Click-Capable Phenanthriplatin Derivatives as Tools to Study Pt(II)-Induced Nucleolar Stress

It is well established that oxaliplatin, one of the three Pt(II) anticancer drugs approved worldwide, and phenanthriplatin, an important preclinical monofunctional Pt(II) anticancer drug, possess a different mode of action from that of cisplatin and carboplatin, namely, the induction of nucleolar stress. The exact mechanisms that lead to Pt-induced nucleolar stress are, however, still poorly understood. As such, studies aimed at better understanding the biological targets of both oxaliplatin and phenanthriplatin are urgently needed to expand our understanding of Pt-induced nucleolar stress and guide the future design of Pt chemotherapeutics. One approach that has seen great success in the past is the use of Pt-click complexes to study the biological targets of Pt drugs. Herein, we report the synthesis and characterization of the first examples of click-capable phenanthriplatin complexes. Furthermore, through monitoring the relocalization of nucleolar proteins, RNA transcription levels, and DNA damage repair biomarker γH2AX, and by investigating their in vitro cytotoxicity, we show that these complexes successfully mimic the cellular responses observed for phenanthriplatin treatment in the same experiments. The click-capable phenanthriplatin derivatives described here expand the existing library of Pt-click complexes. Significantly they are suitable for studying nucleolar stress mechanisms and further elucidating the biological targets of Pt complexes.

NPM1 Average Coefficient of Variation for 1,2 and 3: Figure S1.Average Coefficient of Variation (CV) quantification of NPM1 relocalization induced by 1, 2 and 3.Treatment conditions indicated (either 10 μM or 0.5 μM for Pt complexes, 5 nM for ActD) in A549 cells at 24 hr treatment; CV calculations, and boxplot presentations as described in the Experimental Section.For each treatment data set, average of CV of 3 trials along with std.

In-vitro activity of 3 and Pt controls:
Figure S2.In-vitro proliferation of A549, SK-LU-1 and PANC-1 cells following treatment with 3 and Ptcontrols for 6 days.

Synthesis & Characterization of Ligands:
*Note: While no issues were encountered during our handling of the organic azides and 1, 2 and 3, organic and inorganic azides are known to be neurotoxic and explosive in nature.As such, care should be taken if preparing the compounds described below.
Complete synthetic route for the synthesis of complexes 1-3: After stirring, the reaction mixture was cooled to room temperature and diluted with water (50 mL).The product was then extracted with ethyl acetate (3 x 50 mL), washed with brine (3 x 50 mL) and dried over anhydrous sodium sulphate.The solvent was removed under reduced pressure and the product purified by column chromatography (70: 30 petroleum ether: ethyl acetate).

General Procedure for Reduction of Nitro-group:
n-nitrophenanthridine (1.00 g, 4.46 mmol), iron powder (1.99 g, 35.68 mmol), and ammonium chloride (2.39 g, 44.60 mol) were added to a round bottom flask.Methanol (40 mL) and water (5 mL) were added to the mixture and the resulting suspension heated to 75 o C. The mixture was stirred under reflux overnight and reaction completion was confirmed by TLC the following day.The reaction mixture was allowed to cool to room temperature and the pH of the solution was adjusted to < 5 using HCl (1 M).The solution was extracted with ethyl acetate (2 x 25 mL) and the combined organic layers discarded.The pH of the aqueous phase was then adjusted to > 10 using NaOH (1 M) and the phase again extracted with ethyl acetate (3 x 50 mL).The organic layers were then combined, washed with brine (2 x 25 mL) and dried over anhydrous sodium sulphate.The solvent was removed under reduced pressure to give a crude solid which was purified by column chromatography (70: 30 ethyl acetate: petroleum ether).

General Procedure for Nitro to Azide Transformation:
6M HCl (3.9 mL, 23.4 mmol) was added to phenanthridin-n-amine (0.75g, 3.86 mmol) and the resulting mixture cooled to 0 o C in an ice bath.Sodium nitrite (0.525 g, 7.623 mmol) in water (5 mL) was added dropwise to this mixture with vigorous stirring, and the resulting solution stirred for 30 minutes at 0 o C. Following this a solution of sodium azide (0.99 g, 15.24 mmol) in water (5 mL) was added dropwise and the reaction mixture stirred for a further 4 hour at 0 o C. Following reaction completion, the pH of the solution was adjusted to > 7 with sodium hydroxide (1 M) and the product extracted with ethyl acetate (3 x 50 mL).The organic layers were combined, washed with brine (2 x 25 mL) and dried over anhydrous sodium sulphate.The solvent was removed under reduced pressure and the crude product purified by column chromatography (50: 50 petroleum ether: ethyl acetate).

Single crystal X-Ray diffraction:
The X-ray intensity data for 1 were measured (λ = 1.54178Å) on a Bruker Apex Kappa Duo with an Oxford Cobra Cryosystem low temperature device at 100(2) K using a MiTeGen micromount and NVH immersion oil.See Table S1 for crystal data and structure refinement details.
Bruker APEX 1 software was used to collect and reduce data and correct for Lorentz and polarization effects.Data were corrected for absorption effects using the Multi-Scan method SADABS. 2 Structures were solved with the SHELXT 3 structure solution program using Intrinsic Phasing and refined using Least Squares method on F 2 with SHELXL 4 within the OLEX2 5 package.All non-hydrogen atoms were refined anisotropically.Hydrogen atoms were placed in calculated positions with Uiso dependencies derived from their carrier atoms (riding model).
In 1 the azide group is disordered and modelled over two locations (75:25% occupancy) and refined using geometric (SADI, DFIX) and displacement (SIMU, ISOR) restraints.The methanol OH hydrogen was located on the difference map and refined using geometric restraints (DFIX).
Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no.2296496.Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail:deposit@ccdc.cam.ac.uk).Table S3.Hydrogen Bonds for 1.

Figure S12 .
Figure S12.Individual images of each disordered moiety of 1 with (A) 75% occupied and (B) 25% occupied.Atomic displacement shown at 50% probability and heteroatoms labelled only.

Figure S13 .Figure S14 .
Figure S13.Strong hydrogen bonding network in 1 viewed normal to the c-axis.Hydrogen bonding represented by dotted lines.Atomic displacement shown at 50% probability.

Table S1 .
Crystal data and structure refinement for 1

Table S2 .
Crystal data and structure refinement for 1.