Synthesis, Characterisation and Mechanism of Action of Anticancer 3-Fluoroazetidin-2-ones

Abstract

The stilbene combretastatin A-4 (CA-4) is a potent microtubule-disrupting agent interacting at the colchicine-binding site of tubulin. In the present work, the synthesis, characterisation and mechanism of action of a series of 3-fluoro and 3,3-difluoro substituted β-lactams as analogues of the tubulin-targeting agent CA-4 are described. The synthesis was achieved by a convenient microwave-assisted Reformatsky reaction and is the first report of 3-fluoro and 3,3-difluoro β-lactams as CA-4 analogues. The β-lactam compounds 3-fluoro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxy phenyl)azetidin-2-one 32 and 3-fluoro-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) 33 exhibited potent activity in MCF-7 human breast cancer cells with IC₅₀ values of 0.075 µM and 0.095 µM, respectively, and demonstrated low toxicity in non-cancerous cells. Compound 32 also demonstrated significant antiproliferative activity at nanomolar concentrations in the triple-negative breast cancer cell line Hs578T (IC₅₀ 0.033 μM), together with potency in the invasive isogenic subclone Hs578Ts(i)8 (IC₅₀ = 0.065 μM), while 33 was also effective in MDA-MB-231 cells (IC₅₀ 0.620 μM). Mechanistic studies demonstrated that 33 inhibited tubulin polymerisation, induced apoptosis in MCF-7 cells, and induced a downregulation in the expression of anti-apoptotic Bcl2 and survivin with corresponding upregulation in the expression of pro-apoptotic Bax. In silico studies indicated the interaction of the compounds with the colchicine-binding site, demonstrating the potential for further developing novel cancer therapeutics as microtubule-targeting agents.

Keywords: Reformatsky reaction; antiproliferative activity; breast cancer; combretastatin A-4; fluorinated β-lactams; inhibition of tubulin polymerisation.

Figure 1

Colchicine (1), combretastatins CA-1 (2a), CA-1P (2b), CA-2 (2c), CA-3 (2d), CA-4 (2e), CA-4P (fosbretabulin) (2f), ombrabulin (2g), CB-1 (3a) and CB-2 (3b).

Figure 2

Microtubule-targeting agents 4–15 and target structures.

Scheme 1

Synthesis of 2-azetidinones 26–45. Reagents and conditions: (a) EtOH, reflux, 4 h, 75–91%; (b) TBDMSCl, DBU, CH₂Cl₂, 20 °C, until reaction complete as indicated by TLC, 52%; (c) Zn dust, TMSCl, 40 °C, 15 min, then 100 °C, 2 min, microwave, C₆H₆, 100 °C, 30 min, microwave (26–31, 33–35, 6–58%; 36–41, 43–45, 13–65%); (d) TBAF, THF, 0 °C, 15 min (32, 18%; 42, 21%). Products obtained as racemic mixtures, one enantiomer illustrated.

Figure 3

Preliminary cell viability data for 3-fluoro and 3,3-difluoro β-lactam compounds. (A) 3-Fluoro β-lactam compounds containing different para-substituent ring B 26–29; 3-fluoro β-lactam compounds containing different meta-substituent ring B 30, 32–35; (B) 3-difluoro β-lactam compounds containing different para-substituent ring B 36–39; 3-difluoro β-lactam compounds containing different meta-substituent ring B 40, 42–45 in MCF-7 breast cancer cells. Cell proliferation of MCF-7 cells was determined with an AlamarBlue assay (seeding density 2.5 × 10⁵ cells/mL per well for 96-well plates). Compound concentrations of either 10 μM or 1 μM for 72 h were used to treat the cells with control wells containing vehicle ethanol (1% v/v) and CA-4 (10 μM and 1 μM). The mean value for three experiments is shown with the ± S.E.M. for three independent experiments.

