Fig 1.
Reagents and conditions: (A) Synthesis of MKP101-104, MKP106-117, MKP122: (a) 5-Aminoindoles (0.8–1.0 equiv.), Et3N (0.8–3.0 equiv.), isopropanol, rt, 1–10 h, or 5-hydroxyindole (0.77 equiv.), DBU (1.54 equiv.), MeCN, rt, 1 h; (b) CH3I (1.0–1.5 equiv.), NaH (or Cs2CO3) (1.0–1.2 equiv.), DMF, 0°C (or rt), 1 h; (c) ArNH2 (0.9–1.1 equiv.), 1-butanol, microwave irradiation, 200°C, 30 min. (B) Synthesis of MKP105: (a) 6-Aminoindole (1.5 equiv.), MeOH / H2O (1:3), rt, overnight; (b). CH3I (1.0 equiv.), NaH (1.0 equiv.), DMF, -10°C, 2h; (c) 5-Amino-2-methylbenzenesulfonamide (0.9–1.1 equiv.), 1-butanol, microwave 200°C, 30min. (C) Synthesis of MKP118-121, MKP123: (a) 5-Aminoindole (1.0 equiv.), Et3N (1.0 equiv.), isopropanol, rt, 2 h or 5-hydroxyindole (1.2 equiv.), DBU (2.0 equiv.), MeCN, rt, 1 h; (b) CH3I (1.1 equiv.), NaH (1.1 equiv.), DMF, 0°C rt, 1h; (c) ArNH2 (0.9–1.1 equiv.), 1-butanol, microwave irradiation 200°C, 30 min.
Fig 2.
Structure of pazopanib and its indole derivative MKP101.
Fig 3.
MKP101 inhibits proliferation of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-sensitive non-small cell lung cancer (NSCLC) cells.
(A) Inhibition of HCC827 cell proliferation. Cells were incubated for 72 h with a range of concentrations of MKP101, and cell viability was measured using the WST-1 assay. The IC50 value was 160 nM. (B) Inhibition of EGFR phosphorylation in HCC827 cells. Western blot analysis using antibodies to phosphorylated and total EGFR from a representative experiment is shown. Cells were incubated for 3 h at the indicated concentration. (C) Graph showing the relative intensities of the total and phosphorylated EGFR as determined by band densitometry. IC50, half-maximal inhibitory concentration.
Table 1.
Half-maximal inhibitory concentration (IC50) values (nM) of MKP101 for human kinases.
Table 2.
Kinase profile of MKP101 (1 μM) for the 40 kinases*.
Fig 4.
Pazopanib and MKP101 have similar inhibitory effects on VEGF-induced angiogenesis in HUVECs.
(A) Cytotoxicity of pazopanib and MKP101 in HUVECs. No cytotoxicity was observed at 1 μg/mL pazopanib or 1 μg/mL MKP101. (B-D) Pazopanib and MKP101 block VEGF (50 ng/ml)-induced increases in endothelial proliferation (B), tube formation (C), and migration (D). Cell proliferation and tube formation experiments were performed using 1 μg/mL of pazopanib and 1 μg/mL MKP101. The scratch wound migration assay was performed using 0.5 μg/mL pazopanib and 0.5 μg/mL MKP101. In (A) and (B), the number of cells was expressed as the fold change with respect to the number of cells seeded at day 0. Tube formation responses were compared by normalizing the values relative to those of the corresponding PBS control samples (*p < 0.05 vs. PBS; #p < 0.05 vs. VEGF, mean ± SEM). Scale bar = 100 μm.
Fig 5.
Predicted docking orientation of MKP101 in the epidermal growth factor receptor (EGFR) kinase domain.
The binding poses of (A) MKP101 (carbon atoms in green) and (B) pazopanib (carbon atoms in orange) in the human EGFR kinase domain were compared. The structure of co-crystallized TAK-285 is shown as a reference (carbon atoms in off-white). Hydrogen bonds are displayed as dashed lines. The lipophilic potential surface of the ATP-binding site of EGFR was created using the MOLCAD implemented in Sybyl-X 2.0. A 2D-interaction diagram of the binding model of (C) MKP101 and (D) pazopanib was generated, which displayed amino acid residues within 4.0 Å of the ligand. Acidic, hydrophobic, basic, polar, and other residues at the active site are represented by red, green, purple, blue, and gray spheres, respectively. Hydrogen bonds between the ligand and the backbone are shown in dashed pink lines. The π-π stacking interaction is shown with a green line. The docking models show that MKP101 occupies the ATP-binding site in a manner similar to TAK-285, and the indole ring of MKP101 interacts with the backbone of the Phe856 by hydrogen bonding. However, as expected, pazopanib did not fit well at the ATP binding site. 2D, 2-dimensional; ATP, adenosine triphosphate.
Fig 6.
Structures of MKP102, MKP103, MKP104, and MKP105.
MKP102 and 103 are derivatives that possess a methyl group at the C-3 and C-2 positions of the indole ring, respectively. MKP104 is a derivative with a methyl group on the indole nitrogen. MKP105 is a regioisomer of MKP101.
Table 3.
Activity of MKP compounds for EGFR and HCC827 cells.
Fig 7.
Comparison of the binding configurations of the MKP101 analogues.
(A) MKP102, (B) MKP103, (C) MKP104, and (D) MKP105 (carbon atoms in green) with TAK-285 (carbon atoms in ivory). Hydrogen bonds are displayed as dashed lines. The lipophilic potential surface of the ATP-binding site of EGFR was created using the MOLCAD implemented in Sybyl-X 2.0. In the 2D-interaction diagram, acidic, hydrophobic, basic, polar, and other residues at the active site are represented by red, green, purple, blue, and gray spheres, respectively. Hydrogen bonds between the ligand and backbone are shown in dashed pink lines. The π-π stacking interaction is shown with a green line. Similar to MKP101, the 3-methyl indole moiety of MKP102 occupies a lipophilic pocket that forms a direct hydrogen bond with the backbone of Phe856. However, MKP103 the 2-methyl indole derivative, cannot fit into the lipophilic pocket because of the steric hindrance of the methyl group. The N-methyl indole MKP104 and the 6-amino indole MKP105 lost hydrogen bonding with Phe856 owing to the structural change. ATP, adenosine triphosphate; EGFR, epidermal growth factor receptor; 2D, 2-dimensional.
Fig 8.
Structures of the indole tethered pyrimidine derivatives.
Table 4.
Half-maximal inhibitory concentration (IC50) values of pyrimidine derivatives MKP106–123 against epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR)-2.
Table 5.
Kinase profiles of selected compounds.
Fig 9.
Comparison of the binding poses of MKP123 and MKP122 with MKP101.
Binding poses of (A) MKP123 (carbon atoms in green) and (B) MKP122 (carbon atoms in green) with MKP101 (carbon atoms in ivory). The lipophilic potential surface of the ATP-binding site of EGFR was created using the MOLCAD implemented in Sybyl-X 2.0. 2D-interaction diagram of the binding model of (C) MKP123 and (D) MKP122 displaying the amino acid residues within 4.0Å of the ligand. Acidic, hydrophobic, basic, polar, and other residues at the active site are represented by red, green, purple, blue, and gray spheres, respectively. The hydrogen bond between the ligand and backbone is shown in dashed pink lines. The π-π stacking interaction is shown with a green line. Unlike MKP101, the indole rings of MKP123 and MKP122 occupy a lipophilic pocket without a hydrogen bond with the backbone of Phe856, and their aniline moiety was docked in the hinge region. ATP, adenosine triphosphate; EGFR, epidermal growth factor receptor.