Synthesis and Evaluation of the Tumor Cell Growth Inhibitory Potential of New Putative HSP90 Inhibitors

Abstract

Background: Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones as inhibitors of HSP90.

Methods: In the present work, various compounds with new chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90 inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds (5 and 8) were further studied in various tumor cell lines regarding their potential to cause cell growth inhibition, alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound 8 was studied in A431 squamous cell carcinoma xenografts in nude mice.

Results: Our results indicated that treatment with compounds 5 and 8 decreased the proliferation of tumor cell lines and compound 8 induced apoptosis. In addition, these two compounds were able to downregulate selected proteins known as "clients" of HSP90. Finally, treatment of xenografted mice with compound 5 resulted in a considerable dose-dependent inhibition of tumor growth.

Conclusions: Our results show that two new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising putative HSP90 inhibitors causing tumor cell growth inhibition.

Keywords: (thio)chromenopyridinones; HSP90; antitumor; apoptosis; cell cycle; cell proliferation; naphthoquinones; tumor xenographs.

Scheme 1

Synthesis of N-substituted benzo[g]isoquinoline-5,10-diones. Reagents and conditions: (a) for 2a: benzyl bromide, DMSO, r.t., 48 h, 86%; for 2b: 2-bromomethyl-1,4-difluorobenzene, DMSO, r.t., 24 h, 60%; for 2c: 1-bromomethyl-4-tert-butylbenzene, DMSO, 24 h, r.t., 65%.

Scheme 2

Synthesis of (thio)chromenopyridinones. Reagents and conditions: (a) 3-bromopyridine, KOtBu, 210 °C, 3 h, 74%; (b) LDA, THF, 0 °C, then 1 h at r.t., 90%; (c) benzyl bromide, DMSO, r.t., 48 h, 86%; (d) i: NaNO₂, H₂O, HCl conc., 5 °C; ii: thiophenol, NaOH, H₂O, reflux, 3 h; (e) i: SOCl₂, 1 h, reflux; ii: AlCl₃, PhNO₂, 100 °C, 4 h, 72%; (f) benzyl bromide, DMSO, overnight at r.t., 55%; (g) benzyl bromide, DMSO, overnight at r.t., 34%.

Figure 1

NCI-H460 viable cell number 48 h following treatment with compounds 5 (A) and 8 (B) analyzed with trypan blue exclusion assay. Cells were treated with the GI₅₀ (5.2 μM) and 1.5× GI₅₀ (7.8 μM) of compound 5 and with the GI₅₀ (3.2 μM) and 1.5× GI₅₀ (4.8 μM) of compound 8. Cells were also treated with the corresponding highest concentration of vehicle (solvent) of the compounds (H₂O). Results are presented as % of viable cells in relation to blank cells (treated with medium) and are the mean ± SEM of six independent experiments. * p ≤ 0.001 Blank vs. treatment.

Figure 2

NCI-H460 cell cycle profile 48 h following treatment with compounds 5 (A) and 8 (B), analyzed by flow cytometry. Cells were treated with the GI₅₀ (5.2 μM) and 1.5× GI₅₀ (7.8 μM) of compound 5 and with the GI₅₀ (3.2 μM) and 1.5× GI₅₀ (4.8 μM) of compound 8. Cells were also treated with the corresponding highest concentration of the vehicle (solvent) of the compounds (H₂O). Results represent the mean ± SEM of at least three independent experiments.

Figure 3

NCI-H460 cellular proliferation following 48 h treatment with compounds 5 (A) and 8 (B), analyzed with the BrdU incorporation assay. Cells were treated with the GI₅₀ (5.2 μM) and 1.5× GI₅₀ (7.8 μM) of compound 5 and with the GI₅₀ (3.2 μM) and 1.5× GI₅₀ (4.8 μM) of compound 8. Cells were also treated with the corresponding highest concentration of vehicle (solvent) of the compounds (H₂O). Results represent the mean ± SEM of three independent experiments. * p ≤ 0.001, ** p ≤ 0.05 between Blank vs. treatment.

Figure 4

Analysis of HSP90 client protein levels in squamous-cell carcinoma (A431) and peritoneal mesothelioma (STO) cells after 24 h of treatment with compound 5 (5.4 μM in A431 cells; 2.7 μM in STO cells). Total cellular extracts were obtained 24 h after cell treatment. Actin is shown as a control for protein loading.

Figure 5

Analysis of HSP90 client protein levels in A431 and STO cells after 24 h of treatment with compound 8 (2.6 μM in A431 cells; 2.6 μM in STO cells). Total cellular extracts were obtained 24 h after cell treatment Actin is shown as a control for protein loading.

Figure 6

Antitumor activity of compound 5 in A431 squamous cell carcinoma xenografts in nude mice. Compound 5 dissolved in physiologic saline was administered i.p. (5 mg/kg and 10 mg/kg) for five consecutive days/week. The treatment started when the tumors were just palpable and was repeated for three weeks.