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Although the mechanisms of action of Ru(III) anticancer drugs including NAMI-A and KP1019 are not properly understood, it is reported that they are readily aquated in vivo before reacting to the biological targets and their cytotoxic activities are mainly attributed to their binding with DNA. In the present contribution in order to understand how changing the axial ligands affects the mechanisms of action of Ru(III)-based drugs which are structurally similar to NAMI-A, we study here the aquation reactions (substitution of one chloride anion (Cl−) ligand by a water molecule) of six structurally similar Ru(III) complexes (1–6) including NAMI-A (complex 1) and KP1019 (complex 2) as well as the reactions of their mono-aquated products with the N3, N7 and O6 sites of guanine at the M06-2X/(LanL2DZ+6-311+G**)//M06-2X/(LanL2DZ+6-31G**) level of density functional theory in gas phase and aqueous media. It is found that the aquation reaction of every Ru(III) complex occurs through a single transition state where a chloride ligand of the complex is replaced by a H2O molecule. The activation free energies (ΔGb) and rate constants of aquation reactions show that the complex 6 would get aquated in biological media more readily than other complexes. The rate constant of aquation of complex 6 is 7.98 × 10−3 s−1. It is evident from our calculations that axial ligands play important role in the aquation of Ru(III) complexes structurally similar to NAMI-A, but they do not obey any specific trends. Both the calculated reaction enthalpies (ΔHf) and reaction free energies (ΔGf) show that reactions of Ru(III) complexes (1–6) at the N3, N7 and O6 sites of guanine are exothermic and would occur spontaneously. It is found that KP1019 would react more favourably than NAMI-A at the N3, N7 and O6 of guanine, in agreement with experimental observations. Further, among all the complexes studied here, reactions of complex 6 at the different sites of guanine are most exothermic in aqueous media.

Ruthenium complexes offer the potential of reduced toxicity, a novel mechanism of action, non-cross resistance and a different spectrum of activity compared to platinum containing compounds. Thirteen novel ruthenium(II) organometallic arene complexes have been evaluated for activity (in vitro and in vivo) in models of human ovarian cancer, and cross-resistance profiles established in cisplatin and multi-drug-resistant variants. A broad range of IC50 values was obtained (0.5 to >100 μM) in A2780 parental cells with two compounds (RM175 and HC29) equipotent to carboplatin (6 μM), and the most active compound (HC11) equipotent to cisplatin (0.6 μM). Stable bi-dentate chelating ligands (ethylenediamine), a more hydrophobic arene ligand (tetrahydroanthracene) and a single ligand exchange centre (chloride) were associated with increased activity. None of the six active ruthenium(II) compounds were cross-resistant in the A2780cis cell line, demonstrated to be 10-fold resistant to cisplatin/carboplatin by a mechanism involving, at least in part, silencing of MLH1 protein expression via methylation. Varying degrees of cross-resistance were observed in the P-170 glycoprotein overexpressing multi-drug-resistant cell line 2780AD that could be reversed by co-treatment with verapamil. In vivo activity was established with RM175 in the A2780 xenograft together with non-cross-resistance in the A2780cis xenograft and a lack of activity in the 2780AD xenograft. High activity coupled to non cross-resistance in cisplatin resistant models merit further development of this novel group of anticancer compounds. British Journal of Cancer (2002) 86, 1652–1657. DOI: 10.1038/sj/bjc/6600290 www.bjcancer.com © 2002 Cancer Research UK

The antimetastatic ruthenium(III) complex (H2Im)[trans-RuCl4(HIm)(DMSO)] (NAMI-A) as well as its two analogues (H2Ind)[trans-RuCl4(HInd)(DMSO)] (Ru-Ind) and (HIsq)[trans-RuCl4(Isq)(DMSO)] (Ru-Isq) (HIm–imidazole, HInd–indazole, Isq–isoquinoline, DMSO–dimethyl sulfoxide) were tested for their effect on endothelial cell functions in vitro on human skin microvascular endothelial cells (HSkMEC) and human endothelial progenitor cells (HPEC-CB.2) under normoxic (21 % O2) and hypoxic (1 % O2) conditions. All studied complexes showed very low cytotoxicity profiles towards both mature microvascular and precursor endothelial cells (ECs), independently of oxygen concentration. Among tested compounds Ru-Ind exhibited the highest cytotoxicity. The antiangiogenic activity of ruthenium complexes was evaluated for their influence on pseudo-vessels formation by microvascular endothelial cells (HSkMEC) because of their involvement in melanoma progression. Our studies indicated that Ru-Ind and Ru-Isq exhibited hypoxia- and dose-dependent-inhibition of angiogenesis on Matrigel™. Significant hypoxia-selective downregulation of pseudo-vessels formation by Ru-Isq correlates with efficient inhibition of cell motility. Interestingly, in the applied concentration doses migration of endothelial cells was also inhibited by NAMI-A, but the pseudo-vessels formation on Matrigel™ was unaffected. Angiogenesis-related genes expression profile for both mature and precursor ECs indicated that inhibition of angiogenesis, mainly due to Ru-Isq, as compared to NAMI-A and Ru-Ind correlated with downregulation of CD31 and CD144 expression and upregulation of NOTCH4 expression in mature ECs, which is essential for endothelial cell motility and stalk cells organization control. The hypoxia-selective antiangiogenic activity of Ru-Ind and Ru-Isq, NAMI-A analogues makes them potent antimetastatic therapeutics for their selective action in hypoxia which controls tumor pathologic angiogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s10534-016-9974-9) contains supplementary material, which is available to authorized users.

