimensional pharmacophore map of BRafV600E.

F


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Figure 5. Kinome phylogenetic tree of I-01. The left panel shows the inhibition rate of I-01 against all the targets that belong to the Kinome phylogenetic tree. The atypical protein kinases include ABC1, Alpha, Brd, PDHK, PIKK, RIO, and TIF1 from the top to the bottom accordingly. The right panel demonstrates the 11 kinases that I-01 obtained the most potent activity (IC50 values).

volume 5× SDS-PAGE loading buffer, a boiling water bath is created for 10 min to denaturature protein. The specific signals of bands of interest were quantified by Gel-Pro Analyzer. 2.5.4. Western Blot Detection. Protein samples (30 μg) in 12% SDS-PAGE (polyacrylamide gel electrophoresis) were used for electrophoresis. The protein was transfered from a running gel to a PVDF (poly vinylidene fluoride) membrane with the wet transfer method, and steady flow of 200 mA, with a 1 kDa/min transfer velocity, according to the interest protein molecular weight to determine the transfer membrane time using a 10% nonfat milk blocked PVDF membrane for 1.5 h in 37 °C. Using 5% BSA diluted primary antiphospho-ERK1/2 antibody, 4 °C incubation was conducted overnight. The membrane was washed with 1× TBST three times; each time took 10 min. Using 5% nonfat milk diluted secondary antiphospho-ERK1/2 antibody with HRP tag, room temperature incubation was conducted for 1 h. The membrane was washed with 1× TBST three times; each time took 10 min. Droping an appropriate amount of ECL luminous on a membrane, a Tanon5200 automatic chemiluminescence image analysis system was used.

1000 rpm, discarded supernatant, joined 2 mL EMEM medium supplemented with 10% serum to suspense cells, vaccinating it in culture bottle. Put culture bottle in incubator with 37 °C, saturated humidity, 5% CO2. The next day, discarded the original medium, added 6 mL fresh medium to remove dead cells. Passage when cell numbers around 90%. Discarded supernatant, washed twice with PBS (phosphate buffered saline), added 2 mL 0.25% pancreatic enzyme to digest 2−3 min. When cells becoming round under microscope, discarded pancreatic enzyme, added 2 mL EMEM medium to terminate digestion. Making the adherent cells blow down to a single suspension cells with a pipet gently. According to the speed of cell growth, culture the cell from one to three or four. 2.5.2. Plating Cells and Dosing. When the melanoma cells A375 and SK-MEL-2 in the logarithmic phase, vaccinating 3−5 × 105 cells in 96 cell plate with EMEM medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), culturing cells in incubator with 37 °C, saturated humidity, 5% CO2. When cell confluence reached 80−90%, discarding medium, diluting drugs with culture medium into 0.4, 0.2, 0.1, 0.05, 0.025, dosing drugs in 96 cell plate, using DMSO as a negative control. 2.5.3. Protein Sample Preparation. The cells were treated with compounds or DMSO for 24 h. After treatment, cells were digested with 500 μL 0.25% trypsin, then 500 μL medium were added to terminate digestion. Cells were blown evenly, centrifugalized the mixture 5 min with 3000 rpm, discarded supernatant, put centrifugal tube on the ice, added suitable amount of protein lysis solution RIPA (Radio-immunoprecipitation assay), protease inhibitor PMSF (Phenylmethanesulfonyl fluoride) (1 mL RIPA/10 μL PMSF), and phosphatase inhibitors (1 mL RIPA/10 μL phosphatase inhibitors), cells blown evenly, 4 °C for 30 min, make cells lysate, every 5 min vortex once. Centrifugalizing the lysate 15 min with 12 000 rpm in 4 °C, gathering supernatant, using the BCA protein assay kit to determine the total protein concentration. Adding 1/4

