Modulation of ERCC1-XPF Heterodimerization Inhibition via Structural Modification of Small Molecule Inhibitor Side-Chains

Inhibition of DNA repair enzymes is an attractive target for increasing the efficacy of DNA damaging chemotherapies. The ERCC1-XPF heterodimer is a key endonuclease in numerous single and double strand break repair processes, and inhibition of the heterodimerization has previously been shown to sensitize cancer cells to DNA damage. In this work, the previously reported ERCC1-XPF inhibitor 4 was used as the starting point for an in silico study of further modifications of the piperazine side-chain. A selection of the best scoring hits from the in silico screen were synthesized using a late stage functionalization strategy which should allow for further iterations of this class of inhibitors to be readily synthesized. Of the synthesized compounds, compound 6 performed the best in the in vitro fluorescence based endonuclease assay. The success of compound 6 in inhibiting ERCC1-XPF endonuclease activity in vitro translated well to cell-based assays investigating the inhibition of nucleotide excision repair and disruption of heterodimerization. Subsequently compound 6 was shown to sensitize HCT-116 cancer cells to treatment with UVC, cyclophosphamide, and ionizing radiation. This work serves as an important step towards the synergistic use of DNA repair inhibitors with chemotherapeutic drugs.


Cytotoxicity Analysis
Supplementary Figure 3. Cytotoxicity assay using colony forming assay

HCT-116 Derived XPF Knockout Cells
For CRISPR deletion of XPF from HCT-116 cells, the pSpCas9(BB)-2A-GFP (pX458) vector (Addgene plasmid # 48138) was used. The short guide sequence to target XPF exon one was: 5'-GGGCTAGTAGTGTGCGCCCG-3' and target XPF exon eight was: 5'-CTATATCACTCTTGGAGCGG-3'. All short guide sequences oligonucleotides were synthesized, annealed and cloned into pX458 vector and were confirmed by DNA sequencing at DNA laboratory, University of Calgary. The DNA plasmid (pX458 containing XPF short guide RNA sequence, 5 µg) was transfected into HCT-116 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were harvested and genomic DNA was isolated using the KAPA Express Extract Kit (Kapa Biosystems) according to the manufacturer's instructions. Genomic DNA fragments of XPF around the short guide RNA site were amplified by PCR. The primers used to amplify the genomic region of XPF exon one were: Forward: 5'-CACTAGGAGTCGGCTTCCTT-3', Reverse: 5'-TCTCTGTGTCATCGCGTAGT-3' and exon eight were: Forward: 5'-TCGGGTGAAGGAATAAGGGG-3', Reverse: 5'-ATTTCTTCCGGGCAGCTTTC-3'. PCR products were used for Surveyor nuclease mutation detection assays using the SURVEYOR Mutation Detection Kit (IDT) according to manufacturer's protocol. After transfection, GFP containing cells were sorted into 96-well plates by flow cytometry, at the Flow Cytometry Facility, University of Calgary. Single cells were then expanded for further analysis (i.e., Western blot and DNA sequencing). All CRISPR gene knockout clones were screened by Western blot using anti-XPF antibody (MA12060, ThermoFisher). The membrane was also probed with Mre11 antibody (Novus) as a loading control. For DNA sequencing confirmation of XPF knockout clones, DNA fragments of XPF knockout clones around the short guide RNA site were amplified by PCR. PCR products were subcloned into pEGFP-C2 vector (Clontech) and plasmid DNA from individual clones were sent for Sanger DNA sequencing at DNA laboratory, University of Calgary to confirm all indels.

