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 work flow.

<p>The different steps, the most relevant assay conditions and the go/no-go criteria of the screening campaign are indicated in boxes. The figures on the right refer to the number of compounds screened and that subsequently advanced during the campaign. From 144 compounds, 22 compounds lowered assay signal ≥ 45% for at least one TryS. From these 22, 7 BDA were false positive and the remaining 15 compounds were confirmed as enzyme inhibitors. Two of them are <b>AI</b> with potency in the submicromolar range against <i>Li</i>TryS. AI (P), 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives (P, paullone); APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives; BZ, benzofuroxan derivatives; BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives.</p

Inhibitors of tritryp trypanothione synthetase (TryS) identified in this work and their biological activity.

<p>Inhibitors of tritryp trypanothione synthetase (TryS) identified in this work and their biological activity.</p

Biological activity of compounds against infective <i>Trypanosoma brucei brucei</i> with downregulated expression of trypanothione synthetase (TryS).

<p><b>A)</b> Western blot analysis of cell extracts from 2x10⁷ <i>T</i>. <i>b</i>. <i>brucei</i> from the wildtype (WT), 48 h tetracycline-induced (+) and non-induced (-) TryS-RNAi cell line. Two hundred ng of recombinant <i>Tb</i>TryS was loaded as control. Bands from the molecular weight marker are indicated on left. The picture at the bottom shows the abundance of TryS for each condition as estimated by densitometry and expressed relative to the level of the WT cell line. <b>B)</b> Ponceau staining of the Western blot membrane that served as normalization control of protein load for each condition. <b>C)</b> Cytotoxicity (%) ± S.D. (n = 2) for tetracycline-induced (+) and non-induced (-) TryS-RNAi <i>T</i>. <i>b</i>. <i>brucei</i> treated with 5 μM nifurtimox or 100 nM EAP1-47.</p

Structure of compounds affecting tritryp trypanothione synthetase activity.

<p><b>AI (P)</b>, 4,5-dihydroazepino[4,5-<i>b</i>]indol-2(1<i>H</i>,3<i>H</i>,6<i>H</i>)-one derivatives, paullones derivatives, (FS-554 and MOL2008), five APPDA, 6-arylpyrido[2,3-<i>d</i>]pyrimidine-2,7-diamine derivatives (ZEA10, ZEA35, ZEA40, ZEA41 and ZVR159), eight BDA, <i>N</i>,<i>N'</i>-bis(3,4-substituted-benzyl) diamine derivatives (EAP1-47, EAP1-63, APC1-67, APC1-87, APC1-89, APC1-99, APC1-101 and APC1-111), seven BBHPP, 1-(benzo[<i>d</i>]thiazol-2-yl)-4-benzoyl-3-hydroxy-5-phenyl-1<i>H</i>-pyrrol-2(5<i>H</i>)-one derivatives (AD81, AD84, ADMRC158, ADKPN160, ADKPN161, ADKPN164 and ADKPN165), three BZ, benzofuroxan derivatives (J18, J20 and J31) and one PD, 1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione derivatives [(<i>Z</i>)-8-(2-(2,4-dihydroxybenzylidene)hydrazinyl)-7-(2-hydroxy-3-phenoxy propyl)-1,3-dimethyl-1<i>H</i>-purine-2,6(3<i>H</i>,7<i>H</i>)-dione, TC227]. iPr, tBu, OBn, Mo and Ph, correspond to an isopropyl, tert-butyl, O-benzyl, 4 -morpholinyl and phenyl substitution, respectively.</p

Trypanothione dependent redox metabolism.

<p>The chemical structure of trypanothione (<i>N</i>¹,<i>N</i>⁸-bis(glutathionyl)spermidine; T(SH)₂) is depicted at the center. Synthesis: trypanothione synthetase catalyzes the ligation of two molecules of gluthatione to one of spermidine using the energy provided by two ATP molecules. Regeneration: trypanothione reductase maintains trypanothione in the reduced state at expenses of NADPH, which can be supplied by the oxidative phase of the pentose phosphate pathway <i>via</i> glucose 6-phosphate dehydrogenase. Utilization: reduced trypanothione is involved in multiple functions such as the detoxification of xenobiotics, cell proliferation, defense against oxidants and protein thiol-redox homeostasis. The multipurpose oxidoreductase tryparedoxin plays an important role catalyzing electron transfer from T(SH)₂ to different molecular targets (e.g. peroxidases, ribonucleotide reductase and protein disulfides). G6P: glucose-6-phosphate, 6PGL: 6-phosphogluconolactone, T(SH)₂: reduced trypanothione, TS_2[ox]: oxidized trypanothione, NDPs: nucleosides diphosphate, dNDP: deoxinucleosides diphosphate, E^-: electrophilic species, TS-E: trypanothione-electrophile adduct, ROOH: hydroperoxide, ONOOH: peroxynitrite, NO₂^-: nitrite.</p