New anti-ovarian cancer quinolone derivatives acting by modulating microRNA processing machinery

Abstract

MicroRNAs (miRNAs) play a crucial role in ovarian cancer (OC) pathogenesis and miRNA processing can be the object of pharmacological intervention. By exploiting our in-house quinolone library, we combined a cell-based screening with medicinal chemistry efforts, ultimately leading to derivative 33 with anti-OC activity against distinct cell lines (GI₅₀ values 13.52-31.04 μM) and CC_{50 Wi-38} = 142.9 μM. Compound 33 retained anticancer activity against additional cancer cells and demonstrated a synergistic effect with cisplatin against cisplatin-resistant A2780 cells. Compound 33 bound TRBP by SPR (K _D = 4.09 μM) and thermal shift assays and its activity was TRBP-dependent, leading to modulation of siRNA and miRNA maturation. Derivative 33 exhibited augmented potency against OC cells and a stronger binding affinity for TRBP compared to enoxacin, the sole quinolone identified as a modulator of miRNA maturation. Consequently, 33 represents a promising template for developing novel anti-OC agents with a distinctive mechanism of action.

Fig. 1. Small set of selected in-house quinolones (1–10) and their antiproliferative activity against the SKOV-3 cell line expressed as GI₅₀ values.

Scheme 1. Reagents and conditions: i) ethylamine, Et₂O/EtOH, rt, 15 min; ii) K₂CO₃, DMF, 100 °C, 1 h; iii) 6 N HCl, EtOH, reflux, 1 h; iv) amine, Et₃N, dry DMF or DMSO, 80–100 °C, 1–2 h; v) RANEY®/Ni, H₂, DMF, rt, 30 min–12 h; vi) 10% NaOH, MeOH, reflux, 1 h.

Scheme 2. Reagents and conditions: i) amine, Et₃N, dry DMF or DMSO, 80–110 °C, 90 min–48 h; ii) RANEY®/Ni, H₂, DMF, rt, 1–6 h; iii) 6 N HCl, EtOH, reflux, 9 h (for 15) or 10% NaOH, MeOH, reflux, 30 min–4 h; iv) SnCl₂, EtOH, reflux, 2 h; v) SOCl₂, reflux, 2 h; then, 7 N NH₃ in MeOH, dry DMF, rt, 3 h.

Scheme 3. Reagents and conditions: i) DMF–DMA, AcOH, dry toluene, rt, 2 h; then, ethylamine or cyclopropylamine, rt, 30 min; then, 1.5 M Bu₄NOH, rt, 5 min; ii) amine, Et₃N, dry DMF, 90 °C, 2–3 h; iii) 6 N HCl, EtOH, reflux, 4–16 h or 10% NaOH, MeOH, reflux, 1 h (for 29).

Scheme 4. Reagents and conditions: i) C₂O₂Cl₂, cat. dry DMF, dry CH₂Cl₂, rt, 4 h; then, ethyl 3-(N,N-dimethylamino)acrylate, Et₃N, dry toluene, 90 °C, 4 h; ii) ethylamine or cyclopropylamine, Et₂O, EtOH, rt, 1 h; iii) NaH, dry DMF, 0 °C, 3 h; iv) NaN₃, dry DMF, 90 °C, 24 h; v) Pd/C, H₂, DMF, rt, 3 h; vi) CuBr₂, 5% HBr, 2 M NaNO₂, 0 °C to rt, 30 min; vii) 1,2,3,4-tetrahydroisoquinoline, Pd₂(dba)₃, BINAP, Cs₂CO₃, dry toluene, reflux, 13 h; viii) 6 N HCl, EtOH, reflux, 1–4 h; ix) 1,2,3,4-tetrahydroisoquinoline, Et₃N, dry DMSO, 100 °C, 4 h.

Fig. 2. SPR analyses of enoxacin (ENX), 17 and 33 towards TRBP.

Fig. 3. Cellular thermal shift assays of TRBP in the presence of 33. A) Representative Western blot analysis showing the destabilization effect of 33 on TRBP at different temperatures. Graph showing the quantified levels of TRBP over GAPDH from three independent experiments. B) Western blot analysis showing the concentration dependency of the destabilization effect of 33 on TRBP at 55 °C. Graph showing the quantified levels of TRBP over GAPDH. Error bars represent SE. p-Value was calculated using paired two-tailed Student's t-test (* p < 0.05).

Fig. 4. Compound 33 promotes siRNA/miRNA maturation. A) RNAi enhancing effect of compound 33 and enoxacin at 50 μM. Values are shown as relative quantity with respect to sh-Scr/DMSO treated cells set to a value of 1. ATP5O is used as a reference gene. B) Left graph: Upregulation of miR-21 in HeLa cells upon 6 hours of treatment with 33 and enoxacin at 50 μM. Values are shown as relative quantity with respect to DMSO treated cells set to a value of 1. U6 is used as a reference gene. Upper: Schematic representation of the psi-R21 and psi-CTRL constructs. “miR-21 BS” depicts the binding site of the miRNA. Lower graph: miRNA enhancing effect of compound 33 and enoxacin at 50 μM. Values are shown as the ratio between Renilla (RLuc) and firefly (FLuc) luciferase signals. Error bars represent SE of at least three independent experiments. p-Values were calculated using paired two-tailed Student's t-test (* p < 0.05; ** p < 0.01; ns: not significant).

Fig. 5. Antiproliferative effect after 48 hours of enoxacin at 125 μM and compound 33 A) against WT and KO TRBP HCCLM3 cell lines (30 μM) and B) against WT and KO TRBP SK-Hep-1 cell lines (30 and 50 μM). T-Test results (** P ≤ 0.01, **** P ≤ 0.001).

Fig. 6. Compound 33 inhibited cell growth and induced apoptosis of A2780 cells and sensitized A2780 CIS to cisplatin. A) Left graph: Reduction of number of SKOV-3 and A2780 viable cells upon treatment with compound 33 at 30 μM for 72 hours. The number of viable cells is expressed as percentage with respect to DMSO condition set to a value of 100%; right graph: percentage of SKOV-3 and A2780 dead cells upon treatment described in “A”; B) representative Western blot analysis of protein extracts from SKOV-3 and A2780 cells treated as in “A”.  β-Actin levels are used as a control for protein loading. C) Graph showing the number of viable cells of a culture of A2780 and A2780 CIS treated with 33 (30 μM), cisplatin (CIS, 1 μM) and a combination of the two compounds (33 + CIS). The number of viable cells is expressed as percentage with respect to DMSO condition set to a value of 100%. Error bars represent SE of at least three independent experiments. p-Values were calculated using paired two-tailed Student's t-test (* p < 0.05; ** p < 0.01).