Seiji Komeda

Next-generation anticancer metallodrugs

More than 99% of currently approved clinical drugs are organic compounds. In contrast, the percentage of metal-containing drugs (metallodrugs) is very low. In cancer chemotherapy, however, platinum coordination compounds represented by cisplatin and derivatives thereof are essential anticancer agents with proven effects against a variety of tumors. Because of the proven clinical applications of these platinum-based drugs, the number of research initiatives to identify other metallodrugs that can be used for cancer therapy has increased considerably in the field of inorganic biochemistry. Anticancer platinum compounds continue to be designed and synthesized through several different approaches in order to improve the therapeutic effects and to overcome the disadvantages of current platinum-based drugs. The use of transition metal compounds other than platinum has also attracted attention. Gold coordination complexes, for instance, demonstrate outstanding cytotoxic properties, and certain ruthenium complexes possess a strong ability to inhibit metastases of solid invasive tumors. In this review, the potential of anticancer metallodrugs is described and representative examples from the most recent families of Pt-, Ru-, and Au-based compounds are discussed with respect to their possible modes of action and most probable biomolecular targets.

The phosphate clamp: a small and independent motif for nucleic acid backbone recognition

The 1.7 Å X-ray crystal structure of the B-DNA dodecamer, [d(CGCGAATTCGCG)]2 (DDD)-bound non-covalently to a platinum(II) complex, [{Pt(NH3)3}2-µ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](NO3)6 (1, TriplatinNC-A,) shows the trinuclear cation extended along the phosphate backbone and bridging the minor groove. The square planar tetra-am(m)ine Pt(II) units form bidentate N-O-N complexes with OP atoms, in a Phosphate Clamp motif. The geometry is conserved and the interaction prefers O2P over O1P atoms (frequency of interaction is O2P > O1P, base and sugar oxygens > N). The binding mode is very similar to that reported for the DDD and [{trans-Pt(NH3)2(NH2(CH2)6(NH3+)}2-µ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](NO3)8 (3, TriplatinNC), which exhibits in vivo anti-tumour activity. In the present case, only three sets of Phosphate Clamps were found because one of the three Pt(II) coordination spheres was not clearly observed and was characterized as a bare Pt2+ ion. Based on the electron density, the relative occupancy of DDD and the sum of three Pt(II) atoms in the DDD-1 complex was 1:1.69, whereas the ratio for DDD-2 was 1:2.85, almost the mixing ratio in the crystallization drop. The high repetition and geometric regularity of the motif suggests that it can be developed as a modular nucleic acid binding device with general utility.

A Tetrazolato-Bridged Dinuclear Platinum(II) Complex Exhibits Markedly High in vivo Antitumor Activity against Pancreatic Cancer

