The development of ruthenium anticancer drugs

 

 

Two Pt(II) compounds, commercially known as cisplatin and carboplatin (Figure 1), developed in the early seventies, are currently the most widely used anticancer agents. Today there is hardly any clinical regimen of combination chemotherapy that does not contain either cisplatin or carboplatin.

Notwithstanding the widespread applications of platinum anticancer drugs, there is still a large need for the development of novel metal-based compounds with unprecedented features.

The search for “non-classical” metal antitumor drugs has since long stimulated investigations into the field of non-platinum metal drugs. Non-platinum active compounds are likely to have mechanism of action, biodistribution and toxicity which are different from those of platinum drugs and might therefore be active against human malignancies that are resistant, or have acquired resistance, to them. They might also show a reduced host toxicity. Ruthenium seems to be the most promising among the several metals investigated.

 

Since many years our group has actively worked in the field of inorganic antitumor drugs and has developed several Ru(II) and Ru(III) complexes with coordinated dimethylsulfoxide (dmso), which were shown to possess good antitumor and, above all, antimetastatic properties against animal models. Among these compounds a Ru(III) complex named NAMI-A, [ImH][trans-RuCl4(dmso-S)(Im)] (Im = imidazole) (Figure 2), was selected because of its very good antimetastatic activity and on October 1999 it was introduced into phase I clinical trials at the Netherland Cancer Institute of Amsterdam (NL). NAMI-A (Figure 3) is one of the very few non-platinum antitumor drugs, and the first ruthenium-based compound, to reach clinical phase trials.

Phase I clinical trials have been completed in fall 2002 and we expect to start phase II trials soon.

It is important to stress that metastases of solid tumors represent the main reason of failure in cancer therapy. In fact, while surgery and/or radiotherapy may successfully cure the primary lesions, many human tumors develop distant metastases that bring almost invariably to death. Because of the scattered location of metastases, drug therapy appears to be the best choice and, from the therapeutic standpoint, the development of new compounds endowed with a specific antimetastatic activity is a topic of paramount importance.

            We recently developed new dimeric Ru(III) compounds of formula Na2[{trans-RuCl4(dmso-S)}2(m-L)], in which L = heterocyclic bridging N-ligand (Figure 4). In such dimers, the coordinative environment of each Ru(III) nucleus is very similar to that of NAMI-A. The chemical behavior of the new Ru(III) dimers in physiological solution was also investigated; preliminary in vivo results showed that some of them have an antimetastatic activity comparable to that of NAMI-A at dosages that are 3.5 times lower in terms of moles of Ru.

The interactions of the active Ru complexes with their likely biological targets, i.e. DNA and proteins (in particular transferrin and albumin) were investigated in collaboration with other groups. In general, Ru(III) complexes bind DNA but much more weakly than platinum complexes. Thus the structural and conformational modifications produced on the DNA double helix are significantly smaller. Under physiological conditions, antitumor Ru(III) complexes bind tightly plasma proteins (albumin and transferrin), with a marked preference for surface imidazole groups; thus, very likely, protein binding of ruthenium(III) complexes has a large impact on the biodistribution, the pharmacokinetics and the mechanism of action of these experimental drugs.

 

In vitro and in vivo pharmacological experiments have shown that NAMI-A and other active ruthenium compounds are scarcely cytotoxic, suggesting that their mechanism of action is very likely different from that of platinum drugs and might be unrelated to interactions with DNA. One possibility for explaining the activity of NAMI-A against disseminated tumors is that it interferes with NO methabolism in vivo. Nitric oxide is known to play an important role in many biological functions, and recently it was demonstrated to be involved as mediator in one tumor-induced angiogenic process, which is a key step in the formation of metastases. NO is also known to interact in vivo with iron proteins, thus ruthenium action might also occurr through an iron-mimicking mechanism.

Within this general framework we investigated the reactivity of basic Ru(II)- and Ru(III)-chloride-dmso complexes and of NAMI-A towards NO with the goal of producing spectroscopically and structurally well characterized models. These will be used as reference compounds in subsequent biomimetic studies in physiological conditions. A summary of our results in this field (refs 22, 23), including the reactivity of some of the new Ru-dmso nitrosyls towards heterocyclic nitrogen ligands (N) is reported in Figure 5. The spectroscopic and X-ray structural features for all the new complexes are consistent with the {Ru(NO)}6 formulation, that is a diamagnetic Ru(II) nucleus bound to NO+. Electrochemical measurements on the Ru-NO complexes showed that they are all redox active in DMF solutions and the site of reduction is the NO+ moiety. The reduced complexes are not stable and rapidly release the NO· radical.