3. RESULTS AND DISCUSSION 3.1. Inhibitor Design and Molecular Modeling. In a previous report, we described the synthesis and potency of 3((3-(9H-purin-6-yl)pyridine-2-yl)amino)-N-(4-chloro-3(trifluoromethyl)phenyl)-4-methylbenzamide Y-1 as a BRafV600E inhibitor.33 Y-1 shows potent BRafV600E inhibitory activity (IC50 = 3.15 nM) and potential antiproliferative activity against cell line A375 (IC50 = 8.79 μM). To improve the originality of Y-1,34 we performed structure optimization. Understanding the structure and function of protein active sites is a cornerstone of drug design. By aligning sorafenib (BRafWT IC50 = 22 nM and BRafV600E IC50 = 38 nM)35 with three potent BRafV600E inhibitors which include the cognate ligand of PDB G


Article

Journal of Chemical Information and Modeling Table 1. SAR Exploration of Compounds against BRafV600E Activity

a

Under 0.5 μM concentration of compounds. bNot determination.

3IDP (BRafV600E IC50 = 1.6 nM),34 cognate ligand of PDB 4XV2 (i.e., dabrafenib, BRafV600E IC50 = 5.4 nM),36 and the lead compound Y-1 (BRafV600E IC50 = 3.15 nM),33 we found that all of them occupied an extra hydrophobic pocket (Figure 3A−C) that is confirmed by two binding sites detection methods FTmap24 and Sitemap37 (binding site detection methods). Then, three fragment generation methods38 were employed to decompose several databases,39 and the resulted fragments were

docked to the active binding sites. A N-phenyl-1-(pyrimidin-4yl) cyclopropane-1-carboxamide fragment (the red colored part of I-01 as shown in Figure 4) was found to be in great alignment with the N-phenyl-3-(9H-purin-6-yl)pyridin-2-amine part of compound Y-1 (Figure 3D). Thus, by using a scaffold hopping method, we designed I-01 which adopted a similar binding conformation as sorafenib with the original essential hydrogen bonds kept (two hydrogen bond formed with Cys532 H


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Moreover, I-18 have better physicochemical property (tPSA = 99.56 Å2, LogP = 4.56) than I-01 (tPSA = 107.31 Å2, LogP = 4.34). However, the enzymatic potency was lost as shown in Figure 4. The possible reasons were presented in binding mechanism analysis. Based on the above information, a total of 18 novel compounds were designed and synthesized. 3.2. Chemistry. As summarized in Schemes 1 and 4, 18 compounds were synthesized in total. The synthetic routes are illustrated in Schemes 1−4. The chemical structures of these compounds were confirmed by 1H NMR, 13C NMR, HRMS spectra, and the results are presented in the Supporting Information. 3.3. Investigation of the Enzymatic Activity of Compounds. To further determine the efficacy, the broad kinase spectrum of I-01 was evaluated at 0.5 μM concentration against an RBC (Reaction Biology Corporation) screen panel containing 349 therapeutically important kinases as shown in Figure 5. The kinome phylogenetic tree was generated using KinomeReader.41 The top hits were subjected to follow-up IC50 generation. I-01 was found to inhibit 11 kinases (Raf and RTKs) with IC50 values range from 0.47 to 40.91 nM, including ARaf IC50 = 0.97 nM, BRaf IC50 = 0.99 nM, CRaf IC50 = 0.47 nM, DDR1 IC50 = 2.96 nM, DDR2 IC50 = 2.57 nM, EPHA2 IC50 = 13.50 nM, FGFR2 IC50 = 39.21 nM, FMS IC50 = 3.94 nM, LCK IC50 = 40.91 nM, PDGFRb IC50 = 25.34 nM, and Ret IC50 = 5.24 nM, showing I-01 to be a potent multitargeted kinase inhibitor (right panel of Figure 5). I-01, highly potent against the Pan-Raf and other oncogenic kinase members of the RTKs group with low IC50 values, exhibits excellent target specificity in a selectivity profiling investigation against 349 kinases (Figures 2 and 5). Resistance to BRafV600E and RTKs which involved bypass or restoration of persistent MAPK activation might be overcome by I-01.42 3.3.1. Compounds Screened for Enzymatic Activity against BRafV600E. Compounds were evaluated the potency against BRafV600E using vemurafenib and sorafenib as positive control compounds. Disruption of hydrogen bond interactions by replacement of the L of I-01 with “reverse” amide I-03 or urea I-04 led to inapparent change at enzymatic activity. Structurally, sulfonyl group has similar properties in molecular size and charge distribution with carbonyl, so sulfonyl group

