Synthesis and evaluation of bis(imino)anthracene derivatives as G-quadruplex ligands†

The synthesis of a small number of bis(imino)anthracene derivatives is reported. They were evaluated via NMR for binding efficacy to the G-quadruplex-forming oligonucleotide sequence (TTGGGTT) and show activity against the HeLa cancer cell line. These novel ligands are compared to previously synthesised G-quadruplex ligands that target telomeres and oncogenes.

These cells were kept in media which was changed with fresh media every 2 days. All of the cells were cultured in a humidified incubator whose conditions are 37 o C, 5 % CO 2 , 95 % air.

Cell Passaging
The cells were split when the cells reached 70 % confluency in the flask. First of all, the medium was removed from the flask and then cells were washed with DPBS (1X) (pH 7.4) containing 10 mM sodium dibasic phosphate, 2.7 mM KCI, 2 mM Potassium phosphate, 137 mM NaCI. After that, the cells were detached from the flask with 0.25 % (w/v) trypsin-5mM EDTA in PBS (Phosphate Buffered Saline) solution and incubated for 5 minutes in an incubator with conditions at 37 o C, 5 % CO 2 , 95 % air. Dissociated cells were resuspended with medium containing 10 % Fetal Bovine Serum (FBS) to inhibit the trypsin activity and then centrifuged at 1500rpm for 5 minutes. After centrifugation, the supernatant was discarded, and cells were resuspended with media and seeded into the flasks.

Cell Counting and Viability
In order to determine cell density a hemacytometer was used, with the following procedure. With cell viability calculated with the following equation, where 95% viability was the target for cells; Ligands were drawn using Chemdraw 19.1 PerkinElmer Informatics Inc, with the 3D translation carried out using Avogadro 1.20, which was further used to remove water, add hydrogens dependent on pH 6.7. Force field geometry optimisation was applied to structures before writing as pdb files for UCSF chimera v1.15 software (University of California), or using semi empirical molecular orbital theory (PM6) carried out using the Scigress (Fujitsu) computational chemistry package. Ligand docking was carried out using Autodock Vina using the Chimera interface with the parameters below, and using the CB-Dock web server.
Autodock Vina analysis was undertaken with the below settings applied for the writing of pdbqt files.
All receptor options were set to true except for "Ignore chains of non-standard residues and Ignore all non-standard residues" set to false. Ligand options were set to false. Binding modes = 10, Exhaustiveness of search = 8 and Maximum energy difference was set to 3kcal/mol.
The docking box was chosen to encompass the entire quadruplex model, thus allowing Vina to search the whole of the target space. The docking box was larger than the upper size recommended for use with Autodock Vina, so blind docking was also carried out using the CB-Dock web server, which detects potential docking sites in the target by analysis of the solvent accessible surface and subsequently uses Autodock Vina to generate and score the ligand poses at the detected dicking sites. The results were comparable to those found using Autodock Vina and Chimera which forgoes the cavity detection step.

(E)-N-[2-(1H-indol-3-yl)ethyl]-1-[10-[(E)-2-(1H-indol-3-yl)ethyliminomethyl]-9anthryl]methanimine (2c) 2c
Tryptamine (390mg, 0.00245mol) and 9-10dibromocarbaldehyde (250mg) were stirred in dry DCM (40ml) over molecular sieves. The reaction mixture was refluxed for 12hrs under N 2 environment. The resultant mixture was monitored via TLC which showed the consumption of both starting materials. After TLC analysis, the crude mixture was left to cool down to room temperature. The resulting yellow solid 2c was filtered and it was dried under vacuum before being recrystallised from methanol to give (0.44g, 80%) yield.  N-(5-methylthiazol-2-yl)-1-[10-[(E)-(5-methylthiazol-2-yl)iminomethyl]-9anthryl]methanimine (2e) 2e 2-Amino-5-methylthiazole (500 mg, 0.0048mol) and 9,10-dibromocarbaldehyde (250mg) were stirred in dry DCM (40ml) over molecular sieves. The reaction mixture was refluxed for 12hrs under N 2 environment. The resultant mixture was monitored via TLC which showed the consumption of both starting materials. After TLC analysis, the crude mixture was left to cool down to room temperature. The resulting red solid 2e was filtered and it was dried under vacuum before being recrystallised from methanol to give (0.34g, 75%) yield. 3-Morpholinopropylamine (350 mg, 0.0024mol) and 9,10-dibromocarbaldehyde (250mg) were stirred in dry DCM (40ml) over molecular sieves. The reaction mixture was refluxed for 12hrs under N 2 environment. The resultant mixture was monitored via TLC which showed the consumption of both starting materials. After TLC analysis, the crude mixture was left to cool down to room temperature. The resulting off white solid 2b was filtered and it was dried under vacuum before being recrystallised from methanol to give (0.31g, 60%) yield. Piperonylamine (370 mg, 0.00245mol) and 9,-10-dibromocarbaldehyde (250mg, moles) were stirred in dry DCM (40ml) over molecular sieves. The reaction mixture was refluxed for 12hrs under N 2 environment. The resultant mixture was monitored via TLC which showed the consumption of both starting materials. After TLC analysis, the crude mixture was left to cool down to room temperature. The resulting yellow solid 2a was filtered and it was dried under vacuum before being recrystallised from methanol to give (0.42g, 78%) yield.