A new ruthenium cyclopentadienyl azole compound with activity on tumor cell lines and trypanosomatid parasites

<div><p>As part of our efforts to develop organometallic ruthenium compounds bearing activity on both trypanosomatid parasites and tumor cells, a new Ru(II)–cyclopentadienyl clotrimazole complex, [RuCp(PPh₃)₂(CTZ)](CF₃SO₃), where Cp = cyclopentadienyl, CTZ = clotrimazole, was synthesized and characterized. The compound was evaluated <i>in vitro</i> on <i>T. cruzi</i> (Y strain), the infective form of <i>T. brucei brucei</i> strain 427 (cell line 449), on three human tumor cell lines with different sensitivity to cisplatin (A2780, ovary; MCF7, breast; HeLa, cervix) and on J774 murine macrophages as mammalian cell model. The new compound is more cytotoxic on <i>T. cruzi</i> and on the tumor cell lines than the reference drugs (Nifurtimox and cisplatin, respectively). In addition, complexation of the bioactive CTZ to the {RuCp(PPh₃)} moiety leads to significant increase of the antiparasitic and antitumoral activity. To get insight into the potential “dual” mechanism of antiparasitic action emerging from the presence of Ru(II) and CTZ in a single molecule, the inhibitory effect of this new complex on the biosynthesis of <i>T. cruzi</i> sterols of membrane and the interaction with DNA were studied. Although the tested complex does not affect DNA, it affects the <i>T. cruzi</i> biosynthetic pathway of conversion of squalene to squalene oxide. According to the results reported here, [RuCp(PPh₃)₂(CTZ)][CF₃SO₃] could be considered a prospective antiparasitic and/or antitumoral agent that deserves further evaluation.</p></div

Screening of TGR inhibitors.

<p>The % of inhibition of TR activity of <i>E. granulosus</i> TGR for each compound is shown (compound structures can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035033#pone.0035033.s002" target="_blank">Table S1</a></b>). The screening was carried out with 10 µM inhibitors, except for those marked with an asterisk, which were assayed at 1 µM (these compounds showed non-constant baselines at 10 µM in control experiments without enzyme). A 1 nM TGR was used in all cases. <b>a.</b> Oxadiazole <i>N</i>-oxides, <b>b.</b> Benzofuroxans, <b>c.</b> Thiadiazoles, <b>d.</b> Various compound families: quinoxalines (<b>61</b>, <b>62</b> and <b>65</b>), nitrooxy (<b>64</b>) and oxathiazole (<b>63</b>).</p

Characteristics of active and inactive compounds.

a<p>Highest occupied molecular orbital energy.</p>b<p>Lowest unoccupied molecular orbital energy.</p>c<p>HOMO-LUMO energy gap.</p>d<p>Octanol/water partition coefficient using Ghose-Crippen method.</p>e<p>Solvation energy using Truhlar model SM5.4.</p>f<p>Dipolar moment.</p

<i>F. hepatica</i> newly excysted juveniles viability <i>in vitro</i> after 48 h incubation with TGR inhibitors at 20 µM.

<p>A control with vehicle alone (DMSO) was also included and did not affect NEJ viability.</p

Structures of the families of compounds assayed as inhibitors of flatworm TGR (all compounds assayed are shown in Table S1).

<p>R, R₁-R₄: variable.</p

Effect of preincubation time on TR activity inhibition of TGR.

(*)<p>1.5 µM of compound 1 was incubated with 1 nM enzyme in the presence of NADPH and the reaction started by the addition of DTNB. Control experiments without inhibitors were carried out in parallel to determine the corresponding v_o (initial velocities without inhibitors).</p

Inhibition of TR activity of TGR by active compounds.

<p>Remaining TR activity as a function of inhibitor concentration is plotted for <b>a. 1</b>, <b>b. 2</b>, <b>c. 3</b> and <b>d. 50</b>. 1 nM TGR was used in all assays.</p

Structures of the most potent compounds described herein (2, 23, and 50) and several previously described TGR inhibitors ((I), (II), and (III)) ([13], [14]).

<p>Structural motives are marked.</p

Effect of TGR inhibitors on <i>F. hepatica</i> newly excysted juvenile, tested at 20 µM during 48 h.

<p>a. <b>2</b> (oxadiazole <i>N</i>-oxide), b. <b>1</b> (oxadiazole <i>N</i>-oxide), c. <b>4</b> (oxadiazole <i>N</i>-oxide), d. <b>61</b> (quinoxaline), e. <b>50</b> (thiadiazole), f. control.</p

<i>E. granulosus</i> protoscolex viability <i>in vitro</i> after 48 h incubation with TGR inhibitors at different concentrations.

<p>ND: not determined (since no effect was observed at the highest concentration). <b>42</b> (benzofuroxan control) and <b>19</b> (oxadiazole <i>N</i>-oxide control). A control with vehicle alone (DMSO) was also included and did not affect protoscolex viability.</p>(*)<p>% of inhibition of TR activity at 1 µM not at 10 µM.</p