Imidazolium trans-tetrachloridodimethylsulfoxideimidazolruthenate(III), NAMI-A, a novel antimetastatic ruthenium complex was investigated towards affinity to transferrin (Tf), whether Tf–Ru adducts might be formed after its intravenous injection. Studies were focused on the holotransferrin due to its preferential binding to transferrin receptor. Here, we showed that holotransferrin is able to bind NAMI-A as readily as apotransferrin. The simulation of biological conditions of human serum performed by application of simplified serum models allowed to analyse ruthenium distribution between transferrin and albumin. The presence of physiological concentration of albumin (ca. 18-fold excess over Tf) resulted in a twofold decrease of ruthenium binding to Tf. Interestingly, the introducing of low-molecular-mass components of serum dramatically increased the ruthenation of Tf. Intermolecular competition binding studies between transferrin and albumin showed that both proteins bound similar amount of ruthenium species. Investigation of NAMI-A binding to Tf in human serum showed that this protein was not the major binding partner for Ru complex. However, in spite of many competing proteins still the ruthenation of Tf was observed. The lack of free Ru species (protein unbounded) after incubation with human serum allowed to make an assumption of high affinity of NAMI-A towards serum proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00775-015-1255-5) contains supplementary material, which is available to authorized users.

AIM OF THE STUDY: The tumor metastases targeting ruthenium complex NAMI-A synergistically improves the activity of gemcitabine in combination therapies. High-throughput screening was used to identify other potential drug combinations from a library of FDA approved drugs. Doxorubicin was identified as a hit compound and was therefore evaluated in combination with NAMI-A in vitro and in a preclinical in vivo model. RESULTS: High-throughput screening identified eight structurally diverse compounds that synergize with NAMI-A including doxorubicin. The combination index on MCF-7 cells showed synergism as the concentration of NAMI-A increases independent of the doxorubicin concentration. In MCa mammary carcinoma of CBA mice, NAMI-A (35 mg/kg/day i.p. on days 7-12) followed by doxorubicin (10 mg/kg i.p. on day 16), significantly increased the effects of the individual drugs on metastases with 70 % animals resulting free of macroscopically detectable tumor nodules in the lungs at sacrifice. NAMI-A, unlike doxorubicin, cured 60 % of the treated mice but the combination therapy was toxic to the animals. CONCLUSIONS: The combined therapy of NAMI-A with doxorubicin synergizes on lung metastasis in a preclinical mouse model. The combination therapy at the maximum tolerated doses of the two drugs is toxic. Hence, this combination is not suitable for clinical studies using maximum tolerated doses.

Since the discovery of cis-platinum, many transition metal complexes have been synthesized and assayed for antineoplastic activity. In recent years, ruthenium-based molecules have emerged as promising antitumor and anti-metastatic agents with potential uses in platinum-resistant tumors or as alternatives to platinum. Ruthenium compounds theoretically possess unique biochemical features allowing them to accumulate preferentially in neoplastic tissues and to convert to their active state only after entering tumor cells. Intriguingly, some ruthenium agents show significant activity against cancer metastases but have minimal effects on primary tumors. Two ruthenium-based drugs, NAMI-A and KP1019, have reached human clinical testing. This review will highlight the chemical properties, mechanism of action, preclinical data, and early phase clinical results of these two lead ruthenium compounds. Other promising ruthenium agents will also be reviewed with emphasis on the novel ruthenium compound ONCO4417, and DW1/2 that has demonstrated Pim-1 kinase inhibition in preclinical systems. Further development of these and other ruthenium agents may rely on novel approaches including rational combination strategies as well as identification of potential pharmacodynamic biomarkers of drug activity aiding early phase clinical studies.

