Clickable Cisplatin Derivatives as Versatile Tools to Probe the DNA Damage Response to Chemotherapy

Cisplatin induces DNA crosslinks that are highly cytotoxic. Hence, platinum complexes are frequently used in the treatment of a broad range of cancers. Efficiency of cisplatin treatment is limited by the tumor-specific DNA damage response to the generated lesions. We reasoned that better tools to investigate the repair of DNA crosslinks induced by cisplatin would therefore be highly useful in addressing drug limitations. Here, we synthesized a series of cisplatin derivatives that are compatible with click chemistry, thus allowing visualization and isolation of DNA-platinum crosslinks from cells to study cellular responses. We prioritized one alkyne and one azide Pt(II) derivative, Pt-alkyne-53 and Pt-azide-64, for further biological characterization. We demonstrate that both compounds bind DNA and generate DNA lesions and that the viability of treated cells depends on the active DNA repair machinery. We also show that the compounds are clickable with both a fluorescent probe as well as biotin, thus they can be visualized in cells, and their ability to induce crosslinks in genomic DNA can be quantified. Finally, we show that Pt-alkyne-53 can be used to identify DNA repair proteins that bind within its proximity to facilitate its removal from DNA. The compounds we report here can be used as valuable experimental tools to investigate the DNA damage response to platinum complexes and hence might shed light on mechanisms of chemoresistance.


Synthesis of precursor 1
The reaction was performed on a 10 mmol scale. 2-hydroxy-2,3-diaminopropane (1 equiv) was reacted with di-tert-butyl decarbonate (2.5 equiv) and triethylamine (6 equiv) in 50 ml 1:1 DMF: H2O mixture at room temperature for 5 hours. The reaction was quenched with HCl (1M), washed with NaHCO3 and water. The aqueous phase was extracted three times with dichloromethane (30 ml). The organic phase was dried with Na2SO4 and reduced under pressure to give 1 as a white solid. The compound was used in the following step without further purification.

Synthesis of precursor 2
To a solution of 1 (1 equiv) in dry dichloromethane (50ml) at 0 o C, triethylamine (2 equiv) was added followed by methanesulfonyl chloride (1.3 equiv). The reaction was then allowed to reach room temperature and mixed for 1 hour. The resulting mixture was then quenched with water, HCl and extracted with dichlotomethane. The combined organic fractions were then washed with an aqueous solution of NaHCO3 and dried over Na2SO4. The organic solution was reduced under vacuum to yield product 2 as a yellowish solid. The product was used in the following step without further purification.

Synthesis of precursor 3
To a solution of 2 in DMF (50ml), sodium azide (1.2 equiv) was added, and the resulting mixture was allowed to mix at 100 o C for 2 days. The reaction was then cooled down to room temperature, poured into water and extracted with EtOAc. Evaporation of the organic solvent yielded the desired azide as a white solid. The product was used for the next step without further purification.

Synthesis of precursor 4
In an autoclave, precursor 3 (1 equiv) was dissolved in ethanol (4 ml/mmol). Pd/C (1 mol %) was added to the mixture and resulting mixture was allowed to mix under H2 (50 bar) at room temperature for 24 hours. The resulting mixture was then filtrated over a celite plug and the organic solution reduced under pressure to give 4 as a clean product.

Synthesis of precursor 5
To a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2 equiv) in dry dichloromethane (50ml) was added the corresponding carboxylic acid (1.5 equiv) while stirring in an ice bath. Compound 4 (1 equiv) in dry dichloromethane was added dropwise over approximately 1 hour. The reaction was then removed from the ice bath and stirred for an additional 18 hours at room temperature. The reaction was quenched with H2O (1 mL) while stirring for 15 minutes. The mixture was then filtered, and the filtrate evaporated in vacuo to afford a white solid. The solid was then dissolved in EtOAc and washed with diluted aq. HCl solution (pH 4, 3 x 10 mL) followed by washing with saturated aq. NaHCO3 solution (3 x 10 mL). The organic layer was dried (MgSO4), filtered, and evaporated in vacuo to obtain a white solid. Purification via flash silica gel column chromatography (2:1 CH2Cl2/EtOAc, Rf = 0.23) afforded 5 as a white solid which was used in the next step without further purification.

Synthesis of precursor 6
A solution of 5M HCl in 1,4-dioxane (4 equiv) was cooled on ice and purged with Ar. Precursor 5 (1 equiv) was then added to the reaction mixture while stirring. The ice bath was removed, and the mixture was stirred for 45 minutes at room temperature, during which a white precipitate formed. The precipitate was filtered and washed with 1,4-dioxane and diethyl ether and dried under vacuum to obtain 6 as a white solid which was used in the next without further purification.

Synthesis of platinum complexes 7
The reaction was performed on 0.17 mmol scale of precursor 6. The corresponding carboxylic acid was either commercially available or prepared using known procedures. To a solution of 6 (1 equiv) in dry DMF (1 mL) was added 1,8 diazabicyclo[5.4.0]undec-7-ene (2.2 equiv), then cis-[Pt(DMSO)2Cl2] (1 equiv), then the solution was stirred in the dark at room temperature for approx 48 hours. H2O (4 mL) was then added to the reaction mixture and the clear brown solution was refrigerated for approx 24 hours. The solid that had formed was isolated by filtration, rinsed with excess H2O, and dried in a desiccator.

Characterization of platinum complexes
Pt-51 5-azidopentanoic acid is a known compound and was prepared according to a known procedure(3). Pt-51 was isolated as pale-brown powder (15 mg, 18%

Pt-53
10-undecynoic acid is commercially available and was purchased from Sigma-aldrich. Pt-53 was isolated as pale-brown powder (45 mg, 51%  Additional uncropped images showing clickable Pt-alkyne-53 with AF488-picolyl azide and Ptazide-64 with AF488-alkyne (green) in U2OS cells following a 3-hour treatment with compounds at 5µM or 25µM, respectively. DAPI (blue) was used to counterstain nuclei. Vehicle treated cells (DMF), as well as cells exposed to the CuAAC click reaction without the copper catalyst (-CuSO4), are used as a negative control. Scale bar represents 20 µm. (B) Immunoblot for FANCD2 and Tubulin on protein extracts from wildtype (WT) and FANCD2 deficient (FANCD2 KD) human U2OS cells.