Figure 4

Antiproliferative activity of 3-fluoro β-lactams on MCF-7, MDA-MB-231, Hs578T and Hs578T isogenic subclone Hs578T(i)8 breast cancer cells and non-tumourigenic HEK-293T cells. (A) Antiproliferative activity for 3-fluoro β-lactams 26, 32, 33, 42 and CA-4 in MCF-7 cells. Cells were grown in 96-well plates and treated with indicated β-lactam compounds at 0.01–50 μM for 72 h. Cell viability was expressed as a percentage of vehicle control (ethanol 1% (v/v))-treated cells. The values represent the mean ± S.E.M. for three independent experiments performed in triplicate. (B) Antiproliferative activity of 3-fluoro β-lactam 33 in MCF-7 and MDA-MB-231 cells. (C) Antiproliferative activity of 3-fluoro β-lactam 33 in triple-negative breast cancer cell line Hs578T and its isogenic subclone Hs578T(i)8. (D) Effect of compound 33 on viability of MCF-7 and non-tumourigenic HEK-293T cells. Cells were grown in 96-well plates and treated with compound 33 at 1, 10 and 50 μM for 72 h. Cell viability was expressed as a percentage of vehicle control (ethanol 1% (v/v))-treated cells and was measured by AlamarBlue assay (average of three independent experiments). Two-way ANOVA (Bonferroni post-test) was used to test for statistical significance (***, p < 0.05).

Figure 5

Compound 33 induces apoptosis in MCF-7 breast cancer cells. MCF-7 breast cancer cells were treated with 33 (0.1 and 0.5 µM), CA-4 (50 nM) or vehicle control (0.1% ethanol (v/v)) for 48 h. The percentage of apoptotic cells was determined by staining with Annexin V–FITC and PI. In each panel, the lower left quadrant shows cells that are negative for both PI and Annexin V–FITC, and the upper left shows cells that are only negative PI, which are necrotic. The lower right quadrant shows Annexin-positive cells which are in the early apoptotic stage, and the upper right shows both Annexin- and PI-positive cells, which are in late apoptosis.

Figure 6

β-Lactam 33 decreases the expression of anti-apoptotic proteins Bcl-2 and survivin and increases the expression of pro-apoptotic protein Bax in MCF-7 cells. MCF-7 cells were treated with vehicle control (ethanol 0.1% v/v) or 21 at the indicated concentrations (0.05, 0.1 or 0.5 μM) for 48 h (left) or 72 h (right). Then, the cells were harvested for Western blot analysis to detect the level of the apoptosis-related proteins. Results are indicative of three separate experiments, performed independently. To confirm equal protein loading, each membrane was stripped and re-probed with GAPDH antibody.

Figure 7

Effect of β-lactam compound 33 (10 and 30 µM) and paclitaxel (10 µM) on in vitro tubulin polymerisation. Purified bovine tubulin and GTP were mixed in a 96-well plate. Compounds were added and the reaction was started by warming the solution from 4 to 37 °C. Ethanol (1% v/v) was used as a vehicle control. The effect on tubulin assembly was monitored in a Spectramax 340PC spectrophotometer at 340 nm at 30 s intervals for 30 min at 37 °C. The graph shows one representative experiment. Each experiment was performed in duplicate.

Figure 8

Compound 33 acts as depolymeriser of the microtubule network of MCF-7 breast cancer cells. Cells were treated with vehicle control (0.1% ethanol (v/v)), CA-4 (0.01 μM), paclitaxel (1 μM) or compound 33 (0.1, 0.5 and 1 μM) for 16 h. Cells were fixed in ice-cold methanol and stained with mouse monoclonal anti-α-tubulin–FITC antibody (clone DM1A) (green) and Alexa Fluor 488 dye and counterstained with DAPI (blue). Images were obtained with Leica SP8 confocal microscopy with Leica Application Suite X software, (LAS V 4.13), Wetzlar, Germany. Representative confocal micrographs of three separate experiments are shown. White scale bar indicates 50 μM.

Figure 9

Overlay of the X-ray structure of tubulin co-crystallised with DAMA-colchicine (PDB entry 1SA0) on the best ranked docked poses of the S enantiomers of β-lactams: (A) 32 (3S,4S), (B) 33 (3S,4S), (C) 42 (4S) and (D) 43 (4S). Ligands are rendered as tubes and amino acids as lines. Tubulin amino acids and DAMA-colchicine are coloured by atom type, the novel β-lactam compounds are coloured with a green backbone. The atoms are coloured by element type; carbon = grey, hydrogen = white, oxygen = red, nitrogen = blue, sulphur = yellow, fluorine = green. Key amino acid residues are labelled and multiple residues are hidden to enable a clearer view.