General Information
Reactions were carried out in flame or oven dried glassware under a positive nitrogen atmosphere unless otherwise stated. Transfer of anhydrous solvents and reagents was accomplished with ovendried syringes or cannulae. Solvents and some reagents were distilled before use. Commercially available reagents were used without further purification. Thin layer chromatography was performed on glass plates precoated with 0.25 mm silica gel. Flash chromatography was performed on 230-400 mesh silica gel with the indicated eluents. Nuclear magnetic resonance (NMR) spectra were recorded in indicated deuterated solvents and are reported in ppm in the presence of TMS as internal standard and coupling constants (J) are reported in hertz (Hz). The spectra are referenced to residual solvent peaks: chloroform-d (7.26 ppm, 1 H; 77.26 ppm, 13 C), DMSO-d6 (2.50 ppm, 1 H; 39.51 ppm, 13 C), acetone-d6 (2.05 ppm, 1 H; 206.68 and 29.92 ppm, 13 C) and methanol-d4 (3.31 157 ppm, 1 H; 49.00 ppm, 13 C). Proton nuclear magnetic spectra ( 1 H NMR) and carbon nuclear magnetic resonance spectra ( 13 C NMR) were recorded at 500/400 and 125/100 MHz respectively. Mass spectra were recorded by using electrospray ionization (ESI).

4-Amino-2-((4-(2,2-dimethoxyethyl)piperazin-1-yl)methyl)phenol (18)
MeOH (40 ml) was added to a 3-neck round bottomed flask containing 10% Pd/C (50 mg), before addition of a solution of 17 (1.91 g, 5.87 mmol) in MeOH (50 ml) was added. The reaction vessel was evacuated and backfilled with nitrogen 3 times, then evacuated and backfilled with H2 3 times before being allowed to stir at room temperature for 7 days. After the completion of the reaction, as indicated by TLC, the reaction mixture was filtered over celite and the solvent removed under reduced pressure. The crude product was purified by column chromatography (1.62 g, 93%

2-(4-(5-((6-Chloro-2-methoxyacridin-9-yl)amino)-2-hydroxybenzyl)piperazin-1-yl)acetaldehyde (20)
Compound 19 (321 mg, 0.60 mmol) was dissolved in DCM (25 ml) and cooled to 0 °C. BBr3 (1.32 ml, 1.32 mmol) was added dropwise and allowed to stir at 0 °C for 15 minutes. After completion of the reaction, as indicated by TLC, the reaction was carefully quenched by addition of H2O and the pH adjusted to 8 with a saturated NaHCO3 solution. The reaction mixture was extracted with DCM and the combined organic extracts dried with Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography to afford the product as an orange solid (222 mg, 75%

4-Amino-2-((4-(3,3-diethoxypropyl)piperazin-1-yl)methyl)phenol (23)
MeOH (10 ml) was added to a 3-neck round bottomed flask containing 10% Pd/C (35 mg), before addition of a solution of 22 (438 mg, 1.19 mmol) in MeOH (10 ml) was added. The reaction vessel was evacuated and backfilled with nitrogen 3 times, then evacuated and backfilled with H2 3 times before being allowed to stir at room temperature for 7 days. After the completion of the reaction, as indicated by TLC, the reaction mixture was filtered over celite and the solvent removed under reduced pressure. The crude product was purified by column chromatography (385 mg, 97%

3-(4-(5-((6-Chloro-2-methoxyacridin-9-yl)amino)-2-hydroxybenzyl)piperazin-1-yl)propanal (25)
Compound 24 (177 mg, 0.31 mmol) was dissolved in DCM (10 ml) and cooled to 0 °C. 1M BBr3 in DCM (0.7 ml) was added dropwise and allowed to stir at 0 °C for 15 minutes. After completion of the reaction, as indicated by TLC, the reaction was carefully quenched by addition of H2O and the pH adjusted to 8 with a saturated NaHCO3 solution. The reaction mixture was extracted with DCM and the combined organic extracts dried with Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography to afford the product as an orange solid (153 mg, 97%). The impure mixture obtained was used without further purification.

