Novel 5-Substituted Oxindole Derivatives as Bruton’s Tyrosine Kinase Inhibitors: Design, Synthesis, Docking, Molecular Dynamics Simulation, and Biological Evaluation

Bruton’s tyrosine kinase (BTK) is a non-RTK cytoplasmic kinase predominantly expressed by hemopoietic lineages, particularly B-cells. A new oxindole-based focused library was designed to identify potent compounds targeting the BTK protein as anticancer agents. This study used rational approaches like structure-based pharmacophore modeling, docking, and ADME properties to select compounds. Molecular dynamics simulations carried out at 20 ns supported the stability of compound 9g within the binding pocket. All the compounds were synthesized and subjected to biological screening on two BTK-expressing cancer cell lines, RAMOS and K562; six non-BTK cancer cell lines, A549, HCT116 (parental and p53–/–), U2OS, JURKAT, and CCRF-CEM; and two non-malignant fibroblast lines, BJ and MRC-5. This study resulted in the identification of four new compounds, 9b, 9f, 9g, and 9h, possessing free binding energies of −10.8, −11.1, −11.3, and −10.8 kcal/mol, respectively, and displaying selective cytotoxicity against BTK-high RAMOS cells. Further analysis demonstrated the antiproliferative activity of 9h in RAMOS cells through selective inhibition of pBTK (Tyr223) without affecting Lyn and Syk, upstream proteins in the BCR signaling pathway. In conclusion, we identified a promising oxindole derivative (9h) that shows specificity in modulating BTK signaling pathways.


■ INTRODUCTION
Bruton's tyrosine kinase (BTK) belongs to the Tec family of kinase and is a non-RTK cytoplasmic kinase primarily expressed by hemopoietic lineages, particularly B-cells. 1 BTK has five different domains, and two important phosphorylation sites, tyrosine 233 (Y233) and tyrosine 551 (Y551), that are important for BTK activation are located within the SRC homology and C-terminal kinase domain (Figure 1).Y551 is phosphorylated by spleen tyrosine kinase (Syk) or LYN protooncogene (Lyn), leading to BTK activation. 1 BTK is also activated when PIP3/PI3K is attached to the PH domain with different cell surface receptors. 1BTK is implicated in various human diseases, primarily those related to the immune system, such as X-linked agammaglobulinemia, chronic lymphocytic leukemia (CLL), mantle-cell lymphoma (MCL), follicular lymphoma, non-Hodgkin's lymphomas, Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma, rheumatoid arthritis, and other autoimmune diseases. 1,2Therefore, BTK inhibitors have implications not only in cancer but also in the treatment of severe autoimmune diseases such as multiple sclerosis. 2rrently, there are three covalent irreversible inhibitors of BTK available in the market, namely, ibrutinib, zanubrutinib, and acalabrutinib. 2 Ibrutinib is approved for treating CLL, MCL, and WM, 1,2 whereas the second-generation more selective inhibitors, acalabrutinib and zanubrutinib, are approved for treating CLL, small lymphocytic lymphoma, and relapsed/refractory MCL. 2 However, the anticancer activity of ibrutinib, zanubrutinib, and acalabrutinib that bind to cysteine 481 in the BTK kinase domain is susceptible to resistance caused by the C481S mutation (Figure 1). 3,4dditionally, these drugs result in considerable toxicity after long-term use due to off-target interactions with other kinases containing cysteine motifs. 4There is a growing interest in noncovalent, reversible BTK inhibitors, such as fenebrutinib and vecabrutinib, to overcome drug resistance and minimize off-target effects. 3,4−15 Oxindoles are significant structural motifs in various natural products and pharmaceutical compounds, making them valuable in drug discovery. 16,17Compounds containing the oxindole structure are known for their anticancer, antiinflammatory, antiviral, and antifungal activities. 17,18Importantly, substituted oxindole derivatives exhibit selectivity toward several protein kinases, such as VEGFR-1, VEGFR 2, VEGFR 3, PDGFRα, PDGFRβ, Kit, Flt-3, and CSF-1R. 17−18 Our recent research on BTK inhibitors uncovered the notable selective cytotoxicity of oxindole sulfonamides against BTK-high human B-cell lymphoma cells, with no-to-minimal cytotoxicity in non-BTK cancer and non-cancer cells. 19In this study, we report the design, synthesis, and biological evaluation of a series of novel oxindole derivatives as promising anti-BTK candidates.

