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In Silico Identification of a Novel Hinge-Binding Scaffold for Kinase Inhibitor Discovery Yanli Wang,†,§ Yuze Sun,‡,†,§ Ran Cao,†,§ Dan Liu,† Yuting Xie,† Li Li,† Xiangbing Qi,*,† and Niu Huang*,† †

National Institute of Biological Sciences, Beijing, No. 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing 100084, China

‡

S Supporting Information *

ABSTRACT: To explore novel kinase hinge-binding scaffolds, we carried out structure-based virtual screening against p38α MAPK as a model system. With the assistance of developed kinase-specific structural filters, we identify a novel lead compound that selectively inhibits a panel of kinases with threonine as the gatekeeper residue, including BTK and LCK. These kinases play important roles in lymphocyte activation, which encouraged us to design novel kinase inhibitors as drug candidates for ameliorating inflammatory diseases and cancers. Therefore, we chemically modified our substituted triazole-class lead compound to improve the binding affinity and selectivity via a “minimal decoration” strategy, which resulted in potent and selective kinase inhibitors against LCK (18 nM) and BTK (8 nM). Subsequent crystallographic experiments validated our design. These rationally designed compounds exhibit potent on-target inhibition against BTK in B cells or LCK in T cells, respectively. Our work demonstrates that structure-based virtual screening can be applied to facilitate the development of novel chemical entities in crowded chemical space in the field of kinase inhibitor discovery.

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INTRODUCTION As of June 2017, 30 drugs targeting kinase ATP-binding sites have been approved for clinical use.1,2 However, the discovery of novel and selective kinase inhibitors using high throughput screening (HTS) approaches has become increasingly challenging in an already crowded chemical landscape, since most commercially available chemical libraries have been screened exhaustively against various kinase targets.3,4 Moreover, almost all of the inhibitors that target kinase ATP-binding site contain structural scaffolds forming hydrogen bond (HB) interactions with the kinase hinge backbone, including adenosine-mimic purine analogues, pyrimidine, quinoline, thiazole/imidazole, and indolinone (Figure 1), which has further limited the available chemical space for novel drug discovery.5,6 Given the highly conserved structure of the ATP-binding site across the entire kinase superfamily, especially the hinge region, the lack of target heterogeneity makes it challenging to develop selective kinase inhibitors.7 Lack of selectivity clearly leads to side effects for many kinase inhibitors.8 Collectively, these factors constitute a severe bottleneck for kinase drug discovery. © 2017 American Chemical Society

It is well established that the kinase gatekeeper residue plays a centrally important role in determining the selectivity of kinase inhibitors.9 About 20% of human protein kinases have a small gatekeeper residue (e.g., glycine, alanine, cysteine, serine, or threonine) that directly affects the ability of drugs to access the hydrophobic back pocket. Increased accessibility typically improves inhibitor binding affinity and specificity. A subset of kinases have threonine as their gatekeeper residue which provides additional opportunity for designing kinase inhibitor to form a specific HB interaction. Among these, BTK and LCK are known to be important for the activation of lymphocytes.10−12 BTK is a member of the TEC subfamily that functions as a critical component in the B-cell antigen receptor (BCR) signaling pathway.10,12,13 LCK is a SRC subfamily protein with an essential function in proximal T cell antigen receptor (TCR) signal transduction.14 The p38α MAPK plays a vital role in inflammation by regulating the biosynthesis of Received: July 28, 2017 Published: September 25, 2017 8552

DOI: 10.1021/acs.jmedchem.7b01075 J. Med. Chem. 2017, 60, 8552−8564

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Figure 1. Representative hinge-binding scaffolds collected from literature.5 Hydrogen bonds forming between kinase hinge region and ligand are highlighted in red. The residues of the hinge region are annotated based on its position to the gatekeeper residue (gk).

Figure 2. (A) Workflow of hierarchical virtual screening campaign against p38α MAPK. (B) Predicted binding pose of lead compound in p38α MAPK ATP-site. Potential hydrogen bonds are represented with orange lines. (C) Dose-dependent inhibition of p38α MAPK kinase activity by the lead compound.

tumor necrosis factor (TNF) α, interferon (IFN) γ, and other cytokines via transcriptional and post-transcriptional mechanisms in response to extracellular stimuli.15−17 It has long been anticipated that interventions targeting the activity of BTK, LCK, or p38α MAPK can have strong therapeutic effects.11,18,19 The pharmaceutical industry has undertaken great efforts to develop selective inhibitors against these kinases, and some inhibitors are currently showing promise in both preclinical models and clinical trials.19−22 The BTK inhibitor ibrutinib, 1[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one (PCI-32765),23

was approved by the U.S. FDA for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenstrom’s macroglobulinemia (WM).12,24,25 Ibrutinib also shows significant activities across a variety of inflammatory diseases.23,26 A second-generation BTK inhibitor, acalabrutinib, was shown to be more potent and selective than ibrutinib, and a few other BTK inhibitors have also entered clinical trials (Supporting Information Figure S1).27−29 The oral administration of selective LCK inhibitors, e.g., N-(4-(1-((1s,4s)-4-(4acetylpiperidin-1-yl)cyclohexyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-car8553

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boxamide (A-770041),30 can inhibit IL-2 production and exhibit anti-inflammatory activity in mouse models.18,19 Here, we performed structure-based hierarchical virtual screening to identify novel hinge-binding scaffolds from a chemical library containing more than 100 000 diverse drug-like compounds. We selected p38α MAPK as our starting model system as in our previous computational study31 and discovered a novel lead compound with high ligand efficiency (LE).32,33 Starting with this lead, we then carried out structure-based optimization with a “minimal decoration” strategy against both BTK and LCK, which is to optimize the lead compound by loading the target-specific functional groups onto the privileged structural scaffold based on key molecular interactions. This finally led to the discovery of novel and highly potent inhibitors of BTK and LCK with impressive selectivity. These candidates were further assessed by measuring their on-target activities in cells. The identified compounds could bring practical benefits in developing novel kinase inhibitors for the treatment of inflammatory disease as well as cancers.

structural model (PDB code 1OUY) and formed a stable HB interaction with 1 using its hydroxyl group (Supporting Information Figure S3). 1 has a considerably structural novelty as compared with the currently available small-molecule kinase drugs (Supporting Information Figure S4, Table S1). In addition, none of the structurally similar compounds are reported to have kinase activities based on similarity search in existing patents and literatures in SciFinder database (Supporting Information Table S2), which further demonstrated the novelty of our identified scaffold. 1 was passed through one of the publicly available pan assay interference compounds (PAINS) filters (http://zinc15.docking.org/patterns/home/ ),41 and the promiscuous aggregate-based inhibition was ruled out by the addition of detergent in biochemical assay condition. Therefore, 1 was chosen as our lead compound for further structural optimization. Crystal Structure Determination of p38α MAPK Bound with 1. To validate our binding pose predictions and to facilitate further structural modifications, we determined the crystal structure of p38α MAPK bound with 1 (PDB code 5XYX). The data collection and refinement statistics are summarized in Supporting Information Table S3. The binding pose of 1 in the crystal structure is very similar to our computational prediction (Figure 3A). Specifically, three HB interactions are formed between the ligand 1H-1,2,4-triazol-5amine group and the kinase hinge region, including the hydroxyl group of Thr106, the backbone carbonyl of His107, and the backbone amide of Met109 (Figure 3B). Notably, the predicted conformational change of side chain of Thr106 in p38α MAPK that resulted from the 1 binding was apparent in our crystal complex structure. Our structural study also reveals the unique binding pose of 1H-1,2,4-triazol-5-amine scaffold that is different from that of any of the currently available kinase inhibitors (Figure 3C and Figure 3D). This not only accounts for the high number of hydrogen bonds that 1 forms but also explains its gatekeeper targeting property. Surprisingly, the predicted HB interaction between furan oxygen atom of 1 and the amide group of Gly110 was not present in the crystal complex structure (Figure 3A). Given the previously reported “hinge glycine flip” phenomenon,42 we carefully checked the backbone conformation of Gly110 in our solved crystal complex structure and noted that the carbonyl group of Met109 rather than amide group of Gly110 orients toward the ligand. Thus, the crystal structure strongly suggests that 1 forms a weak hydrogen bond (C−H···O) with the backbone carbonyl group of Met109, with a distance of 2.88 Å between the carbonyl oxygen atom and the furan carbon atom. Although the flipped conformation of hinge glycine complicated the structural comparison between computational models and experimentally determined crystal structure, we found that the predicted ligand binding poses were progressively improved by the increased computational complexity, as exemplified by the gradually reduced rootmean-square deviation (RMSD) in comparison with crystal complex structure, from molecular docking (2.134 Å) to MMGB/SA (1.002 Å) and to MD-PB/SA (0.351 Å) (Supporting Information Figure S5). This finding suggests that using increasingly sophisticated and physically realistic methods progressively improves the accuracy of ligand binding mode prediction, which is consistent with our previous studies.37,38,43 We superimposed the crystal structures of kinases with threonine as gatekeeper residue (including BTK, LCK, FYN, LYN, and BMX) onto our determined crystal complex

