Synthesis of 2,2,6-Trisubstituted 5-Methylidene-tetrahydropyran-4-ones with Anticancer Activity

Abstract

In our continuous search for new, relatively simple 2-alkylidene-1-oxoheterocycles as promising anticancer drug candidates, herein we report an efficient synthesis of 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-ones. These compounds were obtained in a four step reaction sequence, in which starting diethyl 2-oxopropylphosphonate was transformed into 2,2-disubstituted 5-diethoxyphosphoryldihydropyran-4-ones, followed by Michael addition of various Grignard reagents and Horner-Wadsworth-Emmons reaction of the obtained adducts with formaldehyde. Stereochemistry of the intermediate Michael adducts is also discussed. Final 5-methylidenetetrahydropyran-4-ones were tested for their possible antiproliferative effect against three cancer cell lines and 6-isopropyl-2,2-dimethyl-5-methylidenetetrahydropyran-4-one (11c), which showed very high cytotoxic activity against HL-60 human leukemia cells and was three times more active than known anticancer drug carboplatin, was selected for further biological evaluation, in order to disclose its mechanism of action. The obtained results indicated that 11c induced apoptosis in HL-60 cells and caused the arrest of the cell cycle in the G2/M phase, which may suggest its cytotoxic and cytostatic activity.

Keywords: Horner-Wadsworth-Emmons olefination; Michael addition; apoptosis; cell cycle; cytotoxic activity; methylidenedihydropyran-4-ones.

Figure 1

Natural compounds containing 3-alkylidenetetrahydropyran-4-one skeleton.

Scheme 1

Synthesis of diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d and 3-diethoxyphosphoryldihydropyran-4-ones 9a–d.

Scheme 2

Synthesis of 6-substituted 5-diethoxyphosphoryldihydropyran-4-ones 10a–p.

Figure 2

Preferred conformations of trans-, cis- and enol-10f and characteristic coupling constants.

Scheme 3

Synthesis of 2,2,6-trisubstituted-5-methylidenetetrahydropyran-4-ones 11a–p.

Figure 3

Influence of 11c on HL-60 cells after 24 h incubation (A): Metabolic activity, (B): DNA damage measured by H2AX phosphorylation changes and (C): apoptosis induction measured by PARP cleavage. (B,C): Results are expressed as mean ± SEM of a triplicate assay; *** p < 0.001, ** p < 0.01.

Figure 4

Effect of 11c on the cell cycle progression in HL-60 cells. (A): Representative flow cytometry results of the cell cycle changes. (B): Percentage quantification of the total cell populations in each of the cell cycle phases expressed as mean ± SEM of a triplicate assay; *** p < 0.001.