■ RESULTS AND DISCUSSION
Structure-based designing was carried out to generate anticancer compounds against BTK protein.The compound selection process is depicted as a flowchart shown in Figure 3.
Initially, compound 819 was identified as the lead compound from the docking based on the binding free energy, and further modifications were carried out to increase its interactions with the critical amino acids in BTK (PDB-5P9J), as shown in Figure 4.For the purpose of establishing a structure−activity relationship, acid chlorides were introduced at position R (Scheme 1), leading to the synthesis of 10 compounds.It is worth noting that all the 10 compounds (9a−9j) exhibited favorable ADME properties, as detailed in Table 1.
The methyl benzyl group attached to the piperazine ring was replaced with the different acid chlorides, which showed   interaction with ASP-539 amino acid and GLN-412.Replacing the methyl group with hydrogen in the piperazine ring and the oxindole ring methyl group with hydrogen showed better interaction with MET-477 and LEU-528 at the base of the ATP pocket (Figure 4).Amino acid MET-477 forms a hydrogen bond with the carbonyl oxygen of the oxindole ring, and LEU-528 forms a π−σ bond with the phenyl of the oxindole ring in the H1 pocket (Figure 5).Amino acids VAL-416, ALA-428, and LEU-408 form alkyl and π−alkyl bonds with an oxindole ring in the H1 pocket.Other amino acids CYS-481, GLY-480, ALA-478, TYR-476, THR-474, and GLU-475 form van der Waals interactions with the cyclopentylidene moiety in the H2 lipophilic pocket.Amino acid ASN-526 forms a carbon−hydrogen bond with the piperazine ring, oriented toward the bottom of the H3 pocket (Figure 5).In the H3 pocket, we changed 10 different acid chloride substitutions and synthesized analogues with docking scores of more than −10 kcal/mol.
In compound 9b, the cyclohexane group showed van der Waal interaction with ASP-521, ARG-525, ASN-526, TYR-551, and LYS-558 amino acids; and a π−σ bond with VAL-416; and alkyl interactions with LEU-408, LEU-528, and ALA-428.The hydrogen bonds are formed with GLN-412 and MET-477 with 2.89 Å, and the free binding energy is −10.8 kcal/mol.In compound 9f, the bromine group showed π−alkyl interaction with PHE-413, alkyl interaction with LEU-542, and van der Waal interaction with ASP-539 amino acid.Similarly, hydrogen bonds are formed with GLN-412 with 2.94 Å and MET-477 with 3.18 Å and a free binding energy of −11.1 kcal/mol.For compound 9g, the fluorine group forms a π−σ bond with DFG residue ASP-539 in the H3 pocket, an essential amino acid (Figure 5).Similarly, the hydrogen bonds are formed with GLN-412 with 2.91 Å and MET-477 with 3.01 Å, and the free binding energy is −11.3 kcal/mol.
In compound 9h, the aliphatic chain showed van der Waal interaction with only ASP-521, ARG-525, TYR-551, and LYS-558 amino acids; π−σ bond with VAL-416; and alkyl interactions with LEU-408, LEU-528, and ALA-428.The hydrogen bonds are formed with GLN-412 with 2.93 Å and MET-477 with 3.21 Å, as shown in Figure 6, and the free binding energy is −10.8 kcal/mol.The crystal structure of BTK complexed with the cocrystal structure with four compounds, 9b, 9f, 9g, and 9h, is shown in Figure 7.

