Investigation on Human Carbonic Anhydrase IX and XII Inhibitory Activity and A549 Antiproliferative Activity of a New Class of Coumarinamides

Abstract

Background-Aggressive solid tumors are commonly characterized by both basic intracellular pH and acidic extracellular pH, which increase cell survival and proliferation. As carbonic anhydrases IX/XII are involved in this pH regulation, their inhibition is an appealing approach in cancer therapy, avoiding cancer cell survival and proliferation. Substituted coumarins are selective non-classical CA IX and CA XII inhibitors. Methods-In this study, new 7-hydroxycoumarinamides were synthesized and assayed for CA inhibition and antiproliferative activity. Results-All of the coumarinamides showed human CA IX and CA XII selective inhibition over the off-target CA I and CA II isoforms. Coumarin acts as a suicide inhibitor because its heterocyclic ring can be hydrolyzed by CA esterase activity to give the corresponding 2-hydroxycinnamic acid derivative which blocks the entrance of the active site. The 2-hydroxycinnamic acid derivatives deriving from the most potent and selective coumarinamides were docked into CA IX and XII to better understand the activity and selectivity against the two CA isoforms. The most active coumarinamides also produced a decrease of A549 cell proliferation and were able to arrest cells at the G1/S checkpoint. Conclusions-These results may open new perspectives for developing coumarin-based CA IX/XII inhibitors.

Keywords: amides; carbonic anydrase; coumarins; cytostatic activity; enzyme inhibition.

Figure 1

Hydrolysis of coumarin (A) to 2-hydroxycinnamic acid (B).

Scheme 1

General synthetic procedure for 7-hydroxycoumarin amides 4–45. Reagents and conditions are as follows: (i) CH₃COONH₄, water, reflux 15 h, 87% yield; (ii) substituted benzylamines, EDCI, HOBt, dry MeCN, r.t. 6 h, 44–92% yield; (iii) substituted phenylethylamines, EDCI, HOBt, dry MeCN, r.t. 6 h, 70–90% yield; (iv) substituted piperazines, EDCI, HOBt, dry MeCN, r.t. 6 h, 47–82% yield.

Scheme 2

Hydrolysis of coumarins 7, 9, 23 and 38 to 2,4-dihydroxycinnamic acid amides 7A, 9A, 23A and 38A.

Figure 2

Docking poses of the E/Z isomers of hydrolyzed coumarins 7A, 9A, 23A and 38A into hCA IX. (A) (E)-7A, (B) (Z)-7A, (C) (E)-9A, (D) (Z)-9A, (E) (E)-23A, (F) (Z)-23A, (G) (E)-38A, (H) (Z)-38A.

Figure 3

Docking poses of the E/Z isomers of hydrolyzed coumarins 7A, 9A, 23A and 38A into h-CA XII. (A) (E)-7A, (B) (Z)-7A, (C) (E)-9A, (D) (Z)-9A, (E) (E)-23A, (F) (Z)-23A, (G) (E)-38A, (H) (Z)-38A.

Figure 4

Cell viability of BEAS (a) and (b) A549 cells exposed to increasing concentrations (0–150 µM) of selected compounds (4-7, 9, 19, 22, 23, 25, 29, 30, 32, 38, 44 and 45) for 48 h. (c) Cell viability of BEAS and A549 cells exposed to doxorubicin (0-10 µM) after 48 h of exposure. Bar graphs represent cell viability percentages. The untreated control (CTRL = 0 µM) is set as the 100%. Data are presented as means ± standard deviations obtained from one experiment in triplicates (n = 3). * = p < 0.01, ** = p < 0.001, *** = p < 0.0001, **** = p < 0.00001 comparing treated to the untreated control.

Figure 5

(a) Cytotoxicity occurrence in A549 cells exposed to increasing concentrations (0–150 µM) of compounds 7, 9, 23 and 38 after 24 h. The bar graphs show the amount of lactate dehydrogenase (LDH) released from treated A549 cells as a fold increase respective to that secreted by untreated cells (CTRL = 0 µM) after 24 h of exposure. (b) Light phase-contrast images of A549 cells 48 h after treatment. Magnification 10×. Yellow arrows highlight morphological changes in the cell population. Data are presented as means ± standard deviations obtained from one experiment in triplicates (n = 3). ** = p < 0.001, **** = p < 0.00001 comparing treated to the untreated control.

Figure 6

(a) Cell cycle analysis in A549 cells exposed to increasing concentrations (0–150 µM) of compounds 7, 9, 23 and 38 after 48 h. Data are presented as means ± standard deviations from three independent experiments (n = 3). Bars highlight cell percentages in the various phases of the cell cycle (G1, S, and G2) of A549. * = p < 0.01, ** = p < 0.001, *** = p < 0.0001, **** = p < 0.00001 comparing treated with the untreated control (G1 phase). § = p < 0.01, §§ = p < 0.001, §§§ = p < 0.0001, §§§§ = p < 0.00001 comparing treated with the untreated control (S phase). # = p < 0.01, ## = p < 0.001, ### = p < 0.0001, #### = p < 0.00001 comparing treated with the untreated control (G2 phase). (b) The top panel displays the DNA profile of cells 48 h after treatment. Peaks are generated by the emission of PI in the FL3 fluorescence channel. The bottom panel is a dot plot representing the gating strategy.