Methyl 2,2-dimethyl-3-oxopropanoate (27) 1
A solution of oxalyl chloride (0.39 ml, 552 mg, 4.35 mmol) in DCM (9ml) was cooled to -78 °C. A mixture of DMSO (0.4 ml, 443 mg, 5.67 mmol) and DCM (0.5 ml) was added dropwise and allowed to stir for 5 minutes before addition of a solution of methyl 3-hydroxy-2,2-dimethylpropanoate (500 mg, 3.78 mmol) in DCM (2 ml) was added dropwise and allowed to stir for a further 5 minutes before the dropwise addition of triethylamine (2.62 ml, 1.91 g, 18.9 mmol). The reaction mixture was slowly allowed to warm to room temperature. After completion of the reaction, as indicated by TLC, Et2O and H2O were added and the combined organic extracts were dried with MgSO4, filtered, and evaporated. The product was purified by column chromatography (169 mg, 34%

4-Nitro-2-(piperazin-1-ylmethyl)phenol (28)
2-Hydroxy-5-nitrobenzaldehyde (1.0 g, 6.0 mmol) was dissolved in DCM (50 ml). Boc-piperazine (1.2 g, 6.6 mmol) and acetic acid (0.10 ml, 0.11 g, 1.8 mmol) were added and allowed to stir for 30 minutes before addition of NaBH(OAc)3 (1.5 g, 7.2 mmol). The reaction mixture was allowed to stir at room temperature overnight. After the completion of the reaction, as indicated by TLC, the reaction was quenched with satd. NaHCO3 solution, then 1 M NaOH solution. The reaction mixture was then adjusted to pH = 8-9 by addition of 1M HCl. Extraction with DCM and drying of organic extracts with Na2SO4, filtration and removal of solvent under reduced pressure afforded the crude boc-protected product, which was purified by column chromatography (1.74 g, 86%). The bocprotected product was dissolved in DCM (20 ml) and TFA (4 ml) and allowed to stir at room temperature for 2 days. After the completion of the reaction, as indicated by TLC, the reaction mixture was quenched with a saturated solution of NaHCO3 and extracted with DCM. The combined organic extracts were dried with MgSO4, filtered, and the solvent removed under reduced pressure. The crude product was purified by column chromatography (quantitative yield  4,125.4,124.8,121.0,116.5,61.3,53.5,53.4,45.7;HRMS (ESI)

3-(4-(2-Hydroxy-5-nitrobenzyl)piperazin-1-yl)-N,2,2-trimethylpropanamide (31)
Compound 30 (236 mg, 0.78 mmol) was dissolved in DCM (7 ml). DMF (7 drops) then thionyl chloride (0.17 ml, 279 mg, 2.34 mmol) was added and the reaction mixture allowed to stir at room temperature overnight. After the completion of the reaction, as indicated by TLC, the solvent was evaporated under reduced pressure to afford the crude acid chloride as a colourless solid, which was dissolved in DCM (20 ml). Methylamine (2M in THF, 2.0 ml) was added and the reaction mixture was allowed to stir at room temperature overnight. After the completion of the reaction, as indicated by TLC, the reaction was quenched with H2O and extracted with DCM. The combined organic extracts were washed with brine, dried with MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography to afford the desired product (256 mg, 84%

3-(4-(5-Amino-2-hydroxybenzyl)piperazin-1-yl)-N,2,2-trimethylpropanamide (32)
MeOH (5 ml) was added to a 3-neck round bottomed flask containing 10% Pd/C (20 mg), before addition of a solution of 31 (148 mg, 0.42 mmol) in MeOH (5 ml) was added. The reaction vessel was evacuated and backfilled with nitrogen 3 times, then evacuated and backfilled with H2 3 times before being allowed to stir at room temperature for 7 days. After the completion of the reaction, as indicated by TLC, the reaction mixture was filtered over celite and the solvent removed under reduced pressure. The crude product was purified by column chromatography (77 mg, 57%

1-(2-Methoxyethyl)piperazine (TFA salt) (34)
Compound 33 (412 mg, 1.69 mmol) was dissolved in DCM (7.0 ml) and TFA (1.4 ml) and allowed to stir at room temperature overnight. After the completion of the reaction, as indicated by TLC, DCM was evaporated and residual TFA was co-evaporated with toluene to afford the product as a beige solid that was used without further purification (quantitative yield).