■ MOLECULAR DYNAMICS SIMULATIONS
Molecular dynamics (MD) simulations at 50, 20, and 20 ns were performed for complex compounds 9f-5P9J, 9g-5P9J, and 9h-5P9J.Several factors, such as the ligand protonation state, conformation of ligands, water molecules, cofactors, ions, and conformational and solvation entropies, will affect docking predictions in an unexpected pattern.Many reports support the role of MD simulations in filtering docking results. 20,21MD simulations were carried out to determine the interaction stability of the ligand−protein docked complex.The stereochemical solid geometries of the residues were analyzed for the final structure using the Ramachandran map (Figure 8).The residue percentage in the favored region is 95.69% (267 residues), allowed is 3.58% (10 residues), and the outlier is 0.71% (2 residues).
The RMSD of Cα, backbone, and side chains for all complexes showed fluctuations in the range of 0.4−3.2Å, which are in the acceptable range.The results showed that the simulation equilibrated after 4 ns.The RMSD values of Cα and ligand for complex 5P9J-9h are shown in Figure 9A.The protein's secondary structure elements (SSEs) were monitored during the simulations (Figure 9B).The total percentage of SSE for 9f, 9g, and 9h was found to be 42.95,45.71, and 44.49%, respectively.The RMSD values of Cα and ligand and the SSE for complex 5P9J-9f and complex 5P9J-9g are shown in Figures S1A,B   the presence of water, MET-477 bonded 100% through simulation, PHE-413 with 65%, and GLY-412 with 36%.The hydrogen water bonds formed are GLU-475 at 69%, THR-474 at 49%, and CYS-481 at 43%, as shown in Figure 10B.The results showed that the role water molecules play within the binding pocket of the protein 5P9J for compound 9h.The    The 2D diagram of compound 9h is presented with colorcoded rotational bonds.The bar plots gave information about the torsion angle's density and the rotational bond's potential in kcal/mol.The torsion potential relationships of compound 9h with conformational strain were explained, conserving a protein-bound confirmation.The stability of the ligand was analyzed using six parameters: polar surface area, RMSD, solvent-accessible surface area, molecular surface area, IntraHB, and rGyr (radius of gyration), shown in Figure S9.The RMSD of the ligand was shown to be stable, which ranged up to 1.5 Å.The histogram graphs, torsional analysis, and stability analysis of the ligands for compounds 9f and 9g are shown in Figures S10−S15.

■ SYNTHESIS
Commercially available oxindole was used as a starting material for synthesizing 5-substituted oxindole derivatives.To chlorosulfonic acid, oxindole was added portionwise at 0 °C and stirred for 30 min at room temperature (RT).The mixture was heated to 70 °C using an oil bath to afford 2-oxoindoline-5-sulfonyl chloride 2. 22,23 Sulfonyl chloride intermediate 2 was coupled in the presence of pyridine with N-Boc piperazine 3, 24,25 resulting in N-Boc 4-(2-oxoindoline-5-sulfonamido)-piperazine 4. Intermediate 4 in ethanol was subjected to Knoevenagel condensation with cyclopentanone 5 by adding pyrrolidine as a base that affords intermediate 6. 26,27 The protecting group tertiary butyl carbonyl of intermediate 6 was removed by treating it with 4 M HCl in 1,4-dioxane to yield critical scaffold 7 as HCl salt.Analogues 9a−9j were synthesized by amide coupling with different acid chlorides 8a−8j in the presence of di-isopropyl ethyl amine.The liberated hydrochloric acid was neutralized by adding the base di-isopropyl ethyl amine (Scheme 1).

CULTURES
Ten derivatives were tested in a panel of cell lines consisting of ITK-positive T-cell leukemia lines, 28 BTK-positive B-cell leukemia lines, 29 ITK/BTK-negative malignant lines, and two non-malignant fibroblast lines.Although both RAMOS and K562 cell lines are positive for BTK expression, they do not express ITK.However, BTK expression is relatively higher in RAMOS than in K562 cells. 19In contrast, ITK expression is higher in JURKAT than in CCRF-CEM cells, both of which lack BTK expression. 19RAMOS is well known for its high BTK expression. 29Other panel cell lines, including A549,  HCT116, U2OS, MRC-5, and BJ, do not express BTK or ITK. 19he compounds that did not show 50% inhibition of cell proliferation when evaluated at a single dose of 50 μM were not processed for dose−response analysis.The cytotoxicity profiling was done by considering compounds with IC 50 values above 50 μM as inactive, above 30 μM as weakly active, between 10 and 20 μM as moderately active, and below 10 μM as highly active.Based on these standard norms, the effects of four structurally similar compounds (active�9b, 9f, 9g, and 9h) were interesting to observe in BTK-high cell lines (Table 2).These four compounds were inactive in A549, HCT116, U2OS, JURKAT, and non-malignant cells.Of all the 10 compounds, four compounds, 9b, 9f, 9g, and 9h, showed activity in RAMOS cells.
The isopropyl group in compound 9a attached to the piperazine moiety shows no activity.Replacing it with cyclohexyl moiety in compound 9b showed promising activity in RAMOS cells with an IC 50 value of 3.04 ± 0.56 μM.Compounds 9c, 9d, and 9e, having cyclopropyl, benzoyl, and 3-chlorobenzoyl groups attached to the piperazine moiety, did not show any activity in RAMOS cells.Compounds 9f and 9g having 3-bromobenzoyl and 3-fluorobenzoyl groups attached to the piperazine moiety showed promising activity in RAMOS cells with IC 50 values of 2.06 ± 0.43 μM and 2.09 ± 0.47 μM, respectively.Compound 9h with aliphatic group replacement showed promising activity in RAMOS cells with an IC 50 value of 2.75 ± 0.80 μM.Compounds 9i and 9j with pyridine and trifluoro benzoyl moieties attached to piperazine showed no activity in RAMOS cells.The SAR profiling indicates that group cyclopentylidene at the C3 position in compounds 9b, 9f, 9g, and 9h and cyclohexyl in 9b, 3-bromobenzoyl in 9f, 3fluorobenzoyl in 9g, and valeryl in 9h attached to the carbonyl (C�O) group are essential for biological activity in RAMOS cells (Table 2).Compound 9b showed weak activity in CCRF-CEM cells (IC 50 = 35.53± 7.05 μM).Compounds 9g and 9h showed weak activity in CCRF-CEM cells (9g, IC 50 = 46.46 ± 5.81 μM; 9h, IC 50 = 46.15± 3.13 μM).Compound 9j showed moderate activity in U2OS cells (IC 50 = 23.62 ± 6.44 μM), possibly due to non-specific activity.
We also assessed the cytotoxic effects of ibrutinib on our cell line panel.Compared to the derivatives we synthesized, ibrutinib exhibited significant cytotoxic activity against RAMOS cells (IC 50 = 0.29 ± 0.04), in addition to its activity against BTK-null and ITK-positive cancer cells and nonmalignant fibroblast lines (Table 2).This is not surprising given the broad range of kinases inhibited by ibrutinib.However, except for 9j, none of our synthesized compounds that were effective against BTK-high RAMOS cells displayed any toxicity in both malignant and non-malignant cell lines lacking BTK.This data shows the selective cytotoxicity of our derivates on BTK-high cancer cells.