Table 2. Pan-Raf Activity of the Chosen Compounds IC50 (nM) compd

BRafV600E

ARaf

BRafWT

CRaf

I-01 I-15 I-16 I-17 vemurafenib sorafenib

1.91 3.41 3.49 5.31 31.64 38.12

1.12 4.56 8.86 13.3 2.14 7.13

2.41 5.11 5.78 16.1 6.92 22.65

0.49 1.91 1.65 3.02 135.81 6.28

in the hinge region and hydrogen bonds formed with Asp594 and Glu501 in the DFG-out pocket) and a better occupation of the other important binding pockets (Figure 3E). However, by moving the aromatic amide functional group from metaposition of the central benzene ring (I-01) to the para-position (I-02) led to the loss of enzymatic activity which can be initially explained by its almost opposite binding conformation of I-02 (Figure 3F and Figure 4) compared with I-01 (Figure 3E). Before optimizing the lead I-01, we determined the structural features essential for enzymatic activity and identified possible modification sites. The binding mode of I-01 and BRafV600E kinase revealed that I-01 binds to the BRafV600E ATP binding pocket with the purine core forming two essential hydrogen bonds with the hinge region Cys532 (Figures 3 and 4). In addition to these hydrogen bonds, the amide group forms two hydrogen bonds with Asp594 and Glu501 in the DFG-out region. Structure-based optimization of I-01 focused on potency at enzymatic as well as cellular levels. L as the linkage using different groups was attempted. R1 group with different sizes occupied a small hydrophobic pocket typically closed the gatekeeper region. Fragment Q was investigated on rigid and steric factors. R2 substituent in the terminal phenyl ring was directed toward the solvent accessible region. R3 substituent at terminal phenyl ring was exposed to another hydrophobic back pocket, which was formed by a rearrangement of the activation loop and subsequent movement of a phenylalanine side chain of DFG loop.40 4-Substitute-N-methylpicolinamide, the hinge region structure of sorafenib, was introduced to replace purine to afford I-18. I-18 maintained the binding mode of I-01.

Figure 6. RMSD values fluctuation of the compounds (Y-1, I-01, sorafenib, vemurafenib) during the 20 ns MD simulation. I


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Table 3. Time-Average and Standard Deviation of H-Bond, RMSD, Rg of protein, and Predicted Delta G for the Last 5 ns MD average RMSD (Å) compd Y-1 I-01 I-02 I-05 I-09 I-12 I-15 I-18 sorafenib vemurafenib

num of Hbonds 4 4 2 3 4 1 3 1 4 4

± ± ± ± ± ± ± ± ± ±

0 1 1 1 1 1 1 1 1 1

Rg protein (Å) 19.2 19.2 19.2 19.4 19.3 19.3 19.3 19.2 19.3 19.0

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1.3 0.2

complex 11.2 11.2 11.3 10.4 2.6 11.3 11.6 11.3 3.3 2.9

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.2 0.1 0.3 0.1 0.1 0.1 0.5 0.5

protein 2.4 2.5 2.4 2.5 2.6 2.1 2.6 2.5 3.3 2.9

± ± ± ± ± ± ± ± ± ±

0.3 0.3 0.5 0.3 0.3 0.2 0.2 0.4 0.5 0.5

ligand 0.8 0.8 0.9 1.0 1.0 1.4 1.1 0.9 0.7 0.6

± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.2 0.1

average delta G (kcal/mol) −53.2 −51.9 −31.9 −37.2 −52.1 −32.3 −47.3 −31.9 −50.8 −53.1

