S1

Supporting Information

Novel Antitumor Platinum(II) Conjugates Containing the Non-steroidal Anti-inflammatory Agent Diclofenac:

Synthesis and Dual Mechanisms of Antiproliferative Effects Francesco Paolo Intini,†,ǁ Juraj Zajac,‡,§,ǁ

Vojtech Novohradsky,‡ Teresa Saltarella,† Concetta Pacifico,† Viktor Brabec,‡,§ Giovanni Natile,*,† and Jana Kasparkova*,‡

†Department of Chemistry, University of Bari “Aldo Moro”, 70125 Bari, Italy ‡Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 61265 Brno, Czech Republic § Department of Biophysics, Faculty of Science, Palacky University, Slechtitelu 27, 78371 Olomouc, Czech Republic

ǁ F.P. Intini and J. Zajac contributed equally to this work.

Table of Contents

S2: Figure S1. 1H-NMR spectrum of cis-[PtCl2(DCF-en)(NH3)] (1) in DMSO-d6.

S2: Figure S2. 195Pt-NMR-spectrum of cis-[PtCl2(DCF-en)(NH3)] (1) in DMSO-d6. S3: Figure S3. Theoretical and experimental isotope pattern of [M+Cl]– (M=compound 1). S3: Figure S4. 1H-NMR spectrum of trans-[PtCl2(DCF-en)(DMSO)] (2) in DMSO-d6. S3: Figure S5.

195Pt-NMR spectrum of trans-[PtCl2(DCF-en)(DMSO)] (2) in DMSO-d6. S4: Figure S6. Theoretical and experimental isotope pattern of [M–H]– (M = compound 2). S4: Figure S7. Intermolecular hydrogen bond interactions of trans-[PtCl2(DCF-

en)(DMSO)] (2) and its intermolecular π-π stacking interactions. S5: Figure S8. 1D and 2D COSY 1H NMR spectra of cis-[Pt(DCF)2(NH3)2] (3) in DMSO- d6. S6: Figure S9. 1D and 2D NOESY 1H NMR spectra of cis-[Pt(DCF)2(NH3)2] (3) in DMSO-d6.

S6: Figure S10. 195Pt-NMR-spectra of cis-[Pt(DCF)2(NH3)2] (3). S7: Figure S11. Theoretical and experimental isotope patterns of [M + NO3]

– (M = compound 3). S8: Figure S12. Solvolysis of cis-[Pt(NH3)2(DCF)2] (3) in DMSO monitored by 1H NMR spectra. S9: Figure S13. Cell-cycle profiles of HeLa cells treated for 24 h with cisplatin, 3 or free DCF. S10: Figure S14. Changes in the mitochondrial membrane potential (∆ψm) induced by

cisplatin, 3 or free DCF as revealed by the JC-1 assay and imaged by confocal microscopy.

S11: Figure S15. Compound 3 and free DCF inhibit migration of human HeLa cells. S12: Table S1. NMR data for free diclofenac and diclofenac-en ligands and related platinum complexes 1-3. S12: Table S2. Intermolecular hydrogen bonds for complex 2.

S2

Figure S1. 1H-NMR spectrum of cis-[PtCl2(DCF-en)(NH3)] (1) in DMSO-d6.

Figure S2. 195Pt-NMR-spectrum of cis-[PtCl2(DCF-en)(NH3)] (1) in DMSO-d6.

S3

Figure S3. Theoretical (left) and experimental (right) isotope pattern of [M + Cl]– (M = compound 1).

Figure S4. 1H-NMR spectrum of trans-[PtCl2(DCF-en)(DMSO)] (2) in DMSO-d6.

Figure S5. 195Pt-NMR spectrum of trans-[PtCl2(DCF-en)(DMSO)] (2) in DMSO-d6.

S4

Figure S6. Theoretical (left) and experimental (right) isotope pattern of [M – H]– (M = compound 2).