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RESULTS AND DISCUSSION Hierarchical Virtual Screening. Many approaches have been proposed to identify novel inhibitors for protein kinase.34−36 Herein, we used our established pipeline to perform structure-based hierarchical virtual screening against the ATP-binding site of p38α MAPK (Figure 2A).31,37−40 We developed two kinase-specific structural filters to exclude hits that lacked the kinase-binding properties: (1) hydrogen bonding with hinge region backbone atoms, including those of the carbonyl group of His107 and the amide group of Met109; (2) favorable contacts with residues surrounding the hydrophobic back pocket, including Val38, Leu75, Ile84, and Leu104. In total, we docked 107 238 compounds, which generated 380 382 unique binding poses. After exclusion of undesired binding poses with structural filters, 201 285 docking poses were subjected to further minimization-based MM-GB/ SA refining and rescoring. This led to defined 21 893 molecules as qualified hits having favorable binding energies. After a second round of automatic filtering, the top 500 ranked molecules with the lowest energy scores among the 10 381 compounds surviving the structural filters were selected and reevaluated with MD-based MM/PBSA binding energy calculations. Ultimately, eight hits with favorable binding energies and reasonable binding poses were chosen for experimental test (Supporting Information Figure S2A). Four of these were active at the concentration of 50 μM in p38α MAPK activity inhibition assays (Supporting Information Figure S2B). Among the four active hits, N-(2-chloro-6-fluorobenzyl)-3(furan-2-yl)-1H-1,2,4-triazol-5-amine (1) caught our attention, owing to both the unique binding mode of 1H-1,2,4-triazol-5amino moiety and the high number of hydrogen bonds that it forms with the hinge region of p38α MAPK (Figure 2B). The IC50 value of 1 against p38α MAPK is 14.4 ± 1.2 μM (Figure 2C). 1 was predicted to form four hydrogen bonds with the hinge region: (1) the hydroxyl group of the gatekeeper (gk for short) residue Thr106; (2) the backbone carbonyl of His107 (gk + 1); (3) the backbone amide of Met109 (gk + 3); (4) the backbone amide of Gly110 (gk + 4). Most currently available kinase inhibitors form one or two hydrogen bonds with the hinge region, and a few form three, but we are unaware of any compound that forms four hydrogen bonds.6 Interestingly, molecular dynamics simulations showed that the side chain of Thr106 changed its conformation in the starting crystal 8554

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characteristics, 1 holds promise as a starting point for the development of more potent and selective kinase inhibitors. Here, we focused on two important targets, BTK and LCK, both of which have threonine as their gatekeeper residues. Unlike p38α MAPK, both LCK and BTK have an additional residue inserted in their hinge regions that adds to the total space at the opening of ATP-binding site and increases the tolerance of these proteins for inhibitors with relatively bulkier substituents (Figure 4A and Figure 4B). Additionally, the gk + 6 residue (Asp112) in p38α MAPK differs from the counterpart residues (gk + 7) in LCK and BTK, which are Ser323 and Cys481, respectively (Figure 4A and Figure 4B). Notably, Cys481BTK has been exploited for the development of covalent inhibitors against BTK, as exemplified by the clinical drug ibrutinib.44 By superimposing our crystal complex structure with the previously reported PDB structures of LCK (PDB code 3BYM) and BTK (PDB code 3T9T) in complex with other inhibitors, we found that (1) the benzimidazoletriazine based LCK inhibitor, N-phenyl-1-(4-((3,4,5-trimethoxyphenyl)amino)1,3,5-triazin-2-yl)-1H-benzo[d]imidazol-2-amine (AM0) extends outside the hinge region with its bulky 3,4,5-trimethoxyl substituent (Figure 4A), which has been thought to contribute to its potency (IC50 = 6 nM);45 (2) the BTK inhibitor (Z)-4(dimethylamino)-N-(7-fluoro-4-(o-tolylamino)imidazo[1,5-a]quinoxalin-8-yl)-N-methylbut-2-enamide (IAQ) forms a covalent bond with Cys442 (gk + 7) using the acrylamide “warhead”, which is known to be critical for its low nanomolar inhibition potency (IC50 = 1.93 nM) and its selectivity.46 In these complex structures, the aforementioned functional groups (methoxyl, acrylamide) of both the LCK and BTK inhibitors are located at the opening of ATP-binding site, which is the same position that is occupied by the furan moiety of 1 in our crystal complex structure of p38α MAPK. On this basis, we proposed a structurally modifying strategy that includes two stages (Figure 4C): (1) replacing the furan group of 1 with a more easily decorated phenyl group; (2) loading functional groups of interest (e.g., hydroxyl and amine and later methoxyl and acrylamide) on the specific different sites of the phenyl ring. Specifically, we designed 2 by replacing the furan with a phenyl group, which substantially reduced p38 MAPK inhibition activity (27.1 ± 7.4 μM) but left BTK and LCK inhibition activity largely unchanged. In silico modeling indicated that the meta site of phenyl ring orients toward Asp112p38α. We thus introduced HB-donor containing groups (hydroxyl or amine) at this position to promote the formation of additional HB interaction and to facilitate subsequent chemical decoration. As expected, both modifications improve the potency against p38α MAPK (3, 9.2 ± 0.4 μM; 4, 8.8 ± 1.5 μM) compared with the parent compound 2. These modifications also significantly improved compound solubility. Importantly, the inhibition potency of both 3 and 4 against both BTK and LCK was comparable to that observed originally with 1 (Table 1). We determined the cocrystal structure of p38α MAPK in complex with 3 (PDB code 5XYY). Data collection and refinement statistics are summarized in Supporting Information Table S4. Consistently, 3 adopts the same binding pose as 1 (Figure 5A), and the introduced phenol substituent forms an additional HB interaction with the side chain carboxyl group of Asp112. Designing Potent LCK and BTK Inhibitors. We also attempted O- and N-methylation on the 3,4,5-sites of phenyl

