Synthesis, anticancer activity, and molecular modeling of 1,4-naphthoquinones that inhibit MKK7 and Cdc25

Abstract

Cell division cycle 25 (Cdc25) and mitogen-activated protein kinase kinase 7 (MKK7) are enzymes involved in intracellular signaling but can also contribute to tumorigenesis. We synthesized and characterized the biological activity of 1,4-naphthoquinones structurally similar to reported Cdc25 and(or) MKK7 inhibitors with anticancer activity. Compound 7 (3-[(1,4-dioxonaphthalen-2-yl)sulfanyl]propanoic acid) exhibited high binding affinity for MKK7 (K_d = 230 nM), which was greater than the affinity of NSC 95397 (K_d = 1.1 μM). Although plumbagin had a lower binding affinity for MKK7, this compound and sulfur-containing derivatives 4 and 6-8 were potent inhibitors of Cdc25A and Cdc25B. Derivative 22e containing a phenylamino side chain was selective for MKK7 versus MKK4 and Cdc25 A/B, and its isomer 22f was a selective inhibitor of Cdc25 A/B. Docking studies performed on several naphthoquinones highlighted interesting aspects concerning the molecule orientation and hydrogen bonding interactions, which could help to explain the activity of the compounds toward MKK7 and Cdc25B. The most potent naphthoquinone-based inhibitors of MKK7 and/or Cdc25 A/B were also screened for their cytotoxicity against nine cancer cell lines and primary human mononuclear cells, and a correlation was found between Cdc25 A/B inhibitory activity and cytotoxicity of the compounds. Quantum chemical calculations using BP86 and ωB97X-D3 functionals were performed on 20 naphthoquinone derivatives to obtain a set of molecular electronic properties and to correlate these properties with cytotoxic activities. Systematic theoretical DFT calculations with subsequent correlation analysis indicated that energy of the lowest unoccupied molecular orbital E(LUMO), vertical electron affinity (VEA), and reactivity index ω of these molecules were important characteristics related to their cytotoxicity. The reactivity index ω was also a key characteristic related to Cdc25 A/B phosphatase inhibitory activity. Thus, 1,4-naphthoquinones displaying sulfur-containing and phenylamino side chains with additional polar groups could be successfully utilized for further development of efficacious Cdc25 A/B and MKK7 inhibitors with anticancer activity.

Keywords: Cdc25 phosphatase; Cytotoxicity; DFT analysis; Kinase inhibitor; Mitogen-activated kinase kinase 7; Molecular docking; Naphthoquinone.

Fig. 1.

Docking poses of selected naphthoquinones in the MKK7 binding site. Superimposition of docking poses of 7 (red), 22e (magenta), and 22f (violet) (Panel A). Separate poses are shown for compounds 22e (Panel B), and 22f (Panel C). For all panels, except Panel A, residues within 3 Å of each pose are visible. For Panel A, residues within 6 Å of the cavity are visible. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2.

Inhibition of Cdc25B by plumbagin. Cdc25B was incubated with the indicated concentrations of plumbagin, and cleavage of the fluorogenic phosphatase substrate (3-O-methyl fluorescein phosphate) was monitored, as described under Materials and Methods. The data are presented as the mean ± S.D. of triplicate samples from a representative experiment of two independent experiments.

Fig. 3.

Docking poses of plumbagin (compound 2) in the Cdc25B binding site. Residues within 4 Å of the pose are shown.

Fig. 4.

Docking poses of naphthoquinones 22e and 22f in the Cdc25B binding site. For both panels, residues within 4 Å of each pose are shown.

Fig. 5.

Anticancer cell cytotoxicity of plant-derived naphthoquinones shikonin (1) and plumbagin (2). HL60 cells were incubated with the indicated naphthoquinones for 24h, and cell viability was measured, as described. The data are presented as the mean ± S.D. of triplicate samples from a representative experiment of three independent experiments.

Scheme 1.

Reported naphthoquinone-based sulfur-containing analogs and amino derivatives with Cdc25 inhibitory activity [14,17,38].

Scheme 2.

Reagents and conditions for compound synthesis. (a) 22a-f: CaCO₃, 50% EtOH, boiling, 10 h, 80% yield; (b) 22g, 23a, b: CH₃COONa, 50% EtOH, boiling, 12 h, 80% yield; (c) 24: 2 mol. 3-morpholin-4-yl-propylamine, EtOH, boiling, 16 h, 95% yield; (d) 25a: 12 mol piperidine, CH₃OH, boiling, 20 h, 51% yield; (e) 25b: 2 mol CH₃ONa, CH₃OH, boiling, 4 h, 64% yield.