Figure S7. a) Intermolecular hydrogen bond interactions of trans-[PtCl2(DCF-en)(DMSO)] (2). For clarity, hydrogen atoms tied to carbon atoms have been omitted. b) Intermolecular π-π stacking interactions of compound 2.

a b

S5

Figure S8. 1D and 2D COSY 1H NMR spectra of cis-[Pt(DCF)2(NH3)2] (3) in DMSO-d6. The cross-peak labelled A connects two signals falling at 7.47 and 7.12 ppm which were assigned to protons C12/14H (magnetically equivalent) and C13H, respectively. A second cross peak, B, connects two signals close to 7.05 and 6.77 ppm. Finally, cross peak C connects the signal at 6.99 ppm with the most shielded signals falling at 6.26 ppm.

S6

Figure S9. 1D and 2D NOESY 1H NMR spectra of cis-[Pt(DCF)2(NH3)2] (3) in DMSO-d6. The proton at 7.05 ppm has an intense NOE cross peak, a, with the methylene protons (C2H2) at 3.47 ppm, clearly indicating that the signal at 7.05 belongs to C4H. Since the signal at 7.05 is connected to the signal close to 6.77 ppm (COSY experiment, Figure 5), the latter signal was assigned to C5H. The two remaining signals (6.99 and 6.26 ppm) were assigned to C6H and C7H, respectively. It is worth noting that the more shielded signal at 6.26 ppm was assigned to C7H since it points toward the shielding cone of the aromatic ring B (see scheme 4) (Kovala-Demertzi, D.; Mentzafos, D.; Terzis, A. Metal complexes of the anti-inflammatory drug sodium [2-[(2,6-dichlorophenyl)amino]phenyl]acetate (diclofenac sodium). Molecular and crystal structure of cadmium diclofenac. Polyhedron 1993, 12, 1361-1370).

Figure S10. 195Pt-NMR-spectra of cis-[Pt(DCF)2(NH3)2] (3).

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Figure S11. Theoretical (left) and experimental (right) isotope patterns of [M + NO3]– (M =

compound 3).

S8

Figure S12. Solvolysis of cis-[Pt(NH3)2(DCF)2] (3) in DMSO monitored by 1H NMR spectra. Top, plot of the mole fraction of starting complex 3 (χ) versus time (s) for the experiment performed in dmso-d6. χ was calculated from the integrals of the NH signals of DCF belonging to different species (NHA, NHB and NHC for 3, cis-[Pt(NH3)2(DCF)(DMSO-d6)]

+

and free DCF, respectively) falling in the region between 7.5 and 11.0 ppm [χ= NHA/(NHA+NHB+NHC)]. Bottom, plot of −ln χ against time (s).

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0,90

1,00

0 10000 20000 30000 40000

time (s)

mole fraction

y = 9E-05x + 0,0785

R2 = 0,998

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 10000 20000 30000 40000

time (s)

-lnχχ χχ

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Figure S13. Cell-cycle profiles of HeLa cells treated for 24 h with cisplatin, 3 or free DCF at roughly equitoxic concentrations (three-fold values of IC50 found for these compounds in HeLa cells treated for 72 h, Table 2). The cells were stained with propidium iodide and assessed for cell-cycle distribution by FACS analysis. The results of flow cytometric analysis were acquired using ModFit software. The colors represent subG1 phase (blue), G1 phase (red), S phase (blue dashed) and G2/M phase (green) of modeled cell cycle.

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Figure S14. Changes in the mitochondrial membrane potential (∆ψm) as revealed by the JC-1 assay and imaged by confocal microscopy. Treatment with 10 µM cisplatin, 5 µM 3 or 700 µM free DCF for 6 h caused the collapse of mitochondrial membrane potentials in A2780 and A2780cisR cells. After the treatment cells were stained with JC-1 and imaged by confocal microscopy to assess the emission shift from red (590 nm) to green (530 nm) using 488 nm excitation (for other details see Experimental section). The shift in ∆ψm observed by disappearance of red-stained mitochondria (large negative ∆ψm) and an increase in fluorescent green-stained mitochondria (loss of ∆ψm) by 3 is higher compared to that observed by cisplatin. The JC-1 green/red ratios resulting from the mitochondria are shown in Figure 6B.

