Click estradiol dimers with novel aromatic bridging units: synthesis and anticancer evaluation

Figures & data

Figure 1. Chemical structure of estradiol-based antimitotics and proposed mechanism of action. Created with Biorender.com.

Scheme 1. Diazide linkers for CuAAC reaction with EE. Reagents and conditions: (a) NaN₃, DMF, heated, ON; (b) MeI, NaH, DMF, 0 °C, 4 h; (c) 4-DMAP, Ac₂O, DCM, RT, ON; (d) acetone, 2-bromoethanol, K₂CO₃, 60 °C, ON; (e) MeI, DMF, toluene, NaH, 0 °C, 1 h. *Reaction yields for L1 and L10 reported by Thomas et al.¹² were 96%. The yield of L2 by Rasheed et al.¹³ was 95%.

Scheme 2. Structures of estradiol dimers prepared by 1,3-dipolar cycloaddition. CuAAC: CuSO₄·5H₂O (10 mol%), sodium ascorbate (15 mol%), DMF, MW, 80 °C, 2 h.

Table 1. Summary of cytotoxic activities (IC₅₀, µM).^a

Compound	CCRF-CEM	CEM-DNR	K562	K562-TAX	A549	HCT116	HCT116p53-	U2OS	MRC-5	BJ	HUVEC
EE	22.5 ± 1.63	22.97 ± 4.7	8.72 ± 2.17	15.6 ± 6.25	31.47 ± 4.16	30.01 ± 6.89	33.57 ± 11.94	22.4 ± 5.65	>50	>50	n/a
ME	1.61 ± 0.097	1.61 ± 0.2	1.43 ± 0.39	1.32 ± 0.2	2.26 ± 0.15	1.58 ± 0.25	1.99 ± 0.41	2.13 ± 0.3	>50	>50	1.0685 ± 0.18
ED	0.49 ± 0.22	>50	0.58 ± 0.23	>50	>50	0.95 ± 0.11	0.9 ± 0.32	6.23 ± 0.7	1.16 ± 0.25	>50	0.0244 ± 0.017
ED1	1.6 ± 0.27	>50	1.63 ± 0.12	1.58 ± 0.16	1.68 ± 0.2	1.41 ± 0.3	1.5 ± 0.32	1.95 ± 0.44	>50	>50	0.0135 ± 0.002
ED2	1.7 ± 0.19	>50	1.48 ± 0.19	>50	6.69 ± 0.79	3.57 ± 0.66	4.26 ± 0.94	>50	>50	>50	<0.0122
ED3	0.38 ± 0.019	>50	0.33 ± 0.074	>50	0.60 ± 0.047	0.47 ± 0.022	0.45 ± 0.039	0.61 ± 0.033	>50	>50	<0.0122
ED4	1.09 ± 0.11	>50	0.49 ± 0.04	>50	>50	2.06 ± 0.18	2.15 ± 0.1	>50	>50	>50	0.0160 ± 0.006
ED5	0.71 ± 0.04	>50	0.42 ± 0.044	>50	>50	2.02 ± 0.19	1.98 ± 0.17	>50	>50	>50	<0.0122
ED6	1.54 ± 0.25	>50	1.39 ± 0.21	>50	8.6 ± 2.22	1.74 ± 0.11	1.87 ± 0.13	5.06 ± 1.68	>50	>50	<0.0122
ED7	1.29 ± 0.13	>50	0.77 ± 0.13	>50	>50	1.61 ± 0.087	1.79 ± 0.17	3.86 ± 1.39	>50	>50	0.014 ± 0.002
ED8	1.38 ± 0.18	3.44 ± 0.69	0.69 ± 0.15	13.08 ± 3.69	>50	1.77 ± 0.16	2.01 ± 0.12	6.93 ± 1.16	>50	>50	0.079 ± 0.048
ED9	1.69 ± 0.057	>50	1.27 ± 0.12	>50	>50	2.02 ± 0.3	1.96 ± 0.26	5.26 ± 1.69	3.2 ± 0.89	>50	0.038 ± 0.011
ED12	6.14 ± 0.93	>50	4.52 ± 0.64	>50	>50	8.78 ± 0.88	10.52 ± 2.48	16.82 ± 3.46	14.2 ± 3.52	11.3 ± 1.41	0.657 ± 0.132
Colchicine	0.011 ± 0.00061	1.02 ± 0.21	0.014 ± 0.002	1.83 ± 0.19	0.047 ± 0.028	0.024 ± 0.0025	0.027 ± 0.0079	0.028 ± 0.014	0.055 ± 0.029	>50	<0.0122

^aCytotoxic activity was determined by the MTS assay after a 72-h incubation period. The listed IC₅₀ values represent the mean of 3 independent experiments with SD values. Tested cell lines include cancer derived lines such as CCRF-CEM (childhood T-cell acute lymphoblastic leukaemia), CEM-DNR (daunorubicin resistant variant of CCRF-CEM), K562 (chronic myelogenous leukaemia), K562-Tax (paclitaxel-resistant variant of K562), A549 (lung adenocarcinoma), HCT116 (colorectal adenocarcinoma), HCT116p53−/− (p53 gene null variant of HCT116), HeLa (cervical adenocarcinoma), U2OS (osteosarcoma), and normal human cell lines: MRC-5, BJ (normal cycling fibroblasts) and HUVEC (human umbilical vein endothelial cell line). Reference compounds are EE (17α-ethinylestradiol), ME (2-methoxyestradiol), and ED (estradiol dimer ⁹).