 

Moreover, spectroscopic studies indicated that in physiological conditions the active Ru(III) compounds, both monomers and dimers, are easily and quantitatively reduced to Ru(II) species by stoichiometric amounts of biological reducing agents, such as ascorbic acid, cysteine and glutathion. This important feature suggests that NAMI-A and the Ru(III)-dmso compounds might indeed be reduced also in vivo to generate Ru(II) active species.

 

We are also interested in  investigating the factors influencing the orientation and dynamic motions of planar N-donor heterocyclic ligands (L) coordinated to a metal center (M), since such features have broad relevance in metallobiochemistry; in particular, the moiety cis-M(L1)(L2), wherein L1 and L2 are either the same or different planar aromatic N-donor ligands, is common in biological systems and in particular in cross-links formed by DNA with metal anticancer drugs and in metalloenzymes. We have found that superior knowledge of the features of such units can be gained by studying complexes of the bioligand 1,5,6-trimethylbenzimidazole (Me3Bzm), which is a nucleopurine analog, and comparing the results of studies on analogs with the abiological pyridine ligands.

In collaboration with the group of Dr. Marzilli (LSU, Baton Rouge, Louisiana, USA) we have performed an extensive combined X-ray structural and solution NMR investigation of ruthenium(II) and dinuclear rhenium(V) Me3Bzm complexes which allowed us to establish the solution state structures and to detail the dynamic motions of the heterocyclic ligands (refs 1, 3, 13, 17).  The results obtained with Me3Bzm were particularly significant since they indicated that its dynamic behavior was strongly influenced not only by steric factors, but also by electrostatic interactions.

 

Recent Publications

 

1) E. Alessio, E. Zangrando, R. Roppa, L. G. Marzilli «Crystals containing conformers: a rare case in which a solid closely reflects a solution equilibrium mixture.» Inorg. Chem. 1998, 37, 2458-2463.

2) G. Mestroni, E. Alessio, A. Sessanta o Santi, S. Geremia, A. Bergamo,  G. Sava, A. Boccarelli, A. Schettino,  M. Coluccia  «Rhodium(III) analogs of antitumor active ruthenium(III) compounds. The crystal structure of [ImH][trans-RhCl4(Im)2] (Im = imidazole).» Inorg. Chim. Acta, 1998, 273, 62-71.

3) L. G. Marzilli, P. A. Marzilli, E. Alessio «Complexes of lopsided N-donor heterocyclic biologands: has the electrostatic effect of the N2CH proton been overlooked in metallobiochemistry?» Pure & Appl. Chem. 1998, 70, 961-968.

4) G. Sava, I Capozzi, K. Clerici, G. Gagliardi, E. Alessio, G. Mestroni «Pharmacological control of lung metastases of solid tumors by a novel ruthenium complex.» Clin. Exp. Metastasis 1998, 16, 371-379.

5) G. Sava, R. Gagliardi, M. Cocchietto, K. Clerici, I Capozzi, M. Marrella, E. Alessio, G. Mestroni, R. Milanino «Comparison of the effects of the antimetastatic compound ImH[trans-RuCl4(DMSO)(Im)] (NAMI-A) on the arthritic rat and on Mca mammary carcinoma in mice.» Pathol. Oncol. Res. 1998, 4, 30-36.

6) S. Geremia, S. Mestroni, M. Calligaris, E. Alessio  «The first example of a double bridged diruthenium(II) complex containing the rare bridging S,O bidentate dimethyl sulfoxide ligand which defines a stable five-membered ring.» J. Chem. Soc., Dalton Trans. 1998, 2447-2448.

7) G. Sava, K. Clerici, I Capozzi, M. Cocchietto, R. Gagliardi, E. Alessio, G. Mestroni, A. Perbellini  «Reduction of lung metastasis by ImH[trans-RuCl4(DMSO)(Im)]: mechanism of the selective action investigated on mouse tumors.» Anti-Cancer Drugs 1999, 10, 129-138.

8) A. Bergamo, R. Gagliardi, V. Scarcia, A. Furlani, E. Alessio, G. Mestroni, G. Sava  «In vitro cell cycle arrest, in vivo action on solid metastasizing tumors and host toxicity of the antimetastatic drug NAMI-A and of cisplatin.»  J. Parmacol. Exp. Ther. 1999, 298(1), 559-564.