Some platinum(II) coordination complexes are effective anticancer agents. cis-Diamminedichloridoplatinum(II) (cisplatin), a mononuclear platinum(II) complex, is one of the most commonly used anticancer drugs. Although platinum-based chemotherapy can cause serious side effects, its efficacy has prompted the design and synthesis of next-generation anticancer platinum(II) drugs which are effective against cancers that are typically resistant to chemotherapy, such as lung cancer, pancreatic cancer, and platinum-refractory cancer. 3] Lung cancer is the leading cause of cancer deaths worldwide, and non-small-cell lung cancer (NSCLC) accounts for 80 % of lung cancers. Pancreatic cancer remains the fourth leading cause of cancer-related deaths in the United States. Clinical platinum-based drugs show antitumor efficacy by forming Pt–DNA adducts. We and others have reported that azolato-bridged dinuclear Pt complexes such as [{cis-Pt(NH3)2}2(m-OH)(m-pyrazolato)](NO3)2 (1) and [{cis-Pt(NH3)2}2(mOH)(m-1,2,3-triazolato-N1,N2)](NO3)2 (2), interact with DNA through a mechanism different from that of cisplatin and exhibit much higher in vitro cytotoxicity than cisplatin. The chemical structures of 1 and 2 are shown in Figure 1. To further expand drug discovery, we designed two new tetrazolatobridged complexes, [{cis-Pt(NH3)2}2(m-OH)(m-tetrazolatoN1,N2)](ClO4)2 (3) and [{cis-Pt(NH3)2}2(m-OH)(m-tetrazolatoN2,N3)](ClO4)2 (4), which are structural isomers (Figure 1). Herein we report their synthesis, characterization, in vitro cytotoxicity, and preliminary in vivo antitumor efficacy. Complexes 3 and 4 were synthesized by using a modified protocol for azolato-bridged complexes, as previously described (Scheme 1). A slight excess of tetrazole was added to an aqueous solution of the starting material, [cis-Pt(NH3)2(mOH)]2(NO3)2, which was then incubated at 40 8C in the dark to yield 3 and 4 at a molar ratio of 6.5:3.5 (see Supporting Information). This preparation ratio likely resulted from the higher nucleophilicity of N1 or N4 bound to one carbon and one nitrogen atom, compared with N2 or N3 bound to two nitrogen atoms. The two structural isomers were separated and purified by reversed-phase liquid chromatography and characterized by H, C, and Pt NMR spectroscopy and ESI mass spectrometry. For 3, two Pt NMR chemical shifts appeared at 2127 and 2177 ppm, reflecting two slightly different Pt[N3O] environments, because the tetrazolato bridge is arranged in an asymmetric fashion; there is N1,N2 coordination of Pt atoms. In contrast, for 4, a single Pt NMR chemical shift appears at about 2180 ppm, confirming the Pt[N3O] environment and the symmetric structure of the complexes on the tetrazolato bridge; there is N2,N3 coordination of Pt atoms. We tested the cytotoxicity of our azolato-bridged dinuclear Pt complexes, along with cisplatin for comparison, on the H460 human NSCLC cell line; the IC50 values are listed in Table 1. Complex 1 showed no activity at concentrations Figure 1. Structures of (1) [{cis-Pt(NH3)2}2(m-OH)(m-pyrazolato-N1,N2)] 2 + , (2) [{cis-Pt(NH3)2}2(m-OH)(m-1,2,3-triazolato-N1,N2)] 2+ , (3) [{cis-Pt(NH3)2}2(m-OH)(mtetrazolato-N1,N2)] + , and (4) [{cis-Pt(NH3)2}2(m-OH)(m-tetrazolato-N2,N3)] 2 +

Highly efficient DNA compaction mediated by an in vivo antitumor-active tetrazolato-bridged dinuclear platinum(II) complex.

We investigated the effects of antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-tetrazolato-N(1),N(2))](2+) (1) and [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-tetrazolato-N(2),N(3))](2+) (2) on the higher-order structure of a large DNA molecule (T4 phage DNA, 166 kbp) in aqueous solution through single-molecule observation by fluorescence microscopy. Complexes 1 and 2 cause irreversible compaction of DNA through an intermediate state in which coil and compact parts coexist in a single DNA molecule. The potency of compaction is in the order 2 > 1 ≫ cisplatin. Transmission electron microscopic observation showed that both complexes collapsed DNA into an irregularly packed structure. Circular dichroism measurements revealed that the dinuclear platinum(II) complexes change the secondary structure of DNA from the B to C form. These characteristics of platinum(II) complexes are markedly different from those of the usual condensing agents such as spermidine(3+) and [Co(III)(NH(3))(6)](3+). The ability to cause DNA compaction by the platinum(II) complexes is discussed in relation to their potent antitumor activity.