± ± ± ± ± ± ± ± ± ±

4.0 4.7 4.6 4.06 3.8 7.2 5.1 4.1 3.4 3.0

RafV600E IC50 (nM) 3.15 1.91 NA NA 3.89 NA 3.41 NA 38 31

hydrophobic pocket hadn’t enough space to accommodate cyclopentyl. In order to get a better occupation of the solvent accessible region leaning the terminal phenyl ring, the solubilizing morpholine functionality with increasing length of the linker carbon chain was introduced which gave I-15 to I-17. This modification could sustain the BRafV600E inhibitory activity compared to I-01, which is consistent with the hypothesis that the R2 group could occupy a solvent accessible region in BRafV600E. Most of compounds show IC50 values at nanomolar ranges, superior to positive control compounds vemurafenib and sorafenib tosylate. I-01, using amide group as L and cyclopropyl as Q group, shows optimal enzymatic activity. 3.3.2. Pan-Raf Activity of the Chosen Compounds. As mentioned earlier, Pan-Raf inhibitors with similar activity against all subtypes of Raf kinases, might have potency to overcome the resistance of vemurafenib. The chosen compounds I-01 and I-15 to I-17 were subjected to followup Pan-Raf IC50 generation. The results are shown in Table 2. I-15 to I-17 sustained the enzymatic activity compared to I-01. 3.3.3. Binding Mechanism Analysis. Due to the different enzymatic activity of compounds with only a slight difference in structure, molecular dynamics (MD) was conducted to simulate the binding process and calculate the binding energy of these compounds. A total of 10 compounds including sorafenib, vemurafenib, Y-1, I-01, I-02, I-05, I-09, I-12, I-15, and I-18 were investigated. RMSD (root mean standard deviation) variation of protein atoms along time is one of the standard ways to measure the stability of the protein. From Figure 6 and Figure S1 (see the Supporting Information), the blue lines represent the fluctuation of protein while the orange lines demonstrate the change of the small compounds. Both the protein and small compounds reached certain equilibrium during the 20 ns simulation with the protein obtained a higher RMSD than its corresponding ligands. Combined with the time-averaged value of RMSD values with standard deviation are listed in Table 3. The average RMSD for protein were about 2.6 Å (2.1 Å for compound I-12 ∼3.3 Å for sorafenib) with a standard deviation of about 0.4 Å. Similarly, the ligands obtained a smaller RMSD of about 0.9 Å (0.7 Å for vemurafenib ∼1.4 Å for I-12) with a standard deviation of 0.2 Å. This means during the MD simulation process, the small compounds are more stable than the protein. Nonetheless, the radius of gyration (Rg) of the protein were quite steady (in the range of 19.0 to 19.4 from Table 3), indicating the compactness of the protein which is stably folded.

Figure 7. Predicted Delta G and the average number of hydrogen bonds for several compounds.

can be introduced into the drug molecules as the bioisostere of carbonyl to remain or improve activity. Replacement of the amide of I-01 with sulfonyl led to I-05 loss of enzymatic activity (Table 1), which confirmed the importance of amide group for enzymatic activity. Subsequently, the influence of R1 substituent at central phenyl ring was investigated. Halogen atoms, including fluorine and chloride, were employed to afford compounds I-06 and I07. Hydrogen was explored to afford compound I-08. All modifications sustained the BRafV600E inhibitory activity compared to I-01. This result is consistent with the hypothesis that the R1 group would occupy a small hydrophobic pocket typically closed the gatekeeper region of BRafV600E. Q group of different rigidity was investigated. I-09 to I-11 showed comparable BRafV600E inhibitory activity with I-01. This result suggested that the flexible group dimethyl could sustain the BRafV600E inhibition compared to rigid cyclopropyl group. Subsequently, Q of different steric sizes was explored. Expanding the cyclopropyl of I-01 to cyclopentyl in I-12 to I-14 resulted in loss of potency due to steric clash with protein. This result indicates that the Q group occupying a small J


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Figure 8. Binding free energy (Delta G, kcal/mol) fluctuation of the compounds (Y-1, I-01, sorafenib, vemurafenib) during the last 5 ns of production MD simulation.

