Click reaction on in situ generated &#946;-azidostyrenes from cinnamic acid using CAN-NaN<SUB>3</SUB>: synthesis of N-styryl triazoles

N-Styryl triazoles are synthesized in one-pot starting with azido styrene obtained in situ from cinnamic acids and various acetylenes

Phenyl 1,2,3-Triazole-Thymidine Ligands Stabilize G-Quadruplex DNA, Inhibit DNA Synthesis and Potentially Reduce Tumor Cell Proliferation over 3′-Azido Deoxythymidine

<div><p>Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (<b>L1</b>, <b>L2</b> and <b>L3</b>) and non-nucleoside 1,2,3-triazoles (<b>L4–L14</b>) were synthesised using <i>click chemistry</i> and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3′-Phenyl-1,2,3- triazole-thymidine (<b>L2</b>) and 3′-4-Chlorophenyl-1,2,3-triazole-thymidine (<b>L3</b>), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for <b>L3</b> was lowest (50 µM) among the ligands studied. <b>L3</b> terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (<b>L2/L3</b>) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with <b>L3</b>. <i>In vitro</i> DNA synthesis reaction with <b>L3</b> exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T_m of quadruplex DNA increased in the presence of <b>L3</b>, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on <b>L3</b> treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (<b>L2</b>/<b>L3</b>) interact with quadruplex DNA with significantly higher affinity (K_d≈10⁻⁷ M). Interestingly the addition of an electronegative moiety to the phenyl group of <b>L2</b> enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with <b>L3</b>, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.</p></div

Sequences of QF and NQF oligonucleotides used in stop assay.

<p>Sequences of QF and NQF oligonucleotides used in stop assay.</p

DNA fragmentation in B16F10 cells on nucleoside ligands treatment.

<p>B16F10 cells were treated with 200 µM of <b>L1</b>, 125 µM of <b>L2</b> and 50 µM of <b>L3</b> for 24 h. Control cells were treated with equal volume of 1∶1 methanol: water without ligand.</p

Varation of induced tumor size on treatment with nucleoside ligands.

<p>Tumor was induced in C57BL6/J mice by injecting B16F10 cells subcutaneously and treated with 200 µM of <b>L1</b>, 125 µM of <b>L2</b> and 50 µM of <b>L3</b> and an equal volume of 1∶1 methanol: water (without ligand) was treated as vehicle control. Group treated with an equal volume of 1∶1 methanol: water, vehicle control (4 mice) marked •. Group treated with <b>L1</b>, <b>L2</b> and <b>L3</b> (4 mice each) were marked ▪, ▴ and ▾ respectively. Tumor volumes were measured at fixed time points and expressed in mm³. Each experiment was repeated thrice and the mean values were plotted. The set of data with p value≤0.001 and 0.01 were marked with * and # respectively. The statistical data with p value≤0.05 was left unmarked.</p

Effect of nucleoside and non-nucleoside ligands on normal and tumor human cell lines.

<p>+ - Viable cells in the range of 80%–90% ; ++ - Viable cells in the range of 50%–60% ; +++ - Viable cells which are equal or less than 30%.</p

Distribution of cells in various phase of cell cycle in control and L1, L2 and L3 treated B16F10 cells.

<p>Distribution of cells in various phase of cell cycle in control and L1, L2 and L3 treated B16F10 cells.</p

Percentage of alive, early apoptotic, late apoptotic and necrotic cells in control and L1, L2 and L3 treated B16F10 cells.

<p>Percentage of alive, early apoptotic, late apoptotic and necrotic cells in control and L1, L2 and L3 treated B16F10 cells.</p

Structure of ligands used and the MTT assay.

<p><b>A.</b> Structure of nucleoside 1,2,3-triazole ligands <b>L1, L2, L3</b> and the basic structure of non-nucleoside 1,2,3-triazole ligands (<b>L4–L14</b>). The position of various pharmacophores on ligands (<b>L4–L14</b>) are labelled with -R1, -R2 and -R3. <b>B.</b> MTT assay with B16F10 cells on treatment with 50 µM, 100 µM, 150 µM, 200 µM, 250 µM and 300 µM concentration of <b>L1</b>, <b>L2</b> and <b>L3</b> for 24 h and 48 h. The set of data with reducing order of significance (p value≤0.001, 0.01 and 0.05) were marked with *, # and ** respectively.</p