Lead Discovery of Dual G-Quadruplex Stabilizers and Poly(ADP-ribose) Polymerases (PARPs) Inhibitors: A New Avenue in Anticancer Treatment

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1 Supporting Information Lead Discovery of Dual G-Quadruplex Stabilizers and Poly(ADP-ribose) Polymerases (PARPs) Inhibitors: A New Avenue in Anticancer Treatment Erica Salvati, * Lorenzo Botta, Jussara Amato, Francesco Saverio Di Leva, Pasquale Zizza, Antimo Gioiello, Bruno Pagano, Grazia Graziani, Madalena Tarsounas, ± Antonio Randazzo, Ettore Novellino, Annamaria Biroccio and Sandro Cosconati * Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Rome, Italy. Department of Pharmacy, University of Naples Federico II, Naples, Italy. Department of Pharmaceutical Science, University of Perugia, I Perugia, Italy. Department of Systems Medicine, University of Tor Vergata, Rome, Italy. ± Genome Stability and Tumourigenesis Group, CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK. DiSTABiF, University of Campania Luigi Vanvitelli, Caserta, Italy. To whom correspondence should be addressed: sandro.cosconati@unina2.it; erica.salvati@gmail.com. Chemistry. Supplementary Figures. S2 S6 S1

2 Chemistry General Methods. All the compounds were characterized by 1 H NMR and MS analysis. NMR spectra were recorded at a 400 MHz spectrometer. Chemical shifts (δ) are reported relative to CDCl 3 at δ 7.26 ppm, CD 3 OD at 3.30 or (CD 3 ) 2 SO at δ 2.50 ppm and tetramethylsilane at δ 0.00 ppm. Chemical shifts are reported in part per million (ppm) relative to the residual solvent peak. Mass spectral (MS) data were obtained using a LC/MS system with a 0.4 ml/min flow rate using a binary solvent system of 95:5 methyl alcohol/water. UV detection was monitored at 254 nm. Mass spectra were acquired in positive mode scanning over the mass range of The following ion source parameters were used: drying gas flow, 9 ml/min; nebulizer pressure, 40 psig; drying gas temperature, 350 C. N-(2-(4-benzylpiperidin-1-yl)ethyl)-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-2-carboxamide (3): White solid. Yield = 75 %. Pf = 183 C dec. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ: (m, 1H), (m, 1H), 8.26 (s, 1H), 7.83 (d, J = 8.04 Hz, 1H), 7.55 (t, J = 7.56 Hz, 1H), 7.41 (d, J = 8.16 Hz, 1H), 7.35 (t, J = 7.04 Hz, 1H), (m, 2H), (m, 2H), (s, 3H), (m, 3H), 3.42 (t, J = Hz, 2H), (m, 4H), (m, 2H), 2.19 (t, J = Hz, 2H), (m, 4H), (m, 1H). 13 C NMR (100 MHz, CD 3 OD) δ: , , , , , , , , , , , , , , , , , , , , 52.90, 41.51, 35.51, 34.93, MS (ESI): m/z 446 [M+H] + Anal. Calcd. for C 26 H 27 N 3 O 2 S: C, 70.08; H, 6.11; N, Found: C, 70.11; H, 6.14; N, N-(2-(4-benzylpiperidin-1-yl)ethyl)-5-methyl-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-2- carboxamide (4): White solid. Yield = 81 %. Pf = 214 C dec. 1 H NMR (400 MHz, CDCl 3 ) δ: 8.24 (s, 1H), 7.55 (d, J = 8.04 Hz, 1H), 7.56 (t, J = 7.56 Hz, 1H), 7.42 (d, J = 8.72 Hz, 1H), (m, 2H), (m, 3H), 7.10 (d, J = 7.32 Hz, 2H), 3.88 (t, J = 9.0 Hz, 2H), 3.75 (s, 3H), 3.69 (d, J = 8.3 Hz, 2H), 3.26 (t, J = 9.0 Hz, 2H), 2.71 (t, J = Hz, 2H), 2.60 (d, J = 6.96 Hz, 2H), (m, 4H), S2

