Modified carbazoles destabilize microtubules and kill glioblastoma multiform cells

Abstract

Small molecules that target microtubules (MTs) represent promising therapeutics to treat certain types of cancer, including glioblastoma multiform (GBM). We synthesized modified carbazoles and evaluated their antitumor activity in GBM cells in culture. Modified carbazoles with an ethyl moiety linked to the nitrogen of the carbazole and a carbonyl moiety linked to distinct biaromatic rings exhibited remarkably different killing activities in human GBM cell lines and patient-derived GBM cells, with IC₅₀ values from 67 to >10,000 nM. Measures of the activity of modified carbazoles with tubulin and microtubules coupled to molecular docking studies show that these compounds bind to the colchicine site of tubulin in a unique low interaction space that inhibits tubulin assembly. The modified carbazoles reported here represent novel chemical tools to better understand how small molecules disrupt MT functions and kill devastating cancers such as GBM.

Keywords: Carbazole; Colchicine; Gliomas; Microtubules.

Figure 1.. Microtubule targeting agents that bind to the colchicine site and chemical strategy for the synthesis of modified carbazoles.

Combretastatin A-4 (1), carbazole-based analogues of combretastatin (2), nocodazole (3) and scaffold of modified carbazoles (4).

Figure 2:. Antitumor activity of reference MTAs that target the colchicine site, as well as select modified-carbazoles, in T98G GBM cells in culture.

T98G cells in culture were treated with increasing concentrations of A) two MTAs acting through the colchicine site of tubulin, combretastatin A-4 (1) and nocodazole (3), B) compounds 8, 20 and 27, and C) compounds 23 and 25. Cell viability was measured 72 h following treatment using WST-1. Dotted line shows 100% vehicle control. Data are the mean ± SEM of at least three independent experiments performed in triplicate.

Figure 3:. Cell-killing activity of select modified-carbazoles in SF-539 and SF-295 GBM cells in culture.

A) SF-539 cells in culture and B) SF-295 cells in culture were treated with increasing concentrations of compounds 8, 20 and 27. Cell viability was measured 48 h following treatment using Alamar blue and is expressed as optical density (O.D.). Data are gathered at seeding and after 48 h, providing an index of the inhibition of cell proliferation (between both dotted lines) and triggering of cell death (below Seed dotted line). Parameters of cell-killing activity (GI₅₀, Total Growth Inhibition [TGI] and LC₅₀) are in Supplementary Table 1.

Figure 4:. Gas phase pharmacophore overlap model of colchicine, podophyllotoxin, and 20.

Panel A illustrates a three-dimensional stick rendering of the gas phase pharmacophoric overlap model for colchicine (carbons in magenta), podophyllotoxin (carbons in cyan), and 20 (carbons in yellow). For purposes of visual clarity, oxygen atoms are colored red in podophyllotoxin and deeper red for both colchicine and 20. In all three structures, nitrogen atoms are colored blue. The red circles correspond to the two 20 polar hydrogen bond acceptor features in common with colchicine or podophyllotoxin. Panels B, C and D are two-dimensional illustrations of the pharmacophore overlap model depicted in Panel A. Panel B illustrates colchicine. Panel C illustrates podophyllotoxin and Panel D illustrates 20. In Panels B-D, the bolded blue atoms correspond to the steric overlaps of the colchicine and podophyllotoxin atoms that are in common with 20; otherwise, atoms in Panels B-D are colored magenta for colchicine, cyan for podophyllotoxin and yellow for 20. As with Panel A, the red circles in Panels B-D correspond to polar hydrogen bond acceptor features of compound 20 that are in common with colchicine or podophyllotoxin.

Figure 5:. Comparison of modeling and X-ray poses.

Panel A displays the view of the optimized 20 (carbons in yellow) in the colchicine site from the molecular docking studies In Panel B, the pose of the podophyllotoxin-triazole analog (carbons in cyan) from the X-ray 5JCB is shown with 20 overlapped from molecular docking studies. In addition to the high degree of steric congruency with the podophyllotoxin-triazole analog, the ketone oxygens of both 20 and podophyllotoxin-triazole are isosteric. Unique to t 20, compared to podophyllotoxin and colchicine, is the relatively strong hydrogen bond acceptor quinoline N located in the trimethoxy-aryl subsite (near β-Cys-241). 20 can form a direct hydrogen bond (blue dashed lines in both panels) with the side chain S-H of β-Cys-241. The quinoline N of 20 can form a stronger hydrogen bond with a water bridge to polar backbone atoms compared to the ether O’s of the central methoxy groups of podophyllotoxin and colchicine (also see Supplementary Figure 6).

Scheme 1:

Reagents and conditions: a. Cs₂CO₃, alkylbromide, DMF, r.t.; b. acyl chloride, AlCl₃, benzene 0 °C to r.t.; c. i. POCl₃, DMF, μW, 1 h, 100 °C; ii. KMnO₄, water/acetone, reflux; d. EDC, DMAP, DIPEA, amine, DMF. 8: R₁ = H, R₂ = ethyl, R₃ = 4-methylnaphthalenyl; 9: R₁ = H, R₂ = ethyl, R₃ = 4-methylpiperazinyl; 10: R₁ = H, R₂ = ethyl, R₃ = 4-chlorophenyl; 11: R₁ = H, R₂ = ethyl, R₃ = 4-fluorophenyl; 12: R₁ = H, R₂ = ethyl, R₃ = p-tolyl; 13: R₁ = H, R₂ = ethyl, R₃ = methylbenzene; 14: R₁ = H, R₂ = ethyl, R₃ = naphthalenyl; 16: R₁ = H, R₂ = propyl, R₃ = 4-methylnaphthalenyl; 17: R₁ = H, R₂ = 2,2,2-trifluoroethyl, R₃ = 4-methylnaphthalenyl 31: R₁ = MeO-, R₂ = ethyl, R₃ = 4-methylnaphthalenyl;

Scheme 2:

Reagents and conditions: a. Cs₂CO₃, ethylbromide, DMF; b. t-BuLi, THF, −78 to 0 °C; c. PDC, CH₂Cl₂, Molecular sieves r.t.

Scheme 3:

Reagents and conditions: a. Lawesson’s reagent, toluene; b. NaBH₄, MeOH.

Scheme 4:

Reagents and conditions: a. HCl, EtOH; b. NaH, alkylbromide, DMF; c. KOH, H₂O, EtOH, reflux; d. EDC, DMAP, DIPEA, amine, DMF. 35: R₁ = H, R₂ = ethyl, R₃ = 4-methylnaphthalenyl