■ IN VITRO PHARMACOLOGICAL PROPERTIES
We next subjected 9b, 9f, and 9h to in vitro ADME analyses.Results showed that the three selected are stable in the presence of plasma proteins and bind to proteins with >85% affinity (Table 3).However, 9b is metabolized quickly by microsomes, suggesting a high probability that the compound will be primarily metabolized in the liver.Compared to 9b, the intrinsic clearance of 9f and 9h was classified as medium in the microsomal stability assay.The passive diffusion mechanism for the three compounds was classified as low to medium by parallel artificial membrane permeability assay (PAMPA).All the three compounds showed poor permeability in assays with MDCK-MDR1 cells, indicating a low potential for penetration through the blood−brain barrier.Furthermore, 9b showed low permeability in Caco-2 cells, suggesting that it is unsuitable for oral administration due to poor intestinal absorption.However, the medium permeability of 9f and 9h indicates a relatively good human intestinal permeability, suggesting that these compounds are suitable for oral administration.

COMPOUNDS
The effects of 9b, 9f, and 9h at three concentrations were next examined on the activity of BTK tyrosine 223 phosphorylation [pBTK (Tyr223)].Although all the three compounds decreased pBTK (Tyr223) levels, the decrease was significant following cell treatment with 9h at 50 μM concentration (Figure 11A,B).We next checked the effect of these compounds on upstream proteins of the BCR signaling pathway, particularly Lyn and Syk, which are important regulators of BTK signaling. 1Lyn phosphorylation was significantly inhibited by only 9b, whereas none of the compounds affected Syk (Figure 11C,D).
Based on these findings, we next selected 9b and 9h for further analysis in RAMOS cells stimulated with lipopolysaccharide (LPS), a well-known inducer of BTK phosphorylation, which subsequently activates downstream MAPK family proteins, including ERK1/2 and p38. 30,31Additionally, it is established that BTK inhibition blocks the activation of downstream MAPK family proteins. 31The western protein analysis showed that LPS stimulation significantly activated pBTK (Tyr223) signaling (Figure 11E,F).This pBTK (Tyr223) activation was significantly inhibited by only compound 9h, as evidenced by the decrease in pBTK, pERK 1/2 (Thr202/Tyr204), and p-p38 (Thr180/Tyr182) levels (Figure 11F).The effect of 9b was only evidenced on p-p38 (Thr180/Tyr182) levels, although there was no significant effect of 9b on pBTK levels, suggesting that this compound might be non-specific in its activity.Overall, these findings, together with cytotoxicity data, underscore the potency of compound 9h as a significant inhibitor of pBTK activity.