9) G. Sava, E. Alessio, A. Bergamo, G. Mestroni «Sulfoxide ruthenium complexes: non toxic tools for the selective treatment of solid tumour metastases.» Topics in Biological Inorganic Chemistry, Volume 1 “Metallo-pharmaceuticals”, M. J. Clarke and P. J. Sadler eds., Springer, Berlin, 1999, pp.  143-169.

10) E. Iengo, G. Mestroni, S. Geremia, M. Calligaris, E. Alessio  «Novel Ru(III) dimers [Na]2{[trans-RuCl4(Me2SO-S)]2(m-L)} and {[mer,cis-RuCl3(Me2SO-S)(Me2SO-O)]2(m-L)} (L = bridging heterocyclic N-donor ligand) closely related to the antimetastatic complex Na[trans-RuCl4(Me2SO-S)(Him)].» J. Chem. Soc., Dalton Trans. 1999, 3361-3371.

11) G. Sava, R. Gagliardi, A. Bergamo, E. Alessio, G. Mestroni  «Treatment of metastases of solid mouse tumours by NAMI-A: comparison with cisplatin, cyclophosphamide and dacarbazine.» Anticancer Res. 1999, 19,  969-972.

12) E. Alessio, E. Iengo, S. Zorzet, A. Bergamo, M. Coluccia, A. Boccarelli, G. Sava  «Antimetastatic properties and DNA interactions of the novel class of dimeric Ru(III) compounds [Na]2{trans-RuCl4(Me2SO)]2(m-L)} (L = ditopic, non-chelating aromatic N-ligand). A preliminary investigation.» J. Inorg. Biochem. 2000, 79, 157-161.

13) E. Alessio, E. Zangrando, E. Iengo, M. Macchi, P. A. Marzilli, L. G. Marzilli  «Understanding Orientation and Dynamic Motion of Planar Heterocyclic N-Donor Ligands by Exploiting the Symmetry Properties of Mixed-Ligand m-oxo Rhenium(V) Dimers [ReOCl2(L)(L')]-O-[ReOCl2(L)(L')]: a Combined X-ray Structural and Dynamic NMR Investigation.» Inorg. Chem. 2000, 39, 294-303.

14) L. Messori, P. Orioli, D. Vullo, E. Alessio, E. Iengo  «A spectroscopic study of the reaction of NAMI, a novel ruthenium(III) anti-neoplastic complex, with bovine serum albumin. » Eur. J. Biochem. 2000, 267, 1206-1213.

15) E. Gallori, C. Vettori, E. Alessio, F. Gonzalez-Vilchez, R. Vilaplana, P. Orioli, A. Casini, L. Messori  «DNA as a possible target for antitumor Ruthenium(III) complexes.» Arch. Biochem. Biophys. 2000, 376(1), 156-162.

16) M. Cocchietto, G. Salerno, E. Alessio, G. Mestroni, G. Sava  «Fate of the antimetastatic ruthenium complex ImH[trans-RuCl4(DMSO)Im] after acute i.v. treatment in mice.» Anticancer Res., 2000, 20, 197-200.

17) E. Alessio,  E. Iengo, E. Zangrando, S. Geremia, P. A. Marzilli,  M. Calligaris  «Orientation and Restricted Rotation of Lopsided N-donor Heterocyclic Bioligands in Octahedral Ruthenium Complexes.» Eur. J. Inorg. Chem. 2000, 2207-2219.

18) S. Zorzet, A. Bergamo, M. Cocchietto, A. Sorc, B. Gava, E. Alessio, E. Iengo, G. Sava  «Lack of in vitro cytotoxicity, associated to increased G2-M cell fraction and inhibition of matrigel invasion, may predict in vivo-selective antimetastasis activity of ruthenium complexes.» J. Pharmacol. Expl. Ther., 2000, 295, 927-933.

19) B. Serli, E. Iengo, T. Gianferrara, E. Zangrando, E. Alessio  «Novel unsymmetrical Ru(iii) and mixed-valence Ru(iii)/Ru(ii) dinuclear compounds related to the antimetastatic Ru(iii) drug nami-a.» Metal Based Drugs, 2001, 8, 9-18.

20) J. Malina,·O. Novákova,·B. K. Keppler, E. Alessio, V. Brabec  «Biophysical analysis of natural, double-helical DNA modified by anticancer heterocyclic complexes of ruthenium(III) in cell-free media.» J. Biol. Inorg. Chem., 2001, 6, 435-445.