Figure 3. (A) Crystal complex structure of p38α MAPK bound with the lead compound. Protein and the lead compound structures are colored in white and pink, respectively. Hydrogen bonds are represented with orange and cyan lines. The 2Fo − Fc electron density map (contoured at 1σ) around the inhibitor is shown as purple mesh. (B) Schematic representation of the unique binding mode of the lead compound with a focus on the hinge-binding motif 1,2,4-triazol-5amine and (C) active-conformation and (D) inactive-conformation binding modes of representative kinase inhibitors containing a 5amine-pyrazole fragment. Hydrogen bonds between kinase hinge and ligand are highlighted with red circles.

structure of the p38a MAPK; 1 could form key hinge-binding HB interactions without obvious steric clashes with the binding site residues. Therefore, we tested the selectivity of 1 against inhouse kinases with gatekeeper residue of threonine. As expected, 1 showed comparable inhibition activities, including BTK (1.1 ± 0.1 μM), LCK (0.5 ± 0.1 μM), FYN (0.6 ± 0.2 μM), LYN (0.5 ± 0.2 μM), and BMX (18.3 ± 0.2 μM). We also assessed the inhibition activity against in-house kinases with gatekeeper residues larger than threonine (FGFR1, GSK3β, MET, IKKε, AKT1/2, IGF1R, JAK3, ROCK1, SYK, DYRK1A, ITK, and FLT3). 1 showed no significant inhibition against these kinases with larger-sized gatekeeper residues (data not shown). These results support that both the access to back pocket and the HB interaction with threonine gatekeeper are critical for the determination of the selectivity of 1. Structure-Based Lead Optimization against LCK and BTK. Given its small molecular size and unique hinge-binding 8555

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Figure 4. (A) Superposition of crystal complex structures of p38α MAPK bound with 1 and LCK complex (PDB code 3BYM). (B) Superposition of crystal structures of p38α MAPK bound with 1 and BTK complex (PDB code 3T9T). (C) Strategies of structural modification for potent selective kinase inhibitors against LCK and BTK on the structural basis of 1.

306.45 ± 75.02 nM) compared with ibrutinib’s selectivity ratio of 17 (BTK, 0.11 ± 0.01 nM; TEC, 1.86 ± 0.01 nM). Note that the reported BTK/TEC selectivity ratio of ibrutinib was 4.7 (BTK, 1.5 ± 0.2 nM; TEC, 7.0 ± 2.5 nM).48 Besides, 8 also shows considerable selectivity against ITK and ErbB2 (Supporting Information Table S5). We determined the cocrystal structure of BTK bound with 8 (PDB code 5XYZ). Data collection and refinement statistics are summarized in Supporting Information Table S6. As expected, 8 forms three hydrogen bonding interactions with the BTK hinge region, including one hydrogen bond with the side chain of the gatekeeper residue Thr474 and two hydrogen bonds with backbones of the Glu475 and Met477 residues. It also forms a covalent bond with the thiol group of Cys481 using the acrylamide warhead (Figure 5B). We also used LC−MS/MS to confirm the presence of the covalent bond between BTK and 8 (Supporting Information Table S7). Assessing the On-Target Cellular Effects of the Candidate Compounds. Compared to currently available LCK and BTK inhibitors, 7 and 8 exhibit low Tanimoto coefficients (Tc < 0.3) and high ligand efficiency32 (Supporting Information Table S8), highlighting the structural novelty and druggability of our compounds. We assessed the on-target inhibition of BTK and LCK by 1, 7, and 8. First, we examined the effects of BTK inhibition on the phosphorylation level of BTK and its physiological substrate PLCγ2 in Ramos cells. All three compounds inhibited the autophosphorylation of BTK and the phosphorylation of PLCγ2, with varying potencies (Figure 6). Second, we examined the effects of LCK inhibition

group which generates 5, 6, and 7 (Table 1). Encouragingly, 7 exhibited a significantly improved potency against LCK (IC50 = 0.018 ± 0.007 μM) and inferior inhibition activity against p38α MAPK (IC50 > 100 μM). The fact that these modifications substantially reduced the affinity toward p38α MAPK can be attributed to the following: (1) O-/N-methylation eliminates the HB-donor potential and prevents the favorable interaction with Asp112p38α; (2) O-/N-methylation increases the size of the substituent to an extent that exceeds the tolerance of the p38α MAPK hinge region, which lacks the one-residue insertion compared with LCK. Unfortunately, the poor water solubility of 7 might lead to the failed attempts in obtaining the crystals of LCK bound with 7. Covalent kinase inhibitors developed to target BTK have shown great promise in treating cancers and inflammatory diseases.23,46−48 However, the first-generation BTK inhibitor (ibrutinib) was shown to irreversibly inhibit other off-target kinases at clinically relevant concentration (e.g., TEC, ITK, and ErbB2/HER2).48,49 This could be responsible for certain side effects of ibrutinib including bleeding, rash, atrial fibrillation, and diarrhea.48−50 Although it is not fully elucidated, the mechanism of bleeding may be related with the inhibition of TEC. Starting from 5, we introduced the acrylamide moiety as the “warhead” to the meta-substitution position of the phenyl ring, generating 8, which exhibits significantly improved BTK inhibition (IC50 = 5 nM). We further assessed the selectivity of 8 against several off-targets of ibrutinib (33P filtration binding assay, Reaction Biology Corp.). Encouragingly, 8 has a BTK/ TEC selectivity ratio of 160 (BTK, 1.91 ± 0.02 nM; TEC, 8556

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Table 1. SAR Results for the Chemical Modifications on 1 against p38α MAPK, BTK, and LCKa

a

The IC50 values were determined for each compound (mean ± SEM) from three independent tests.

Figure 5. (A) Crystal structure of p38α MAPK in complex with 3. (B) Crystal structure of BTK in complex with 8. Protein and ligand structures are colored in white and hot pink, respectively. Hydrogen bonds are represented with cyan dash lines. The 2Fo − Fc electron density map (contoured at 1σ) around the inhibitor is shown as purple mesh.

on the phosphorylation level of ZAP70 and its physiological substrate LAT in Jurkat cells. 7 significantly inhibited the phosphorylation of both ZAP70 and LAT, while 1 and 8 showed only moderate inhibition, even at high concentrations (Figure 6). These cellular assay results suggest the reasonability of regarding 7 primarily as an LCK inhibitor and regarding 8 primarily as a BTK inhibitor. In addition, the inhibition activity of all three inhibitors are dose-dependent, and the cellular

inhibition activities of all three compounds are consistent with their in vitro determined molecular potencies.

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CONCLUSION Here, we employed in silico screening techniques to search for novel kinase inhibitors using p38α MAPK as a model system. The identified lead compound 1 is structurally dissimilar to any known kinase hinge binders and is characterized by a unique 8557