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Figure S15. Compound 3 and free DCF inhibit migration of human HeLa cells. Sub-cytotoxic, roughly equitoxic concentrations of cisplatin, 3 and free DCF [15, 2.3, or 150 µM, respectively; these concentrations correspond approximately to 0.65 % values of IC50 found for these compounds in HeLa cells treated for 72 h (Table 2)] were added to the “wounded” cell monolayer. Cell migration was photographed at 24 h and compared to the 0 h control. Scale bars represent 200 µm.

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Table S2. NMR Data for Free Diclofenac (DCF) and Diclofenac-en (DCF-en) Ligands and Related Platinum

Complexes 1-3. Chemical shifts are expressed in ppm: 1H and 13C values are referenced to residual solvent peaks in the case of DMSO-d6. Displacements of protons and carbons of the DCF and DCF-en groups in complexes 1-3 with respect to the free ligand are also reported.

Compound H2 (∆δ)

H4 (∆δ)

H5 (∆δ)

H6 (∆δ)

H7 (∆δ)

NH9 (∆δ)

H12/1 (∆δ)

H13 (∆δ)

NH1‘ (∆δ)

H2‘ (∆δ)

H3‘ (∆δ)

NH24‘

DCF-en 3.57 7.18 6.85 7.00 6.28 8.45 7.51 7.15 8.33 3.06 2.58 1.50 Complex 1 3.57 7.18 6.85 7.04

(0.04) 6.29 (0,01)

8.31 (0.14)

7.51 7.16 (0.01)

8.47 (0.14)

3.34 (0.28)

2.63 (0.05)

4.92 (3.42)

Complex 2 3.56 (0.01)

7.17 (0.01)

6.85 7.04 (0.04)

6.29 (0,01)

8.29 (0.16)

7.51 7.16 (0.01)

8.44 (0.11)

3.34 (0.28)

2.69 (0.11)

5.15 (3.65)

DCF 3.347 7.04 6.72 6.91 6.22 10.38 7.43 7.05 Complex 3 3.47

(0.10) 7.05 (0.01)

6.77 (0.05)

6.99 (0.08)

6.26 (0.04)

8.23 (2.15)

7.47 (0.04)

7.12 (0.07)

Compound C1 (∆δ)

C2 (∆δ)

C3 (∆δ)

C4 (∆δ)

C5 (∆δ)

C6 (∆δ)

C7 (∆δ)

C8 (∆δ)

C10 (∆δ)

C12/14 (∆δ)

C13 (∆δ)

C11/15 (∆δ)

C2‘ (∆δ)

C3‘ (∆δ)

DCF-en 171.3 39.3 125.3 130.1 120.3 126.8 115.6 142.6 136.8 128.7 124.7 128.9 42.2 40.9 Complex 1 171.9

(0.6) 39.5 (0.2)

125.5 (0.2)

130.4 (0.3)

120.7 (0.4)

127.3 (0.5)

115.9 (0.3)

143.3 (0.7)

137.2 (0.4)

129.1 (0.4)

125.2 (0.5)

129.5 (0.6)

38.9 (3.3)

45.2 (4.3)

Complex 2 172.0 (0.7)

39.5 (0.2)

125.5 (0.2)

130.4 (0.3)

121.3 (1.0)

127.2 (0.4)

116.0 (0.4)

143.3 (0.7)

137.5 (0.5)

128.9 (0.2)

125.23 (0.6)

129.7 (0.8)

38.7 (3.5)

44.0 (3.1)

DCF 174.8 44.6 128.6 129.6 119.5 125.3 115.2 143.1 138.1 128.9 123.4 128.6 Complex 3 179.1

(4.3) 41.3 (3.3)

126.4 (2.2)

130.3 (0.7)

120.1 (0.6)

126.5 (1.2)

115.6 (0.4)

143.1 137.6 (0.5)

128.8 (0.1)

124.6 (1.2)

129.5 (0.9)

Table S2. Intermolecular Hydrogen Bonds for Complex 2.

D—H···A DH···A (Å) D···A (Å) D—H···A (°) Symmetry codes N2—H2a···O2 2.05(7) 2.95(1) 175(5 -x,+y+1/2,-z+1/2 N1—H1N1a···Cl2 2.58(1) 3.22(1) 128(1) -x,+y+1/2,-z+1/2 N1—H2N1a···Cl1 2.49(1) 3.39(1) 173(1) -x,+y-1/2,-z+1/2