21) G. Sava, A. Bergamo, S. Zorzet, B. Gava, C. Casarsa, M. Cocchietto, A. Furlani, V. Scarcia, B. Serli, E. Iengo, E. Alessio G. Mestroni “Influence of chemical stability on the activity of the antimetastasis ruthenium compound NAMI-A.” Eur. J. Cancer, 2002, 38, 427-435.

22) B. Serli, E. Zangrando, E. Iengo, E. Alessio “Novel mono- and dinuclear ruthenium nitrosyls with coordinated pyrazine.”  Inorg. Chim. Acta, 2002, 339, 265-272.

23) B. Serli, E. Zangrando, E. Iengo, G. Mestroni, L. Yellowlees, E. Alessio “Synthesis, Structural, Spectroscopic, and Electrochemical Characterization of Novel Ruthenium-Dimethylsulfoxide Nitrosyls.” Inorg. Chem., 2002, 41, 4033 - 4043.

24) I. Turel, M. Pecanac, A. Golobic, E. Alessio, B. Serli “Novel Ru(III)-DMSO complexes of the antiherpes drug acyclovir.” Eur. J. Inorg. Chem., 2002, 1928-1931.

25) A. Bergamo, B. Gava, E. Alessio, G. Mestroni, B. Serli, M. Cocchietto, S. Zorzet, G. Sava “Ruthenium-based NAMI-A type complexes with in vivo selective metastasis reduction and in vitro invasion inhibition unrelated to cell cytotoxicity.” Int. J. Oncol. , 2002, 21, 4033 - 4043.

26) B. Serli, E. Zangrando, T. Gianferrara, L. Yellowlees, E. Alessio “Coordination and release of NO by ruthenium-dimethylsulfoxide complexes-implications for anti-metastases activity.” Coord. Chem. Rev. , 2003, 245, 73 - 83.

27) E. Zangrando, B. Serli, L. Yellowlees, E. Alessio “The first examples of four and five O-bonded dmso ligands on a ruthenium centre.” Dalton Trans. , 2003, , 4391 - 4392.

28) A. Bergamo, G. Stocco, B. Gava, M. Cocchietto, E. Alessio, B. Serli, E. Iengo, G. Sava “Distinct effects of dinuclear ruthenium(III) complexes on cell proliferation and on cell cycle regulation in human and murine tumor cell lines.” J. Pharmacol. Exp. Ther., 2003, 305, 725 - 732.

29) A. Bergamo, G. Stocco, C. Casarsa, M. Cocchietto, E. Alessio, B. Serli, S. Zorzet, G. Sava “Reduction of in vivo lung metastases by dinuclear ruthenium complexes is coupled to inhibition of in vitro humour invasion.” Int. J. Oncol. , 2004, 24, 373 - 379.

30) I. Turel, M. Pečanac, A. Golobič, E. Alessio, B. Serli, A. Bergamo, G. Sava “Solution, solid state and biological characterization of ruthenium(III)-DMSO complexes with purine base derivatives.” J. Inorg. Biochem. , 2004, 98, 393 - 401.

31) E. Alessio, G. Mestroni, A. Bergamo, G. Sava “Ruthenium Anticancer Drugs.” in "Metal Ions and Their Complexes in Medication and in Cancer Diagnosis and Therapy", Vol. 42 of Met. Ions Biol. Syst., A. Sigel and H. Sigel, eds., M. Dekker: New York Int. J. Oncol. , 2004, , 323 - 351.

32) M. Bacac, A. C. G. Hotze, K. van der Schilden, J. G. Haasnoot, S. Pacor, E. Alessio, G. Sava, J. Reedijk “The hydrolysis of the anti-cancer ruthenium complex NAMI-A affects its DNA binding and antimetastatic activity: an NMR evaluation.” J. Inorg. Biochem. , 2004, 98, 402 - 412.

33) A. H. Velders, A. Bergamo, E. Alessio, E. Zangrando, J. G. Haasnoot, C. Casarsa, M. Cocchietto, S. Zorzet, G. Sava “Synthesis and chemical-pharmacological characterization of the antimetastatic NAMI-A-type Ru(III) complexes (Hdmtp)[trans-RuCl4(dmso-S)(dmtp)],(Na)[trans-RuCl4(dmso-S)(dmtp)] and [mer-RuCl3(H2O)(dmso-S)(dmtp)] (dmtp=5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine).” J. Med. Chem. , 2004, 47, 1110 - 1121.

 

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