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Figure 6. On-target inhibition of BTK and LCK by 1, 7, and 8 in B and T cells. Human BTK kinase domain (residues 382−659) was inserted into modified pFastBac-HTB (a PreScission-cleavable 6*His tag was introduced into the vector). The inserted genes were sequenced to ensure the sequence was correct. The expression and purification of wild type and mutant p38α MAPK were carried out as previously reported.44 The protein (purity >98%) was concentrated to 17 mg/mL in storage buffer (50 mM TrisHCl (pH 7.4), 150 mM NaCl, 5% glycerol, 10 mM MgCl2, 5 mM DTT). The expression and purification of BTK kinase domain were performed as previously described.60 The protein was then concentrated to 6 mg/mL in stock buffer (20 mM Tris, pH 8.0, 50 mM NaCl, 3 mM DTT). The samples were immediately fresh frozen in liquid nitrogen and stored at −80 °C. Kinase Assays. The enzymatic activities of LCK, BTK, FYN, LYN, and BMX were measured in a 384-well format with an HTRF KinEASE-TK assay kit (Cisbio Inc.) according to product instructions. Briefly, analyte enzymes were initially incubated with different concentrations of compounds or vehicle control. The enzymatic reactions were started by adding a mixture of ATP and TK substrate− biotin. After incubation at room temperature for 40 min, the reaction was stopped and antibody detection mixture was added. Next, following a 30 min incubation at room temperature, the fluorescence at 620 and 665 nm was measured on an Envision plate reader (PerkinElmer). Specifically, for BTK inhibitor evaluation, 1 μg/mL enzyme, 18 μM ATP, and 0.3 μM TK substrate−biotin were used. For the LCK assay, 0.1 μg/mL LCK (Thermo Fisher Scientific), 2 μM ATP, and 0.1 μM TK substrate−biotin were used. For the FYN assay, 0.5 μg/mL FYN (Thermo Fisher Scientific), 10 μM ATP, and 0.6 μM TK substrate−biotin were used. In the LYN assay, 0.2 μg/mL LYN (Thermo Fisher Scientific), 1 μM ATP, and 0.1 μM TK substrate− biotin were used. In the BMX assay, 1 μg/mL BMX (Thermo Fisher Scientific), 14 μM ATP, and 0.3 μM TK substrate−biotin were used. The enzymatic activity of p38α MAPK was measured in a 384-well format with a Z′-LYTE kinase assay kit-Ser/Thr 15 peptide (Thermo Fisher Scientific) as specified in a previous report.61 0.1% (v/v) Triton X-100 was added in p38α kinase activity assay to prevent the nonspecific inhibition due to aggregation.62,63 The inhibition selectivity of 8 and ibrutinib against BTK and TEC was evaluated commercially (Reaction Biology Corporation) using a miniaturized radioisotope (33P) based assay platform.64 Briefly, after compounds were incubated with specific kinase/substrate pairs for 20

gatekeeper residue of threonine targeting mode of action. As expected, 1 has inhibition activity against multiple kinases that share the same common gatekeeper residue of threonine, including p38α MAPK (14.4 μM), BTK (1.1 μM), and LCK (0.5 μM), among several others. Impressed with the attractive properties of 1 (low MW, high LE, and amenability to modification), we used it as a starting scaffold and attempted to make minimal decorations to improve the binding affinity against selected kinases like LCK and BTK that have threonine as their gatekeeper residues. Encouragingly, 7 and 8 were more potent and more highly selective than the lead compound against LCK (18 nM) and BTK (8 nM). The potent on-target inhibition effects of these compounds (1, 7, and 8) observed in cellular assays highlight the promise of these novel compounds as candidate drugs for the treatment of cancers and inflammatory diseases. Moreover, our work underscores both the power and flexibility of in silico approaches for drug discovery and represents a successful example of how a “minimal decoration” strategy can be used to turn an initial lead compound into attractive candidate drugs.

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EXPERIMENTAL SECTION

Computational Details. The crystal structure of p38α MAPK (PDB code 1OUY) was selected as the starting model. Hierarchical virtual screening was conducted using our previously reported computational pipeline.31,39,40 First, we docked our in-house compound library against the ATP-binding site of p38α MAPK using DOCK 3.5.54.51−53 This generated multiple poses for each compound. Second, we carried out MM-GB/SA refinement and rescoring using the protein local optimization program (PLOP).37,54−56 During this process, the protein was kept rigid and the binding energy was calculated according to the equation Ebind = ER*L − EL − ER. The top ranking compounds with the lowest binding energies were further evaluated with a MD-based MM-PB/SA method implemented in the AMBER10.0 suite.57 AMBER99SB58 and general Amber force field (GAFF)59 were applied to the receptor and the ligands, respectively. Protein Expression and Purification. Human p38α MAPK was cloned into the pET28a vector using the NcoI/NotI restriction sites. 8558

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Scheme 1. General Route for the Synthesisa

Reagents and conditions: (a) (1) when X = Cl, aminoguanidine nitrate, acyl chloride, NaOH, MeCN, 0 °C, then H2O reflux; (2) when X = OH, carboxylic acid, CDI, aminoguanidine nitrate, base, solvent, 0−100 °C; (b) (1) 2-chloro-6-fluorobenzaldehyde, solvent, reflux; (2) NaBH3CN, acetic acid, room temperature; (c) trifluoroacetic acid, THF, 0 °C; (d) hydrazine hydrate, Raney Ni, ethanol, 60 °C; (e) acryloyl chloride, trimethylamine, THF, 0 °C. a

min, the reaction was started by addition of 33P ATP. Reactions are spotted onto P81 ion exchange paper 2 h later. Then kinase activity was then detected using a filter-binding method. Crystallography. We used the hanging drop vapor diffusion method for protein crystallization. For apo p38α MAPK protein crystallization, the reservoirs contained an aqueous solution of 0.2 M MgCl2, 0.1 M Bis-Tris, pH 6.5, and 25% PEG3350. The complex crystals were obtained by soaking apo crystals in reservoir solution containing 1−2.5 mM compound for 12 h at 20 °C. For crystallization of the BTK complex, the protein (6 mg/mL) was incubated with 0.9 mM 8 for 30 min on ice. Crystallization was performed using a hanging drop procedure where the reservoir solution contained 100 mM Tris (pH 8.5), 25% (w/v) polyethylene glycol 3350, and 0.2 mM sodium chloride. The resultant crystals were cryoprotected using reservoir solution supplemented with 15−20% v/v glycerol and were then flash cooled in liquid nitrogen. Diffraction data were collected at the Shanghai Synchrotron Research Facility beamline BL17U.65 The data were processed using HKL-2000.66 The structures of the p38α MAPK complex (both 1 and 3) and the BTK complex (8) were determined by molecular replacement using the p38α and BTK structures (PDB codes 1OUY, 3T9T) as the searching models. After initial refinement, candidate inhibitor compounds were modeled into the electron density using COOT.67 The complex structures of the p38α MAPK mfc1 p38α MAPK mfc2, and BTK were finally refined 2.61 Å, 1.70 Å, and 2.64 Å, resolution, using REFMAC.68 B and T Cell Activation and Phospho-Blots. The activation of human Ramos B and Jurkat T cells was performed using previously reported methods.23 Briefly, Ramos B cells or Jurkat T cells (2 × 106) were incubated with or without varying concentrations of candidate inhibitor compounds for 1.5 h at 37 °C in a 5% CO2 incubator. Goat anti-human IgM F(ab′)2 (40 μg/mL, Thermo Fisher Scientific) or Dynabeads human T-activator CD3/CD28 (Thermo Fisher Scientific) was then added to stimulate respectively Ramos B cells or Jurkat T cells for 5 min at 37 °C. The cells were then centrifuged at 300g, washed once with cold DPBS, and lysed on ice for 20 min in RIPA