3 (m, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ: , , , , , , , , , , , , , , , , , 43.31, 40.36, 28.45, 22.09, 21.43, MS (ESI): m/z 460 [M+H] + Anal. Calcd. for C 27 H 29 N 3 O 2 S: C, 70.56; H, 6.36; N, Found: C, 70.60; H, 6.34; N, N-(2-(4-benzylpiperidin-1-yl)ethyl)-5-oxo-4,5-dihydrothieno[3,2-c]isoquinoline-2-carboxamide (5): White solid. Yield = 67 %. Pf = 194 C dec. 1 H NMR (400 MHz, CDCl3) δ: 8.37 (d, J = 7.60 Hz, 1H), 7.82 (s, 1H), 7.66 (t, J = 8.16 Hz, 1H), 7.57 (d, J = 7.32 Hz, 1H), 7.51 (t, J = 7.72 Hz, 1H), (m, 2H), (s, 3H), 3.80 (t, J = 9.1 Hz, 2H), (m, 2H), 3.26 (t, J = 8.0 Hz, 2H), (m, 2H), (m,4h), (m, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ: , , , , , , , , , , , , , , , , , 57.34, 54.09, 46.96, 43.71, 37.36, MS (ESI): m/z 446 [M+H] + Anal. Calcd. for C 26 H 27 N 3 O 2 S: C, 70.08; H, 6.11; N, Found: C, 70.07; H, 6.13; N, N-(2-(4-benzylpiperidin-1-yl)ethyl)-4-methyl-5-oxo-4,5-dihydrothieno[3,2-c]isoquinoline-2- carboxamide (6): White solid. Yield = 84 %. Pf = 185 C dec. 1 H NMR (400 MHz, (CD3)2SO) δ: (m, 1H), 8.29 (d, J = 7.80 Hz, 1H), 8.02 (s, 1H), 7.86 (d, J = 7.76 Hz, 1H), 7.78 (t, J = 6.88 Hz, 1H), 7.58 (t, J = 7.52 Hz, 1H), (m, 2H), (m, 3H), 3.91 (t, J = 7.52 Hz, 2H), 3.75 (s, 3H), 3.17 (d, J = 10.8 Hz, 2H), 2.83 (t, J = 7.52 Hz, 2H), 2.50 (d, J = 6.88 Hz, 2H), 2.22 (t, J = 7.52 Hz, 2H), (m, 4H), (m, 1H) 13 C NMR (100 MHz, CDCl 3 ) δ: , , , , , , , , , , , , , , , 58.89, 53.72, 42.66, 37.30, 35.92, 31.96, MS (ESI): m/z 460 [M+H] + Anal. Calcd. for C 27 H 29 N 3 O 2 S: C, 70.56; H, 6.36; N, Found: C, 70.60; H, 6.37; N, (4-benzylpiperidin-1-yl)ethanamine (16). Yield = 73 %. Pf = 86 C: 1 H NMR (CD 3 OD) δ 7.79 (t, J=8 Hz, 3H), 7.78 (d, 1H), (m, 2H), (m, 2H), 3.81 (t, J = 6.56 Hz, 2H), 3.02 (d, J = S3

