Insights on Cancer Cell Inhibition, Subcellular Activities, and Kinase Profile of Phenylacetamides Pending 1 H-Imidazol-5-One Variants

Abstract

Structural changes of small-molecule drugs may bring interesting biological properties, especially in the field of kinase inhibitors. We sought to study tirbanibulin, a first-in-class dual Src kinase (non-ATP competitive)/tubulin inhibitor because there was not enough reporting about its structure-activity relationships (SARs). In particular, the present research is based on the replacement of the outer ring of the biphenyl system of 2-[(1,1'-biphenyl)-4-yl]-N-benzylacetamide, the identified pharmacophore of KX chemotype, with a heterocyclic ring. The newly synthesized compounds showed a range of activities in cell-based anticancer assays, agreeing with a clear SAR profile. The most potent compound, (Z)-N-benzyl-4-[4-(4-methoxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-1H-imidazol-1-yl]phenylacetamide (KIM-161), demonstrated cytotoxic IC₅₀ values at 294 and 362 nM against HCT116 colon cancer and HL60 leukemia cell lines, respectively. Profiling of this compound (aqueous solubility, liver microsomal stability, cytochrome P450 inhibition, reactivity with reduced glutathione, and plasma protein binding) confirmed its adequate drug-like properties. Mechanistic studies revealed that this compound does not depend on tubulin or Src kinase inhibition as a factor in forcing HL60 to exit its cell cycle and undergo apoptosis. Instead, KIM-161 downregulated several other kinases such as members of BRK, FLT, and JAK families. It also strongly suppresses signals of ERK1/2, GSK-3α/β, HSP27, and STAT2, while it downregulated AMPKα1 phosphorylation within the HL60 cells. Collectively, these results suggest that phenylacetamide-1H-imidazol-5-one (KIM-161) could be a promising lead compound for further clinical anticancer drug development.

Keywords: imidazolone; leukemia; multi-kinase downregulation; phosphokinase profiling; scaffold hopping; src kinase; structure–properties relationship; tirbanibulin.

FIGURE 1

Rational design of imidazolone analogs of KX chemotype. (A) Examples of ATP-competitive kinase inhibitors with altered kinase specificity after minor structural changes. Replacing 3-cyanoquinoline with quinazoline caused a shift in kinase inhibition specificity from Src to VEGFR. The benzoyl moiety in midostaurin conferred some specificity toward mutated FLT3 kinase compared to the non-specific staurosporine. (B) The black motif is considered a fixed part, common among the prototypes (KX1-136 and KX2-391) and the newly designed compounds. The red parts are variables.

SCHEME 1

Conditions: (A) acetic anhydride, sodium acetate, heat; (B) pyridine, heat; (C) acetonitrile, heat.

FIGURE 2

Dose–response curve of 4c (KIM-161) against (A) solid tumor cell lines and (B) leukemia cell lines.

FIGURE 3

Effect of KIM-161 (1 µM) on HL60 cell cycle compared to untreated cells (control) for 48 h.

FIGURE 4

Caspase-3 activity profile after treatment of HL60 cells with KIM-161 (1 µM) compared to untreated cells (control) for 48 h.

FIGURE 5

The LFC heat map for peptides shows the values of differentially inhibited phosphorylated peptides (KIM-161 vs. DMSO control). The treatment effects are log-ratios [log2(treatment) − log2(control)]. Black are peptides which did not pass the threshold of inhibition. DMSO concentration 2% v/v; compound concentration log −9 = 0.001 μM; log −8 = 0.01 μM; log −7 = 0.1 μM; log −6 = 1 μM; log −5 = 10 μM, and log −4.3 = 50 μM).

FIGURE 6

Mean kinase statistics (represented by bar length) and mean specificity score (represented by color scheme) for the top 20 affected PTKs after treatment of HL60 cells with KIM-161 at 0.1 µM.

FIGURE 7

Kinome tree: The top predicted kinases that have been changed after treatment of HL60 cells with KIM-161 are represented on the phylogenetic tree of the human protein kinase family. The size of the dot indicates total kinase score, and the color denotes kinase statistics (red: higher in on-chip–treated lysates, blue: lower in on-chip–treated lysate). Kinase statistics cut-off ≤ −1.2 was applied to select top altered.

FIGURE 8

Phosphokinase proteome profile of KIM-161 in HL60 cell line. (A) Cells lysates were prepared after 10 h treatment with KIM-161 and applied to the membranes prespotted with the different antibodies. (B) The bar graph shows variation of phosphokinases in HL60 cells upon KIM-161 exposure relative to the untreated control cells. The height of each bar represents the average intensity of certain protein signals in treated cells/untreated × 100. Therefore, the dotted line represents the control percentage (100%).

FIGURE 9

Tubulin polymerization assay for KIM-161 at 10 and 30 µM compared to paclitaxel (10 µM) and KX2-391 (10 µM) as positive controls. The negative control (vehicle) contained DMSO (1% v/v). The effect on the tubulin assembly was monitored turbidometrically at 340 nm every 30 s for 60 min at 37°C. Fold inhibition of tubulin polymerization was calculated using the V _max value for each reaction. The results represent the mean for three separate experiments.

FIGURE 10

Effect of antioxidant pretreatment on viability of HCT116 treated with KIM-161. Cells were pretreated with NAC (5 mM) for 1 h, followed by KIM-161 at concentrations 0.5, 1.0, and 5.0 µM for 48 h. Cell viability was expressed as a percentage of vehicle control and was measured by the MTT assay (average of two independent experiments). Statistical significance was determined by two-way ANOVA using Šídák’s multiple comparisons test. * = p < 0.05; *** = p ≤ 0.001; **** = p < 0.0001.