buffer (Sigma-Aldrich) containing phosphatase inhibitor cocktail 2 and a protease inhibitor cocktail (both from Sigma-Aldrich). Samples were then centrifuged at 20 000g for 20 min at 4 °C. The supernatants were collected and analyzed by immunoblotting with phospho-specific antibodies against the BTK pY223, PLCγ2 pY1217, ZAP70 pY319, or LAT pY191 epitopes, respectively. These same blots were then stripped of the phospho-specific antibodies and reanalyzed using antibodies against the BTK, PLCγ2, ZAP70, or LAT proteins to measure total protein levels. All of the antibodies were obtained from Cell Signaling Technology. Chemistry. General synthetic routes to compounds 1−8 were provided in Scheme 1. General Chemistry Information. All solvents and reagents were purchased from commercial sources and used as received. Yields were not optimized. All reactions were monitored by thin layer chromatography (TLC) analysis. LC−MS analysis was performed on a Waters UPLC system operating in APCI (+ or − ) or ESI (+ or − ) ionization mode. Analytes were eluted using a linear gradient with a mobile phase of water/acetonitrile and detected at 220 nm. HRMS analysis was performed on Agilent 1290 UHPLC-Agilent 6540 QTOF. Analytes were eluted using a linear gradient with a mobile phase of water/acetonitrile containing 0.1% formic acid. Column chromatography was carried out on silica gel (200−300 mesh). Analytical HPLC was performed using a corona charged aerosol detector or photodiode array detector with a BEH C18 (1.7 μm, 2.1 mm × 150 mm, Waters, America) at a temperature of 50 °C and a flow rate of 0.3 mL/min. Mobile phases A and B under neutral conditions were water and acetonitrile, respectively. The ratio of mobile phase B was increased linearly from 20% to 100% over 3.5 min and then maintained at 100% over the next 1.5 min. All final test compounds were purified to >95% chemical purity as measured by analytical HPLC. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Mercury plus 400 (400 MHz). All 1H NMR spectra were consistent with the proposed structures. All proton shifts are given in parts per million (ppm) downfield from TMS (δ) as the internal standard in deuterated solvent, and coupling constants (J) are in hertz (Hz). NMR 8559

DOI: 10.1021/acs.jmedchem.7b01075 J. Med. Chem. 2017, 60, 8552−8564

Journal of Medicinal Chemistry

Article

mixture was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 100:1 to 10:1) to give 3-(5-amino1H-1,2,4-triazol-3-yl)phenol as a white solid (2.67g, yield 41.9%). Mass spectrum (ESI) m/z calcd for C8H8N4O [M − H]− 175.07, found 175.10. 1H NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H), 9.47 (s, 1H), 7.36−7.29 (m, 2H), 7.17 (t, J = 8.0 Hz, 1H), 6.77−6.67 (m, 1H), 6.00 (s, 2H). Step 2. The mixture of 3-(5-amino-1H-1,2,4-triazol-3-yl)phenol (5.0 g, 28.38 mmol), 2-chloro-6-fluorobenzaldehyde (5.0 g, 31.53 mmol), and p-toluenesulfonicacid (500 mg, 2.90 mmol) in isopropanol (50 mL) was heated to reflux overnight. The solution was allowed to cool to room temperature before NaBH3CN (4.0 g, 63.65 mmol) and acetic acid (2 mL) were added. Then, the resulting mixture was stirred at room temperature for 10 h before being quenched with water. The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 100:1 to 10:1) to give 3-(5-((2-chloro-6-fluorobenzyl)amino)-1H1,2,4-triazol-3-yl)phenol (3) as a white solid (1.2 g, yield 13.3%). HPLC purity: 95.04%. HRMS m/z calcd for C15H12ClFN4O [M − H]− 317.0684, 319.0684, found 317.1660, 319.2549. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 9.43 (s, 1H), 7.35 (s, 4H), 7.13− 7.27 (m, 2H), 6.90 (s, 1H), 6.75 (s, 1H), 4.51 (s, 2H). 3-(3-Aminophenyl)-N-(2-chloro-6-fluorobenzyl)-1H-1,2,4-triazol-5-amine (4). Step 1. A suspension of aminoguanidinium nitrate (2.58 g, 18.82 mmol) and NaOH (789.79 mg, 19.75 mmol) in methanol (30 mL) was stirred at room temperature for 3 h, the solvent was evaporated off, and the residue was resolved by N,Ndimethylformamide (DMF) (30 mL). A solution of 3-nitrobenzoic acid (3.0 g, 17.95 mmol) and N,N-diisopropylethylamine (DIPEA) (2.55 g, 19.73 mmol) in N,N-dimethylformamide (DMF) (30 mL) was treated with N,N′-carbonyldiimidazole (CDI) (3.2 g, 19.73 mmol) at 0 °C. The solution was stirred at room temperature for 3 h. Then, it was added dropwise to the suspension. After the addition, the reaction mixture was stirred at room temperature for 3 h. The solvent was removed by rotatory evaporation, and the residue was dissolved in water (100 mL) and heated to reflux for 18 h. After cooling to room temperature, it was filtered, and the solid was recrystallized from methanol to give 3-(3-nitrophenyl)-1H-1,2,4-triazol-5-amine as a yellow solid (3.0 g, yield 81.5%). Mass spectrum (ESI) m/z calcd for C8H7N5O2 [M + H]+ 206.06, found 206.20. 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 8.65−8.60 (m, 1H), 8.28 (d, J = 7.8 Hz, 1H), 8.22−8.16 (m, 1H), 7.71 (t, J = 8.0 Hz, 1H), 6.22 (s, 2H). Step 2. The mixture of 3-(3-nitrophenyl)-1H-1,2,4-triazol-5-amine (300 mg, 1.46 mmol) and 2-chloro-6-fluorobenzaldehyde (300 mg, 1.89 mmol) in anhydrous toluene (10 mL)/DMSO (0.1 mL) was heated to reflux for 15 h. The solution was allowed to cool to room temperature before NaBH3CN (200 mg, 3.18 mmol) and acetic acid (1 mL) were added. Then, the resulting mixture was stirred at room temperature for 3 h before being quenched with water. The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give N-(2-chloro-6-fluorobenzyl)-3-(3-nitrophenyl)-1H1,2,4-triazol-5-amine as a light-yellow solid (150 mg, yield 29.5%). Mass spectrum (ESI) m/z calcd for C15H11ClFN5O2 [M + H]+ 348.06,350.06, found 348.10, 350.10. 1H NMR (400 MHz, CD3OD) δ 8.82 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.29−8.22 (m, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.40−7.27 (m, 2H), 7.14 (t, J = 8.8 Hz, 1H), 4.70 (s, 2H). Step 3. The mixture of N-(2-chloro-6-fluorobenzyl)-3-(3-nitrophenyl)-1H-1,2,4-triazol-5-amine (50 mg, 0.14 mmol) and hydrazine hydrate (0.5 mL, 80% in water) in ethanol (10 mL) was heated to 60 °C. Then, Raney Ni (10 mg) was added. The resulting mixture was stirred at this temperature for 3 h. After cooling to room temperature, it was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give 3-(3-aminophenyl)-N-(2-chloro-6-fluorobenzyl)-1H-1,2,4-triazol-5-amine (4) as a white solid (36 mg, yield 78.8%). HPLC purity: 100%. HRMS m/z calcd for C15H13ClFN5 [M + H]+ 318.0844, 320.0844, found