Figure 2. Effect of cytotoxic compounds on cell cycle (A), mitosis (B), sub-G₁ fraction (C), induction of polyploidy (D), and DNA/RNA synthesis (E, F) as assessed by flow cytometry in CCRF-CEM lymphoblasts (% of positive cells). Experiments were conducted at concentrations corresponding to 1 × IC₅₀ and 5 × IC₅₀ values. DNA fragmentation was assessed using a logarithmic model expressing the percentage of particles with propidium iodide content lower than cells in the cell cycle’s G₀/G₁ phase (<G₁). Detailed numerical values can be found in the Supplementary Material Table S1.

Figure 3. Estradiol dimers inhibit tubulin polymerisation in vitro (A). Tubulin assembly was measured using compounds at a concentration of 10 μmol/L, or an equivalent volume of DMSO. Inhibition of polymerisation velocity relative to DMSO control reaction measured at a concentration of 10 μmol/L. Data are represented as mean values with standard deviation (SD) calculated from three independent experiments (B).

Table 2. Inhibition of tubulin assembly in comparison to colchicine as reference compound.

Compound	Colchicine	ED	ED3	ED5
IC50 (µmol/L)	1.09 ± 0.33	2.18 ± 1.80	2.24 ± 1.54	2.01 ± 0.21

The listed IC₅₀ values with SD represent the mean of two independent experiments.

Figure 4. Estradiol dimers inhibit the formation of mitotic spindle. Immunofluorescent imaging of U2OS labelled with α-tubulin – Alexa Fluor-488 antibody (green fluorescence) following a 24-h incubation with DMSO (control), ED (comparative control), ED3, ED5 (all dimers concentrations were set to 10 µM), and colchicine (1 µM). Condensed chromosomes were visualised using Hoechst 33342 (blue fluorescence). Scale bar 2 µm.

Figure 5. Estradiol dimers inhibit the endothelial cell tube formation angiogenesis. Figure illustrates the network formation of HUVEC primary endothelial cells in the presence of DMSO (control), 60 nM suramine, 60 nM colchicine or 5 × IC₅₀ concentration of ED, ED3 and ED5. The cells and nuclei were visualised using DIC and Hoechst 33342 (blue fluorescence) or calcein (green fluorescence). Scale bar: 200 µm.

Figure 6. Docking poses of compounds ED3 (magenta) and ED5 (yellow) within the tubulin structure 4O2B (α chain is green, β – orange).

Figure 7. Calculated protein–ligand contacts were identified in at least 10% of MD trajectories for ED (panel A), ED3 (panel B), and ED5 (panel C). Contacts were analysed exclusively for the stable part of trajectories, 50–150 ns.

Figure 8. Molecular structures of previously reported estradiol dimers (panel A) and correlation plot between calculated binding free energies and tubulin polymerisation speed (panel B). The shaded region depicts the confidence interval at 0.95 significance level.

Table 3. Binding free energies of estradiol dimers from this (ED, ED3, ED5) and the previous study (D1, D2, D3, D9)¹⁰ were calculated by MM-PBSA.

Dimer	ΔG, kcal/mol	−TΔS, kcal/mol	Tubulin polymerisation inhibition, %
ED	−16.8	16.5	55.6
ED3	−2.4	18.2	31.6
ED5	−24.0	15.0	70.2
D1^a	−22.6	16.8	65.0
D2^a	−19.4	13.0	79.1
D3^a	−2.9	28.8	36.8
D9^a	−5.6	35.6	49.6

^a Values were calculated from Ref. ¹⁰

Thomas JR, Liu XJ, Hergenrother PJ. Size-specific ligands for RNA hairpin loops. J Am Chem Soc. 2005;127(36):12434–12435.

 PubMed Web of Science ®Google Scholar

Rasheed OK, Lawrence A, Quayle P, Bailey PD. A modular approach to functionalised dyes. Synlett. 2015;27(6):905–911.

 Web of Science ®Google Scholar

Jurášek M, Černohorská M, Řehulka J, Spiwok V, Sulimenko T, Dráberová E, Darmostuk M, Gurská S, Frydrych I, Buriánová R, et al. Estradiol dimer inhibits tubulin polymerization and microtubule dynamics. J Steroid Biochem Mol Biol. 2018;183:68–79.

 PubMed Web of Science ®Google Scholar

Jurášek M, Řehulka J, Hrubá L, Ivanová A, Gurská S, Mokshyna O, Trousil P, Huml L, Polishchuk P, Hajdúch M, et al. Triazole-based estradiol dimers prepared via CuAAC from 17α-ethinyl estradiol with five-atom linkers causing G2/M arrest and tubulin inhibition. Bioorg Chem. 2023;131:106334.

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Data availability statement

The datasets presented in this study are available from the corresponding author upon reasonable request.