■ CONCLUSIONS
As an initial effort, we screened the zinc database using oxindole as a core moiety to identify new BTK inhibitors.One compound with good binding energy with BTK protein and good ADME properties was chosen to design a focused library.Compound 819 was taken as the lead and further modified for better interactions with the BTK protein, and 10 analogous (9a−9b) were synthesized.The cytotoxic activity of the compounds was examined in a panel of cancer and non-cancer cell lines.Notably, four molecules, 9b, 9f, 9g, and 9h, exerted good anticancer activity in the micromole range in BTK-high RAMOS lymphoma cells.MD simulations for compounds 9f, 9g, and 9h were conducted, and the RMSF values were below 2 Å.The RMSD and the ligand-RMSF percentage for Cα indicated the stability of compounds 9f, 9g, and 9h with 5P9J, and the protein-bound conformations were confirmed by torsional analysis.Compound 9h showed more protein−ligand contacts in the simulations.All the three compounds exhibited low permeability in assays conducted with MDCK-MDR1 cells, indicating limited potential for crossing the blood−brain barrier.Compound 9b displayed low permeability in Caco-2 cells, suggesting that it may not be suitable for oral administration due to poor intestinal absorption.However, the medium permeability observed for 9f and 9h in Caco-2 cells suggests that these compounds have relatively good human intestinal permeability, indicating that they are suitable for oral administration.The antiproliferative activity of 9h corresponded to its pBTK (Tyr223) inhibitory activity in RAMOS cells.Moreover, this compound did not affect Lyn and Syk, two proteins upstream of BTK in the BCR signaling pathway, suggesting that the anti-pBTK activity of 9h is due to its activity on BTK.According to our present findings, 9h exhibits promising specificity and efficacy in modulating BTK signaling pathways, warranting further investigation.

■ MATERIALS AND METHODS
Selection of the Target Molecule.The crystal structure of BTK [Protein Data Bank (PDB) ID: 5P9J, resolution: 1.08 Å, R-value free: 0.224, and R-value work: 0.204, no mutations] was obtained from RCSB PDB. 32By removing its cocrystallized ligand and water molecules and adding missing residues, the protein was prepared using Swiss PDB Viewer.The SDF files from the ZINC15 database were used as they are for virtual screening.The ligand preparation for the designed molecules was drawn in ChemDraw and saved as SDF files.The schematic representation of the workflow is shown in Figure 3.
Structure-Based Pharmacophore Selection.Oxindole was taken as the core moiety to search the molecules from the ZINC15 database, where around 550 molecules were selected and docked with the BTK protein (5P9J) using the PyRx Virtual screening tool. 33,34The top 19 molecules were selected, considering docking scores above 10 kcal/mol as criteria.From those 19 molecules, we have designed new molecules based on the pharmacophoric features, which play an essential role in the macromolecule ligand recognition and biological activity shown in Figure 4.
Pharmacokinetics and Drug-Likeness Prediction.The compounds finalized after docking using the PyRx Virtual screening tool were further proceeded to predict their pharmacokinetics and drug-likeness.The physicochemical properties, pharmacokinetics, Log P, water solubility, and AMES toxicity were predicted by Swiss ADME.The druglikeness properties were checked with Lipinski violations using Swiss ADME 35,36 (Table 1).
MD Simulation.The MD studies were conducted for the complex structures of the 5P9J protein with selected compounds 9f, 9g, and 9h using Desmond Software Release 2018-4 for academic licensing (Schrodinger, LLC, New York, NY, USA) to check the stability of binding for all the complexes. 37The simulations used the 0.15 M NaCl and SPC water model to mimic a physiological ionic concentration.Energy minimization was conducted for 100 ps.The MD simulations were run for 20 ns at 300 K and standard pressure (1.01325 bar), with a dimension buffer of 10 Å × 10 Å × 10 Å with an orthorhombic box and an NPT ensemble.The energies were recorded at intervals of 1.2 ps.The MD-simulated net charge system of the protein−ligand complex was neutralized by adding Na + or Cl − ions.The Nose−Hoover chain and Martyna−Tobias−Klein algorithms were used to maintain the temperature of all MD systems at 300 K and pressure at 1.01325 bar.
Chemistry.All the solvents and reagents used for synthesis were purchased from commercial sources (Sigma-Aldrich, Avra, TCI).All reactions were observed by thin-layer chromatography using Merck classic aluminum silica plates with a thickness of 200 μm, size 20 × 20 cm, and were checked in Ultraviolet−visible spectroscopy at 254 nm.All compounds were purified using column chromatography with silica gel (60−100#) as the stationary phase.Proton 1 H and 13 C NMR spectra were recorded on an SA-AGILENT 400 MHz NMR and an (Ascend) AVANCE NEO 600 MHz FT-NMR spectrometer.Proton NMR chemical shifts are reported using tetramethylsilane (TMS) as a standard reference in parts per million (δ).ESI spectra were recorded on Micro mass, Quattro LC using ESI+ software with a capillary voltage of 3.98 kV and an ESI mode positive-ion trap detector.IR spectra were recorded on an FT-IR spectrometer (Shimadzu FT-IR 8300 spectrophotometer), and peaks were reported in cm −1 .Melting points were measured in degrees centigrade (°C) using a MP apparatus and reported.