data were reported as follows: chemical shift, integration, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; td, triplet of doublets; ddd, doublet of doublet of doublets; and brs, broad singlet), and coupling constants. N-(2-Chloro-6-fluorobenzyl)-3-(furan-2-yl)-1H-1,2,4-triazol-5amine (1). Step 1. A suspension of aminoguanidinium nitrate (22.0 g, 160.5 mmol) in acetonitrile (200 mL) was cooled to 0 °C. NaOH (20.0 g, 500.0 mmol) was added in portions, and the mixture was stirred at room temperature for 3 h. Then, furan-2-carbonyl chloride (20.0 g, 153.2 mmol) was added dropwise at 0 °C. After the addition, the mixture was stirred at room temperature for 6 h. The solvent was then removed by rotatory evaporation. The residue was dissolved in water (200 mL) and heated to reflux for 5 h. The solvent was removed under reduced pressure to give a crude product, which was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give 3-(furan-2-yl)-1H-1,2,4-triazol-5-amine as a white solid (8.5 g, yield 37%). Mass spectrum (ESI) m/z calcd for C6H6N4O [M + H]+ 151.05, found 151.10. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 12.07 (s, 1H), 7.68 (s, 1H), 6.71−6.61 (m, 1H), 6.54 (s, 1H), 6.08 (s, 2H). Step 2. A stirred mixture of 3-(furan-2-yl)-1H-1,2,4-triazol-5-amine (8.5 g, 56.6 mmol) and 2-chloro-6-fluorobenzaldehyde (17 g, 107.2 mmol) in anhydrous toluene (100 mL) was heated to reflux for 8 h. After cooling to room temperature, NaBH3CN (5.0 g, 79.6 mmol) and acetic acid (20 mL) were added. Then, the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with water (10 mL), and the solvent was removed under reduced pressure. The residue was purified by reverse phase flash column chromatography (C18) to give N-(2-chloro-6-fluorobenzyl)-3-(furan-2-yl)-1H-1,2,4triazol-5-amine (1) as a white solid (3.2 g, yield 19.3%). HPLC purity: 98.58%. HRMS m/z calcd for C13H10ClFN4O [M + H]+ 293.0527, 295.0527, found 293.2722, 295.2885. 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 7.70 (s, 1H), 7.37 (dd, J = 20.6, 7.3 Hz, 2H), 7.24 (t, J = 8.7 Hz, 1H), 6.94 (s, 1H), 6.73 (s, 1H), 6.55 (s, 1H), 4.50 (s, 2H). N-(2-Chloro-6-fluorobenzyl)-3-phenyl-1H-1,2,4-triazol-5-amine (2). Step 1. To a solution of aminoguanidinium nitrate (531 mg, 3.88 mmol) in H2O (3 mL) was added NaOH (155.2 mg, 3.88 mmol) at 0 °C before benzoyl chloride (545 mg, 3.88 mmol) was added. The mixture was heated to reflux for 5 h before the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give 3-phenyl-1H-1,2,4-triazol-5-amine as a white solid (40 mg, yield 6.5%). Mass spectrum (ESI) m/z calcd for C8H8N4 [M + H]+ 161.05, found 161.10. Step 2. A stirred mixture of 3-phenyl-1H-1,2,4-triazol-5-amine (40 mg, 0.25 mmol) and 2-chloro-6-fluorobenzaldehyde (79 mg, 0.5 mmol) in ethanol (15 mL) was heated to reflux for 8 h. After cooling to room temperature, NaBH3CN (75 mg, 1.2 mmol) and acetic acid (0.5 mL) were added. Then, the mixture was stirred at room temperature for 3 h before it was quenched with water (10 mL) and the solvent was removed under reduced pressure. The residue was purified by reverse phase flash column chromatography (C18) to give N-(2-chloro-6-fluorobenzyl)-3-phenyl-1H-1,2,4-triazol-5-amine (2) as a white solid 11 mg, yield 14.6%). HPLC purity: 96.63%. HRMS m/z calcd for C15H12ClFN4 [M − H]− 301.0735, 303.0735, found 301.2102, 303.1912. 1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 7.91 (m, 2H), 7.49 (m, 1H), 7.45−7.31 (m, 4H), 7.23 (m, 1H), 4.52 (s, 2H). 3-(5-((2-Chloro-6-fluorobenzyl)amino)-1H-1,2,4-triazol-3-yl)phenol (3). Step 1. A suspension of aminoguanidinium nitrate (5.46 g, 39.82 mmol) and K2CO3 (5.5 g, 39.82 mmol) in N,N-dimethylformamide (DMF) (50 mL) was stirred at room temperature for 1 h. A solution of 3-hydroxybenzoic acid (5.0 g, 36.2 mmol) in N,Ndimethylformamide (DMF) (30 mL) was treated with N,N′carbonyldiimidazole (CDI) (6.46 g, 39.82 mmol) at 0 °C. Then, the solution was stirred at room temperature for 1 h. Then, the solution was added dropwise to the suspension. After the addition, the reaction mixture was stirred at room temperature for 3 h. The mixture was then heated to 100 °C for 5 h. After cooling to room temperature, the 8560

DOI: 10.1021/acs.jmedchem.7b01075 J. Med. Chem. 2017, 60, 8552−8564

Journal of Medicinal Chemistry

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318.1504, 320.0482. 1H NMR (400 MHz, CD3OD) δ 7.33 (ddd, J = 19.0, 9.5, 6.9 Hz, 2H), 7.27−7.19 (m, 2H), 7.19−7.08 (m, 2H), 6.78 (d, J = 7.8 Hz, 1H), 4.64 (s, 2H). N-(2-Chloro-6-fluorobenzyl)-3-(3-(methylamino)phenyl)-1H1,2,4-triazol-5-amine (5). Step 1. The mixture of 3-aminobenzoic acid (500 mg, 3.65 mmol) and triethylamine (1.11 g, 10.97 mmol) in methanol (6 mL) was allowed to cool to 0 °C before di-tert-butyl dicarbonate (880 mg, 4.03 mmol) was added. Then, the resulting mixture was stirred at room temperature for 10 h before being quenched with water. The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 100:1 to 10:1) to give 3-((tertbutoxycarbonyl)amino)benzoic acid as a white solid (800 mg, yield 92.5%). Mass spectrum (ESI) m/z calcd for C12H15NO4 [M − H]− 236.10, found 236.10. Step 2. The mixture of 3-((tert-butoxycarbonyl)amino)benzoic acid (1.0 g, 4.21 mmol) in N,N-dimethylformamide (DMF) (10 mL) was allowed to cool to 0 °C before sodium hydride (505.7 mg, 12.64 mmol, 60% dispersion in mineral oil) was added. The mixture was stirred at this temperature for 1 h before iodomethane (1.26 g, 8.80 mmol) was added. The resulting mixture was stirred at room temperature for 5 h before being quenched with water. The solvent was removed under reduced pressure, and the residue was dissolved in methanol (10 mL)/water (1 mL) before sodium hydroxide (337.2 mg, 8.43 mmol) was added. After the starting material was consumed by TLC analysis, the mixture was adjusted to pH 3−4 by 3 M HCl and extracted with ethyl acetate (50 mL). The ethyl acetate solution was dried and concentrated to give 3-((tert-butoxycarbonyl)(methyl)amino)benzoic acid as a white solid (950 mg, yield 89.7%). Mass spectrum (ESI) m/z calcd for C13H17NO4 [M − H]− 250.12, found 250.10. 1H NMR (400 MHz, DMSO-d6) δ 13.06 (s, 1H), 7.83 (t, J = 1.8 Hz, 1H), 7.75−7.72 (m, 1H), 7.53 (ddd, J = 8.0, 2.3, 1.2 Hz, 1H), 7.49−7.43 (m, 1H), 3.20 (s, 3H), 1.40 (s, 9H). Step 3. A suspension of aminoguanidinium nitrate (360 mg, 2.63 mmol) and K2CO3 (660 mg, 4.78 mmol) in N,N-dimethylformamide (DMF) (3 mL) was stirred at room temperature for 1 h. A solution of 3-((tert-butoxycarbonyl)(methyl)amino)benzoic acid (600 mg, 2.39 mmol) in N,N-dimethylformamide (DMF) (30 mL) was treated with N,N′-carbonyldiimidazole (CDI) (426 mg, 2.63 mmol) at 0 °C. The solution was stirred at room temperature for 1 h. Then it was added to the suspension. The resulting mixture was stirred at room temperature for 3 h, then it was heated to 100 °C for 3 h. The mixture was cooled to room temperature, filtered, and concentrated. The residue was purified by column chromatography (silica gel, dichloromethane/ methanol = 100:1 to 10:1) to give tert-butyl (3-(5-amino-1H-1,2,4triazol-3-yl)phenyl)(methyl)carbamate as a yellow oil (220 mg, yield 31.8%). Mass spectrum (ESI) m/z calcd for C14H19N5O2 [M + H]+ 290.15, found 290.20. 1H NMR (400 MHz, CDCl3) δ 7.85−7.80 (m, 1H), 7.73−7.67 (m, 1H), 7.61 (s, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.23− 7.18 (m, 1H), 7.04 (s, 2H), 3.23 (s, 3H), 1.46 (s, 9H). Step 4. The mixture of tert-butyl (3-(5-amino-1H-1,2,4-triazol-3yl)phenyl)(methyl)carbamate (200 mg, 0.69 mmol), 2-chloro-6fluorobenzaldehyde (200 mg, 1.26 mmol), and p-toluenesulfonic acid (11.9 mg, 0.069 mmol) in isopropanol (5 mL) was heated to reflux overnight. The solution was allowed to cool to room temperature before NaBH3CN (120 mg, 1.91 mmol) and acetic acid (0.5 mL) were added. Then, the resulting mixture was stirred at room temperature for 5 h before being quenched with water. The solvent was removed under reduced pressure. The crude tert-butyl (3-(5-((2chloro-6-fluorobenzyl)amino)-1H-1,2,4-triazol-3-yl)phenyl)(methyl)carbamate was used directly without further purification. Step 5. The mixture of crude tert-butyl (3-(5-((2-chloro-6fluorobenzyl)amino)-1H-1,2,4-triazol-3-yl)phenyl)(methyl)carbamate (100 mg) in tetrahydrofuran (THF) (5 mL) was cooled to 0 °C before trifluoroacetic acid (10 mL) was added. Then, the resulting mixture was stirred at room temperature for 5 h. The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 100:1 to 10:1) to give N-(2-chloro-6-fluorobenzyl)-3-(3-(methylamino)phenyl)-1H-1,2,4-triazol-5-amine (5) as a white solid (45 mg, yield