4 11.06 Hz, 2H), 2.62 (t, J = 6.52 Hz, 2H), 2.50 (d, J = 6.76 Hz, 2H), 1.97 (t, J = Hz, 2H), (m, 4H), (m, 1H); 13 C NMR (100 MHz, (CD 3 OD) δ: , , , , , , , , 55.74, 53.41, 42.54, 37.57, 34.40, MS (ESI): m/z 349 [M+H] + Calcd. for C 22 H 24 N 2 O 2 : C, 75.83; H, 6.94; N, Found: C, 75.87; H, 6.91; N, Methyl 4-bromo-1-oxo-1,2-dihydroisoquinoline-3-carboxylate (18). White solid. Yield = 56 %. Pf = 186 C: 1 H NMR (400 MHz, (CDCl 3 ) δ: (m, 1H), 8.47 (d, J = 7.80 Hz, 1H), 8.28 (d, J = 8.24 Hz, 1H), 7.85 (t, J = 7.56 Hz, 1H), 7.69 (t, J = 7.56 Hz, 1H), 4.04 (s, 3H). 13 C NMR (100 MHz, (CDCl 3 ) δ: , , , , , , , , , , , , , MS (ESI): m/z 283 [M+H] + Calcd. for C 11 H 8 BrNO 3 : C, 46.84; H, 2.86; N, Found: C, 46.87; H, 2.85; N, bromo-1-oxo-1,2-dihydroisoquinoline-3-carbaldehyde (13). White solid. Yield = 71 % over 2 steps. Pf = 185 C: 1 H NMR (400 MHz, (CDCl 3 ) δ: (s, 1H), 8.49 (d, J = 7.44 Hz, 1H), 8.21 (d, J = 8.12 Hz, 1H), 7.87 (t, J = 7.24 Hz, 1H), 7.75 (t, J = 7.92 Hz, 1H). 13 C NMR (100 MHz, (CDCl 3 ) δ: , , , , , , , , , , , , , MS (ESI): m/z 253 [M+H] + Calcd. for C 10 H 6 BrNO 2 : C, 47.65; H, 2.40; N, Found: C, 47.67; H, 2.43; N, Methyl 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-2-carboxylate (7): White solid. Yield = 86 %. Pf = 221 C. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ: (s, 1H), 8.07 (s, 1H), 7.89 (d, J = 7.84 Hz, 1H), 7.55 (t, J = 7.44 Hz, 1H), 7.40 (d, J = 8.24 Hz, 1H), 7.25 (t, J = 7.48 Hz, 1H), 3.88 (s, 3H). 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ: , , , , , , , , , , , , MS (ESI): m/z 260 [M+H] + Anal. Calcd. for C 13 H 9 NO 3 S: C, 60.22; H, 3.50; N, Found: C, 60.23; H, 3.47; N, 5.42 S4

5 Methyl 5-methyl-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-2-carboxylate (8): White solid. Yield = 53 %. Pf = 270 C dec. 1 H NMR (400 MHz, CDCl 3 ) δ: 8.37 (s, 1H), 7.84 (d, J = 7.64 Hz, 1H), 7.60 (t, J = 8.00 Hz, 1H), 7.44 (d, J = 8.64 Hz, 1H), 7.30 (t, J = 7.44 Hz, 1H), 3.96 (s, 3H), 3.77 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ: , , , , , , , , , , , , , , , , 52.26, MS (ESI): m/z 274 [M+H] + Anal. Calcd. for C 14 H 11 NO 3 S: C, 61.52; H, 4.06; N, Found: C, 61.49; H, 4.09; N, Methyl 5-oxo-4,5-dihydrothieno[3,2-c]isoquinoline-2-carboxylate (9): White solid. Yield = 78 %. Pf = 190 C. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ: (s, 1H), 8.28 (d, J = 7.60 Hz, 1H), 7.92 (d, J = 7.72 Hz, 1H), 7.81 (t, J = 6.64 Hz, 1H), 7.62 (t, J = 7.16 Hz, 1H), 7.58 (s, 1H), 3.86 (s, 3H). 13 C NMR (100 MHz, (CD 3 ) 2 SO) δ: , , , , , , , , , , , , MS (ESI): m/z 260 [M+H] + Calcd. for C 13 H 9 NO 3 S: C, 60.22; H, 3.50; N, Found: C, 60.19; H, 3.48; N, 5.39 Methyl 4-methyl-5-oxo-4,5-dihydrothieno[3,2-c]isoquinoline-2-carboxylate (10): White solid. Yield = 67 %. Pf = 227 C dec. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ: 8.31 (d, J = 8.08 Hz, 1H), 8.02 (s, 1H), 7.94 (d, J = 7.68 Hz, 1H), 7.81 (t, J = 7.52 Hz, 1H), 7.63 (t, J = 7.80 Hz, 1H), 3.89 (s, 3H), 3.70 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ: , , , , , , , , , , , , 52.65, MS (ESI): m/z 274 [M+H] + Anal. Calcd. for C 14 H 11 NO 3 S: C, 61.52; H, 4.06; N, Found: C, 61.55; H, 4.10; N, S5