We focused on the number of hydrogen bonds and binding free energy calculation to analyze the reason why some compounds (e.g., I-02, I-05, I-12, and I-18) do not exhibit any enzymatic activity although they are only slightly different with other compounds with very high enzymatic activity. From Table 3 and Figure 7a, we could observe that most of the compounds achieved more than three hydrogen bonds with BRafV600E protein with three of them (i.e., sorafenib, vemurafenib, and I-01) have four hydrogen bonds. However, the other three compounds (i.e., I-02, I-12, and I-18) without BRafV600E enzymatic activity only obtain an average of two, one, and one hydrogen bond(s), respectively. Although I-05 also does not show any BRafV600E enzymatic activity, it got an average of three hydrogen bonds. The average binding free energy from the last 5 ns MD production process is shown in Table 3 and Figure 7b, and the fluctuation of binding free energy along time is depicted in Figure 8 and Figure S2 (see the Supporting Information). The binding free energy values for the compounds with high enzymatic activity are relatively low (less than −50.00 kcal/mol except I-15 of −47.34 kcal/mol); however, those for the compounds without enzymatic activity are higher than −40.00 kcal/mol (i.e., −31.90, −37.19, −32.34, and −31.89 kcal/mol for I-02, I-12, I-15, and I-18, respectively). In total, the difference of the number of hydrogen bonds and binding free energy can explain why those

Table 4. Compounds Screened for Cellular Potency against Different Melanoma Cells compd I-01 I-03 I-04 I-06 I-07 I-08 a b

A375 activity %a IC50 (μM) 58.4 98.7 62.3 100.6 102.6 85.1

13.2 80.5 25.8 NDb ND 58.7

compd I-15 I-16 I-17 vemurafenib sorafenib

A375 activity % IC50 (μM) 14.7 14.6 17.2 11.1 29.6

5.4 4.2 6.4 0.7 13.6

SK-MEL-2 IC50 (μM) 3.4 0.9 1.2 5.6 11.4

Cellular activity of compounds under the concentration of 10 μM. Not determination.

Figure 9. Cellular activity of compounds under the concentration of 10 μM.

Figure 10. (A) ERK kinase inhibition in A375. (B) ERK kinase inhibition in SK-MEL-2. K


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4. CONCLUSION Depending on FTmap and Sitemap, we found that all of DFGout conformation Raf inhibitors occupied an extra hydrophobic pocket. Through fragment-based approach, we determined cyclopropyl formamide fragment could occupy this pocket properly like other positive control compounds. The obtained compound I-01 with DFG-out conformation inhibits all subtypes of Raf proteins. Based on the structure homology of Raf and RTKs, we evaluated the broad kinase spectrum of I-01. The results displayed that I-01 also inhibits eight oncogenic kinase members of the RTKs groups in the low nanomolar range. Since I-01 can avoid the repair and compensatory mechanism leading to resistance, we develop derivatives I-01 to I-18 with activity against multiple targets of Pan-Raf and RTKs. I-16 inhibits all subtypes of Raf proteins with IC50 value of 3.49 nM (BRafV600E), 8.86 nM (ARaf), 5.78 nM (BRafWT), and 1.65 nM (CRaf), respectively, it was more potent than positive controls (vemurafenib and sorafenib). In particular, I-16 exhibits the lower IC50 values 4.25 μM, 0.93 μM against A375 and SK-MEL-2, which also displays better antiproliferative activity against four cell lines HepG2, SW579, MV4-11, and COLO-205 compared to positive control (these cell lines are extraordinarily expressed BRAFV600E or BRafWT). Then, further mechanism investigation in terms of the ERK inhibition in human melanoma A375 and SK-MEL-2 cell lines by Western blot was investigated. The results reveal that our compounds inhibit the proliferation of melanoma A375 cells through ERK pathway, without paradoxical activation of ERK in BRafWT type melanoma SK-MEL-2 cells. Our results support the hypothesis that the Pan-Raf and RTKs inhibition might be a tractable strategy to overcome the intrinsic and acquired resistance of melanoma caused by the current BRafV600E inhibitor therapy. Moreover, with this multitargeted profile, the compounds could be used as a therapeutic aid for patients developing resistance while being treated with currently marketed kinase inhibitors.