19.6%,two steps). HPLC purity: 99.84%. HRMS m/z calcd for C16H15ClFN5 [M + H]+ 332.1000, 334.1000, found 332.4052, 334.4059. 1H NMR (400 MHz, DMSO-d6, mixture of rotamers, 2:1) δ13.17 (s, 0.5H), 12.07 (s, 1H), 7.35 (s, 3H), 7.23 (s, 1.5H), 7.12 (s, 4.5H), 6.85 (s, 1H), 6.48−6.52 (m, 1.5H), 6.13 (s, 0.6H), 5.71−5.75 (m, 1.5H), 4.51 (s, 3H), 2.69 (s, 4.5H). 3-(3-Amino-4-methoxyphenyl)-N-(2-chloro-6-fluorobenzyl)-1H1,2,4-triazol-5-amine (6). Step 1. A suspension of aminoguanidinium nitrate (139 mg, 1.01 mmol) and KOH (57 mg, 1.02 mmol) in methanol (2 mL) was stirred at room temperature for 1.5 h and concentrated. The residue was diluted with N,N-dimethylformamide (DMF) (2 mL). A solution of 4-methoxy-3-nitrobenzoic acid (197 mg, 1.0 mmol) in N,N-dimethylformamide (DMF) (5 mL) was treated with N,N′-carbonyldiimidazole (CDI) (190 mg, 1.17 mmol) at 0 °C. The solution was stirred at room temperature for 1.5 h. Then it was added dropwise to the suspension. After the addition, the reaction mixture was stirred at room temperature for 2 h. The solvent was removed by rotatory evaporation, the residue was dissolved in water (50 mL) and heated to reflux for 5 h. After cooling to room temperature, it was filtered, and the solid was recrystallized from methanol to give 3-(4-methoxy-3-nitrophenyl)-1H-1,2,4-triazol-5amine as a light-yellow solid (110 mg, yield 46.8%). Mass spectrum (ESI) m/z calcd for C9H9N5O3 [M + H]+ 236.07, found 236.20. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.26 (d, J = 1.4 Hz, 1H), 8.11 (dd, J = 8.8, 2.1 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 6.14 (s, 2H), 3.95 (s, 3H). Step 2. The mixture of 3-(4-methoxy-3-nitrophenyl)-1H-1,2,4triazol-5-amine (100 mg, 0.43 mmol) and 2-chloro-6-fluorobenzaldehyde (150 mg, 0.95 mmol) in anhydrous toluene (10 mL) was heated to reflux for 15 h. The solution was allowed to cool to room temperature before NaBH3CN (45 mg, 0.72 mmol) and acetic acid (1 mL) were added. Then, the resulting mixture was stirred at room temperature for 3 h before being quenched with water. The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give N-(2-chloro-6-fluorobenzyl)-3-(4-methoxy-3-nitrophenyl)-1H-1,2,4-triazol-5-amine as a light-yellow solid (70 mg, yield 43.6%) that was used directly in the next step. Mass spectrum (ESI) m/z calcd for C16H13ClFN5O3 [M + H]+ 378.07, 380.07, found 378.30, 380.30. Step 3. The mixture of N-(2-chloro-6-fluorobenzyl)-3-(4-methoxy-3nitrophenyl)-1H-1,2,4-triazol-5-amine (70 mg, 0.19 mmol) and hydrazine hydrate (1.0 mL, 80% in water) in ethanol (10 mL) was heated to 60 °C. Then, Raney Ni (10 mg) was added. The resulting mixture was stirred at room temperature for 3 h. After cooling to room temperature, it was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give 3(3-amino-4-methoxyphenyl)-N-(2-chloro-6-fluorobenzyl)-1H-1,2,4-triazol-5-amine (6) as a white solid (50 mg, yield 77.6%). HPLC purity: 96.1%. HRMS m/z calcd for C16H15ClFN5O [M + H]+ 348.0949, 350.0949, found 348.4669, 350.4831. 1H NMR (400 MHz, CD3OD) δ 7.36−7.23 (m, 4H), 7.11 (t, J = 8.3 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 4.63 (s, 2H), 3.88 (s, 3H). N-(2-Chloro-6-fluorobenzyl)-3-(3,4-dimethoxyphenyl)-1H-1,2,4triazol-5-amine (7). Step 1. A suspension of aminoguanidinium nitrate (13.55 g, 98.83 mmol) and NaOH (240.6 mg, 6.02 mmol) in methanol (100 mL) was stirred at room temperature for 3 h. The solvent was evaporated off, and the residue was dissolved in N,Ndimethylformamide (DMF) (100 mL). A solution of 3,4-dimethoxybenzoic acid (15.0 g, 82.34 mmol) in N,N-dimethylformamide (DMF) (150 mL) was treated with N,N′-carbonyldiimidazole (CDI) (14.69 g, 90.59 mmol) at 0 °C. Then, the solution was stirred at room temperature for 3 h, and the solution was added dropwise to the suspension. After the addition, the reaction mixture was stirred at room temperature for 3 h. The solvent was then removed by rotatory evaporation, the residue was dissolved in water (150 mL) and heated to reflux for 18 h. After cooling to room temperature, it was filtered, and the solid was recrystallized from methanol to give 3-(3,4dimethoxyphenyl)-1H-1,2,4-triazol-5-amine as an off-white solid (9.38 8561