6 A B C D Figure S1. (A, C) Normalized CD-melting curves of Tel23 G4 in the absence (black) and in presence of: (A) 3 (cyan), 4 (red), 5 (blue), 6 (green); and (C) 7 (cyan), 8 (red), 9 (blue), 10 (green). (B, D) CD spectra of Tel23 before (solid lines) and after (dashed lines) melting experiment (at 20 and 90 C, respectively) in the absence (black) and in presence of: (B) 3 (cyan), 4 (red), 5 (blue), 6 (green); and (D) 7 (cyan), 8 (red), 9 (blue), 10 (green). S6

7 A B C D Figure S2. (A, C) Normalized CD-melting curves of Tel26 G4 in the absence (black) and in presence of: (A) 3 (cyan), 4 (red), 5 (blue), 6 (green); and (C) 7 (cyan), 8 (red), 9 (blue), 10 (green). (B, D) CD spectra of Tel26 before (solid lines) and after (dashed lines) melting experiment (at 20 and 90 C, respectively) in the absence (black) and in presence of: (B) 3 (cyan), 4 (red), 5 (blue), 6 (green); and (D) 7 (cyan), 8 (red), 9 (blue), 10 (green). S7

8 A B C D Figure S3. (A, C) Normalized CD-melting curves of Tel23-p G4 in the absence (black) and in presence of: (A) 3 (cyan), 4 (red), 5 (blue), 6 (green); and (C) 7 (cyan), 8 (red), 9 (blue), 10 (green). (B, D) CD spectra of Tel23-p before (solid lines) and after (dashed lines) melting experiment (at 20 and 95 C, respectively) in the absence (black lines) and in presence of: (B) 3 (cyan), 4 (red), 5 (blue), 6 (green); and (D) 7 (cyan), 8 (red), 9 (blue), 10 (green). S8

9 Figure S4. CD spectra (left) and CD-melting curves (right) of d(cgcgaattcgcg) 2 duplex in the absence (black) and in presence of 3 (blue), 4 (red), and 6 (green). S9

10 A B C Figure S5. Normalized CD-melting curves of (A) Tel23, (B) Tel26, and (C) Tel23-p G4s in the absence (black) and in presence of 19 (red), and 20 (blue). S10

11 Figure S6. MST time traces recorded by adding increasing concentrations of compound 4 to the labeled telomeric G4 (top) and the corresponding binding curve (bottom). Error bars represent standard error of three measurements. S11

12 Figure S7. MST time traces recorded by adding increasing concentrations of compound 8 to the labeled telomeric G4 (top). The compound did not show binding to the G4 (bottom). S12

13 Figure S8. FRET-melting curves of F21T (0.2 µm) in the absence (black line) and in presence of compound 4 without (red line) and with 5.0 µm (blue line) and 10.0 µm (green line) double-stranded DNA (ds12) competitor. S13

14 120 % of PARP1 activity Olaparib 20 (IC 50 (IC = = nm) 6.7 nm) 5(IC 50 = 223 nm) [nm] Figure S9. Cell-free PARP1 activity assay. Recombinant PARP1 was exposed to graded concentrations (2.5 nm 1 µm) of compound 5 or 20. Then PARP1 activity was assayed in a reaction mixture containing 32 P-NAD +, cleaved DNA and histones. Inhibition of PARP1 activity was measured by quantifying radiolabeled poly ADP-ribose polymers. Bars indicate means ± SD. S14

15 % of γh2ax positive DMSO hrs 6 hrs 12 hrs 24 hrs Figure S10. Timing of DDR recovery after compounds removal. BJ-EHLT cells were exposed to 1 µm of the indicated compounds for 24 hours in quadruplicate samples. At the end of treatment, one sample for each condition was fixed (black bars) and the others underwent medium replacement and were fixed at the indicated time points (white bars). All the samples were processed for immunofluorescence against γh2ax and counterstained with Hoechst. Histograms in A report the percentage of γh2ax positive nuclei in each sample. Histograms show the mean values of three independent experiments. Bars indicate means ± SD. S15