compounds although has similar binding modes with the actives but do not show any enzymatic activity to some extent. 3.4. Antiproliferative Activity against Various Cell Lines. As described above, BRafV600E and BRafWT are important targets in developing small-molecular inhibitors for cancer therapies, especially melanoma, colon cancer, leukemia, hepatoma, and thyroid carcinoma.43 A375,44 SK-MEL-2,45 and COLO-20546 cell lines have extraordinary expression of BRafV600E. MV4-11,47 HepG2, and SW57948 cell lines have extraordinary expression of BRafWT. Although other substituted compounds display similar enzyme inhibitory performances to I-01, their cell activity is completely different. Replacement of the amide function of I-01 with either a “reverse” amide or urea linkage, which lead to decreased potency at cellular level (Table 4). Compared to I-01, I-03 and I-04 showed a 6/2−fold loss of A375 inhibition. This result shows that the amide as the L would be better suited to antiproliferative activity. Subsequently, the influence of R1 substituent on central phenyl ring was researched. I-08 shows loss of potency in a range similar to halogen-substituted compounds I-06 to I-07. I-01 with a methyl substituent displayed optimal A375 antiproliferative activity. It should be noted that I-15 to I-17 introduced different hydrophilic group R2 to the solvent accessible region successfully improved the cellular potency against various cell lines, which is probably due to enhanced water solubility and improved cellular permeability. The antiproliferative activities of I-15 to I-17 are not only with potency against A375 and SKMEL-2 cell lines as shown in Table 4 but also against HepG2, SW579, MV4-11, and COLO205 cell lines as shown in Figure 9. I-16 displays potent antiproliferative activities against A375 and SK-MEL-2 cell lines with IC50 values as 4.25 or 0.93 μM, respectively. The result proves that I-16 is superior than sorafenib against this two cell lines. Although it is not comparable with vemurafenib against A375, it is superior to vemurafenib against SK-MEL-2. As mentioned earlier, vemurafenib shows paradoxical activation of MAPK pathway in wildtype BRaf cells, I-16 might have potency to overcome the resistance of vemurafenib. I-16 also exhibits comparable antiproliferative activities against HepG2, SW579, MV4-11, and COLO205 cell lines to positive control compounds. Thus, I-16 may be a promising compound to be developed as a candidate for therapy. 3.5. Western Blot: ERK Kinase Inhibition in A375 and SK-MEL-2 Cells. We tested I-16 for its ability to inhibit P-ERK in cancer cells by Western blot analysis (Figure 10). I-16 effectively and dose-dependently inhibits P-ERK in the A375 cell line with BRafV600E at concentration as low as 400 nM, which is as potent as vemurafenib. In the SK-MEL-2 cell line with BRafwt, I-16 effectively and dose-dependently inhibits PERK at concentration as low as 400 nM. However, vemurafenib activates P-ERK with the increase of its concentration. A key finding from this class of compounds is that, unlike vemurafenib, they dramatically inhibit the proliferation of melanoma A375 cells through ERK pathway, without paradoxical activation of the MAPK in SK-MEL-2 cells. Our results support the hypothesis that the Pan-Raf and RTKs inhibition might be a tractable strategy to overcome the intrinsic resistance of colon cancer and the acquired resistance of melanoma caused by the current BRafV600E inhibitor therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00795. Figure S1. RMSD value fluctuation of the compounds during the 20 ns MD simulation. Figure S2. Distribution of predicted binding energy (Delta G, kcal/mol) for the 5 ns production MD. Figure S3. The IC50 curves for compound I-01. Figure S4. The IC50 curves for compound I-16 and the chemical synthesis and structure identification of compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-25-86185201. Fax: +86-25-86185182. E-mail: twf@ cpu.edu.cn (W.T.). *Tel.: +86-25-86185180. Fax: +86-25-86185179. E-mail: [email protected] (T.L.). ORCID

Weifang Tang: 0000-0002-5527-897X Author Contributions

∥ Y.Z. and L.W. contributed equally to this work and should be considered as cofirst authors.

Notes

The authors declare no competing financial interest. L


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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (No. 21572273/B020601). A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions and College Student Innovation Project for the R&D of Novel Drugs (No. J1030830). We also express our gratitude to Dr. Xiazhong Ren, Dr. Huajun Yang, Dr. Chunlan Dong (Crown Bioscience Corporation), and Dr. Jamie Planck (RBC, Pennsylvania, USA) for their helpful support in biological evaluation.

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