DOI: 10.1021/acs.jmedchem.7b01075 J. Med. Chem. 2017, 60, 8552−8564

Journal of Medicinal Chemistry

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ORCID

g, yield 51.7%). Mass spectrum (ESI) m/z calcd for C10H12N4O2 [M + H]+ 221.10, found 221.40. 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 7.46−7.39 (m, 2H), 6.98 (d, J = 8.8 Hz, 1H), 5.93 (s, 2H), 3.77 (d, J = 3.4 Hz, 6H). Step 2. The mixture of 3-(3,4-dimethoxyphenyl)-1H-1,2,4-triazol-5amine (8 g, 36.33 mmol), 2-chloro-6-fluorobenzaldehyde (8 g, 50.46 mmol), and p-toluenesulfonic acid (625.5 mg, 3.63 mmol) in isopropanol (40 mL) was heated to reflux overnight. The solution was allowed to cool to room temperature before NaBH3CN (5.0 g, 79.56 mmol) and acetic acid (1 mL) were added. Then, the resulting mixture was stirred at room temperature for 5 h before it was quenched with water. The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, dichloromethane/methanol = 50:1 to 10:1) to give N-(2-chloro-6fluorobenzyl)-3-(3,4-dimethoxyphenyl)-1H-1,2,4-triazol-5-amine (7) as a white solid (4.5 g, yield 34.1%). HPLC purity: 100%. HRMS m/z calcd for C17H16ClFN4O2 [M + H]+ 363.0946, 365.0946, found 363.1035, 365.1004. 1H NMR (400 MHz, DMSO-d6, mixture of rotamers 2:1) δ 13.13 (s, 0.5H), 12.06 (s, 1H), 7.50−7.41 (m, 3H), 7.40−7.29 (m, 3H), 7.28−7.15 (m, 2H), 7.05 (d, J = 8.2 Hz, 0.5H), 6.98 (d, J = 8.8 Hz, 1H), 6.87 (t, J = 5.4 Hz, 1H), 6.12 (t, J = 6 Hz, 0.5H), 4.53 (d, J = 4.9 Hz, 2H), 4.45 (d, J = 5.2 Hz, 1H), 3.78 (d, J = 6.9 Hz, 9H). N-(3-(5-((2-Chloro-6-fluorobenzyl)amino)-1H-1,2,4-triazol-3yl)phenyl)acrylamide (8). The mixture of 3-(3-aminophenyl)-N-(2chloro-6-fluorobenzyl)-1H-1,2,4-triazol-5-amine (4) (12.0 mg, 0.038 mmol) and trimethylamine (5.0 mg, 0.049 mmol) in tetrahydrofuran (THF) (0.5 mL) was allowed to cool to 0 °C before acryloyl chloride (3.45 mg, 0.038 mmol) was added. Then, the resulting mixture was stirred at room temperature for 3 h before it was quenched with water. The solvent was removed under reduced pressure. The residue was purified by reverse phase HPLC (C18) to give N-(3-(5-((2-chloro-6fluorobenzyl)amino)-1H-1,2,4-triazol-3-yl)phenyl)acrylamide (8) as a white solid (2.6 mg, yield 18.5%). HPLC purity: 98.78%. HRMS m/z calcd for C18H15ClFN5O [M + H]+ 372.0949, 374.0949, found 372.1031, 374.1007. 1H NMR (400 MHz, CD3OD) δ 8.12 (s, 1H), 7.77 (s, 1H), 7.73−7.63 (m, 1H), 7.40 (s, 1H), 7.37−7.27 (m, 2H), 7.14 (t, J = 8.1 Hz, 1H), 6.46 (dd, J = 17.0, 9.6 Hz, 1H), 6.38 (dd, J = 17.0, 2.3 Hz, 1H), 5.79 (dd, J = 9.6, 2.3 Hz, 1H), 4.67 (s, 2H).

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Xiangbing Qi: 0000-0002-7139-5164 Niu Huang: 0000-0002-6912-033X Author Contributions §

Y.W., Y.S., and R.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the staff at the Shanghai Synchrotron Radiation Facility for their assistance in data collection. We also thank Dr. She Chen and Lin Li in the proteomics center at National Institute of Biological Sciences, Beijing for the assistance with LC−MS/MS experiments. Financial support from the Chinese Ministry of Science and Technology “973” Grant 2014CB849802 (to N.H.) is gratefully acknowledged. Computational support was provided by Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) under Grant U1501501.

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ABBREVIATIONS USED MAPK, mitogen-activated protein kinase; BTK, bruton tyrosine kinase; LCK, lymphocyte-specific protein tyrosine kinase; BCR, B cell antigen receptor; TCR, T cell antigen receptor; HB, hydrogen bond; CLL, chronic lymphocytic leukemia; MCL, mantle cell lymphoma; WM, Waldenstrom’s macroglobulinemia; LE, ligand efficiency; HTS, high throughput screening; RMSD, root-mean-square deviation; GK, gatekeeper; MMPBSA, molecular mechanics Poisson−Boltzmann surface area; PAINS, Pan Assay Interference Compounds

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(1) Hantschel, O. Unexpected off-targets and paradoxical pathway activation by kinase inhibitors. ACS Chem. Biol. 2015, 10 (1), 234− 245. (2) Gunosewoyo, H.; Yu, L.; Munoz, L.; Kassiou, M. Kinase targets in CNS drug discovery. Future Med. Chem. 2017, 9 (3), 303−314. (3) Goldstein, D. M.; Gray, N. S.; Zarrinkar, P. P. High-throughput kinase profiling as a platform for drug discovery. Nat. Rev. Drug Discovery 2008, 7 (5), 391−397. (4) Akritopoulou-Zanze, I.; Hajduk, P. J. Kinase-targeted libraries: the design and synthesis of novel, potent, and selective kinase inhibitors. Drug Discovery Today 2009, 14 (5−6), 291−297. (5) Ghose, A. K.; Herbertz, T.; Pippin, D. A.; Salvino, J. M.; Mallamo, J. P. Knowledge based prediction of ligand binding modes and rational inhibitor design for kinase drug discovery. J. Med. Chem. 2008, 51 (17), 5149−5171. (6) Xing, L.; Klug-Mcleod, J.; Rai, B.; Lunney, E. A. Kinase hinge binding scaffolds and their hydrogen bond patterns. Bioorg. Med. Chem. 2015, 23 (19), 6520−6527. (7) Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P. Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2011, 29 (11), 1046−1051. (8) Zhang, J.; Yang, P. L.; Gray, N. S. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 2009, 9 (1), 28−39. (9) Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Through the “gatekeeper door”: exploiting the active kinase conformation. J. Med. Chem. 2010, 53 (7), 2681−2694. (10) Hendriks, R. W.; Yuvaraj, S.; Kil, L. P. Targeting Bruton’s tyrosine kinase in B cell malignancies. Nat. Rev. Cancer 2014, 14 (4), 219−232. (11) Gaestel, M.; Kotlyarov, A.; Kracht, M. Targeting innate immunity protein kinase signalling in inflammation. Nat. Rev. Drug Discovery 2009, 8 (6), 480−499.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01075. Structure and activity of tested compounds, representative binding modes of known kinase inhibitors, structural comparison between the predicted binding mode and crystal structure, statistics of crystal structures, chemical similarity analysis, and 1H NMR, HPLC, and HRMS results of representative compounds (PDF) Compound characterization checklist (XLS) Molecular formula strings and some data (CSV) Accession Codes

PDB codes for p38α MAPK bound with 1 and 3 are 5XYX and 5XYY. PDB code for BTK bound with 8 is 5XYZ. Authors will release the atomic coordinates and experimental data upon article publication.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*X.Q.: phone, 86-10-80726688-8655; fax, 86-10-80708048; email, [email protected]. *N.H.: phone, 86-10-80720645; fax, 86-10-80720813; e-mail, [email protected]. 8562

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