Following up on an earlier theoretical report by Knapp-Mohammady (Phys Lett A 372:18811884, 2008) on the ground state of the neutral Ru complex NAMI-A (trans-imidazoledimethylsulfoxide-tetrachlororuthenate), we first report here a quantum-chemical study of the effect of both oxidation and reduction of the parent molecule to form the anionic and cationic species. The new structures are compared with the equilibrium nuclear structure reported earlier for the neutral complex. We anticipate that one such Ru cluster, with potential as an anti-cancer drug, will interact via an appropriate receptor, rather than directly with DNA. A receptor for NAMI-A binding in here proposed to be the steroid hormone, estrogen, C18H24O2. The biomolecular structure of the dicomplex is predicted from restricted HartreeFock theory and density functional theory (DFT) calculations. The vibrational frequencies of NAMI-A and the dicomplex with estrogen are also reported. Some maps of the ground-state electron-density for the three neutral biomolecular species are finally presented. The use of vibrational spectroscopy, vibrational absorption (VA) and vibrational circular dichorism (VCD) are advocated to be measured, simulated and be used to understand the nature of the interaction of the Ru complex NAMI-A in complex with estrogen. Our aim in presenting these spectral simulations is to motivate the measurement of the VA and VCD spectra of estrogen, the Ru complex NAMI-A and finally of the estrogenRu NAMI-A complex. It should also be instructive to measure the VA and VCD spectra of estrogen and the estrogen receptor, both alone, together and finally together in the presence of the Ru NAMI-A complex to substantiate our claim that the Ru complex NAMI-A ties up estrogen, and hence prevents estrogen binding to the estrogen receptor.

Imidazolium-trans-dimethylsulfoxideimidazoletetrachlororuthenate (NAMI-A) is a ruthenium compound effective on solid tumor metastases. In this study, we evaluated the effects of different routes of administration of NAMI-A on the distribution to primary tumor, lungs and kidneys in BD2F1 hybrids with Lewis lung carcinoma or in CBA inbred mice with MCa mammary carcinoma. NAMI-A concentration and the percentage of cumulative dose (%Dtot) retained in these tissues is independent of the animal strain and of the tumor model used. Also the presence of the tumor does not change the distribution of NAMI-A in the lungs and in the kidneys. A dose-dependent antimetastatic effect is evident with intraperitoneal (i.p.) treatments at three different doses. Treatment of tumor bearing mice with NAMI-A administered i.p., per os or by aerosol showed a similar effect on lung metastases, although the concentration of ruthenium reached in the lungs was markedly different. On the basis of the data obtained, we can conclude that the antimetastatic effects are related to the amount of NAMI-A administered, rather than to the lung's concentration of the compound.

The binding to human lactoferrin of three Ru(III) complexes with anti-tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during transferrin-mediated delivery to cells. The complexes, HIm[RuIm2Cl4], HInd[RuInd2Cl4] and (HInd)2 [RuIndCl5], where Im = imidazole and Ind = indazole, were diffused into crystals of apo-lactoferrin (apoLf). X-ray diffraction data were collected to 2.6 A, 2.2 A and 2.4 A respectively. The binding sites for the Ru complexes were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallographically to final R factors of 0.202 (for 8.0 to 2.3 A data) and 0.192 (for 8.0 to 2.4 A data) respectively. Two types of binding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at His 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic ligands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine whether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His residues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific binding sites on proteins.

The antitumor and antimetastatic effects of a ruthenium(III) complex, Na[trans-RuCl4(DMSO)Im], have been examined in mice bearing MCa mammary carcinoma. Na[trans-RuCl4(DMSO)Im] is capable of reducing primary tumor growth and of prolonging the survival time with different schedules of administration. However, better effects were obtained (a) with treatments started soon after tumor implantation, (b) with daily administration rather than with treatments at 4-day intervals and (c) using relatively low daily doses. The prolongation of survival time in tumor-bearing hosts, greater than that obtained with cisplatin, cannot be simply related to the effect on primary tumor growth, always less than that of cisplatin. Rather, emphasis is placed on the reduction of lung metastasis formation, obtained either by i.v. injection of tumor cells (lung colonization or spontaneously from i.m. tumor implants, which is significantly greater than the reduction of primary tumor growth. The antimetastatic effect depends on the treatment schedule and is greater with low doses given daily than with high doses administrated with drug-free intervals. Metastasis reduction involved mainly large metastatic nodules, reducing the average weight of each metastasis by 8-fold for spontaneous metastases and by 5-fold for lung colonies. The antimetastatic action of Na[trans-RuCl4(DMSO)Im] thus indicates the possibility that related analogs can represent a new generation of antitumor compounds capable of selectively interacting with metastasis formation of solid tumors.