Figure 2 .
Figure 2. Structures of commercially marketed drugs containing oxindole as the core moiety approved for treating canine and human tumors, Parkinson's disease in humans, and heart conditions in dogs, hypertension, schizophrenia, and bipolar disorder.

Figure 3 .
Figure 3. Schematic representation of workflow of the study.

Figure 4 .
Figure 4. Structure-based pharmacophore modeling of compound 819 (A) to 9g (B) for better interaction; the blue color ring shows the replacement of methyl benzyl with 3-fluoro benzoyl, the pink color ring shows the replacement of the methyl group with hydrogen, and the red color ring shows the replacement of the methyl group with hydrogen.(A) Interaction profile of compound 819.(B) Interaction profile of 9g.Scheme 1. Synthesis of 5-Substituted Oxindole Derivatives; Conditions: (i) ClSO 3 H, 70 °C, 2 h; (ii) Pyridine, 1,4-Dioxane, rt, 2 h; (iii) Piperazine, EtOH, rt, 2 h; (iv) 4 M HCl in 1,4-Dioxane, rt, 16 h; and (v) DIPEA, CH 2 Cl 2 , rt, 3 h and S2A,B, respectively.The protein-RMSF was monitored to analyze the local changes along the protein chain.The ligand-RMSF was examined to study the fluctuations at the atom level, as shown in Figure 10A.The brown line indicates "Fit on Protein", and the pink line indicates "Fit on Ligand".Compound 9h interacted with GLY-412 and MET-477, making hydrogen bonds without water.In

Table 1 .Figure 5 .
Figure 5. Schematic representation of the interaction of BTK with 9g.

Figure 9 .
Figure 9. (A) RMSD plot of protein (5P9J) and ligand (9h).(B) SSE of the protein is shown with helices in blue and beta strands in orange.

Figure 10 .
Figure 10.(A) Ligand-RMSF plot for compound 9h-protein 5P9J, where the brown line indicates the ligand fluctuations regarding the binding site residues on the target protein and the pink line indicates the fluctuations where the ligand in each frame was aligned on the ligand in the first reference frame.(B) Compound 9h shows interacting residues.

Figure 11 .
Figure 11.Compound effect on BTK signaling.(A) RAMOS cells were treated with 9b, 9f, and 9h for 24 h at indicated concentrations, and whole protein extracts were probed for phosphorylated and total BTK, ERK, and p38 by western blotting.(B) Ratio of pBTK/total BTK, pERK/total ERK, and p-p38/total p38 band intensities.Mean ± SEM, n = 2−3, one-way ANOVA, Dunnett's multiple comparison test.(C) Effect of 9b, 9f, and 9h on Lyn and Syk phosphorylation in RAMOS cells after 24 h of treatment.(D) Ratio of pLyn/total Lyn and pSyk/total Syk bands shown in panel C. Mean ± SEM, n = 2, one-way ANOVA, Dunnett's multiple comparison test.(E) RAMOS cells were stimulated with LPS for 10 min and then treated with 9b and 9h at 50 μM concentration for 3 h, and changes in BTK, ERK1/2, and p38 phosphorylation were probed by western blotting of the whole protein extract.(F) Ratio of pBTK/total BTK, pERK/total ERK, and p-p38/total p38 bands shown in panel E. Mean ± SEM, n = 2−3, one-way ANOVA, Dunnett's multiple comparison test.Images of all uncropped blots are shown in the Supporting Information (Figure S17).

Table 2 .
Biological Activity of 10 Compounds in ITK/BTK-Negative, ITK-Positive, and BTK-Positive Cancer Cell Lines and Non-malignant Fibroblast Lines a a IC 50 values are in μM.Mean ± SD, n ≥ 6. Representative dose−response curves of 9b, 9f, 9g, 9h, and 9j are shown in the Supporting Information.

Table 3 .
Pharmacological Properties of 9b, 9f, and 9h Determined by In Vitro and In Vivo ADME Assays