Synthesis of antitumor azolato-bridged dinuclear platinum(II) complexes with in vivo antitumor efficacy and unique in vitro cytotoxicity profiles

We synthesised four tetrazolato-bridged dinuclear Pt(ii) complexes, [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)](n+), where R is CH3 (1), C6H5 (2), CH2COOC2H5 (3), or CH2COO(-) (4) and n = 2 (1-3) or 1 (4). Their structures were characterised by (1)H, (13)C, and (195)Pt NMR spectroscopy, mass spectrometry, and elemental analysis, and the crystal structure of 1 was determined by X-ray crystallography. The cytotoxicities of the complexes to human non-small-cell lung cancer (NSCLC) cell lines sensitive and resistant to cisplatin were assayed. Complex 1 was more cytotoxic than cisplatin in both PC-9 and PC-14 NSCLC cell lines, and cross-resistance to 1 in the cisplatin-resistant cells was largely circumvented. Complex 3 was moderately cytotoxic, whereas 2 and 4 were only marginally cytotoxic. We also determined the growth inhibitory activities of 1 and 3, as well as prototype azolato-bridged complexes [{cis-Pt(NH3)2}2(μ-OH)(μ-pyrazolato)](2+) (AMPZ), [{cis-Pt(NH3)2}2(μ-OH)(μ-1,2,3-triazolato-N1,N2)](2+) (AMTA), [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N1,N2)](2+) (5-H-X), and [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N2,N3)](2+) (5-H-Y), against a panel of 39 human cancer cell lines (JFCR39). The average 50% growth inhibition concentrations of the complexes against the JFCR39 cell lines ranged from 0.933 to 23.4 μM. The cytotoxicity fingerprints of the complexes based on the JFCR39 cytotoxicity data were similar to one another but completely different from the fingerprints of clinical platinum-based anticancer drugs. Complex 3 exhibited marked antitumor efficiency when tested in vivo on xenografts of PANC-1 pancreatic cancer in nude mice. The high potency of 3 confirmed that the tetrazolato-bridged structure exhibits high in vivo antitumor efficacy.

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH3)2}2(µ-OH)(µ-1,2,3-ta-N1,N2)](NO3)2 (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.

An in vivo highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex largely circumvents in vitro cisplatin resistance: two linkage isomers yield the same product upon reaction with 9-ethylguanine but exhibit different cytotoxic profiles

Cytotoxicity assays of azolato-bridged dinuclear Pt(II) complexes, [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-azolato)](2+), where the azolato was pyrazolato (1), 1,2,3-triazolato-N1,N2 (2), tetrazolato-N1,N2 (3), or tetrazolato-N2,N3 (4), were performed in cisplatin-sensitive and -resistant human non-small-cell lung cancer cell lines (PC-9 and PC-14). These complexes largely circumvented the cisplatin resistance in both cell lines, with resistance factors for 1-4 in the range of 0.5-0.8 and 0.9-2.0 for PC-9 and PC-14 cells, respectively. Complex 4 exhibited approximately 10 times the cytotoxicity of 3. When 3 and 4 were reacted with 2 molar equiv. of 9-ethylguanine (9EtG), they yielded an identical product, [{cis-Pt(NH(3))(2)(9EtG-N7)}(2)(μ-tetrazolato-N1,N3)](3+), that had N1,N3 platinum coordination through a Pt(II) migration process on the tetrazolate ring. The second-order rate kinetics of these isomers were almost the same as each other and faster than those of 1 and 2. The cytotoxicity of azolato-bridged complexes, except for 3, increases as their kinetic rates in the 9EtG reaction increase.

DNA conformation and repair of polymeric natural DNA damaged by antitumor azolato-bridged dinuclear PtII complex

Design of new antitumor Pt drugs is currently also focused on those new Pt complexes which form on DNA major adducts that can hardly be removed by DNA repair systems. An attempt of this kind has already been done by designing and synthesizing new antitumor azolato-bridged dinuclear Pt(II) complexes, such as [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-pyrazolate)](2+) (AMPZ). This new Pt(II) complex exhibits markedly higher toxic effects in some tumor cell lines than conventional mononuclear cisplatin. The primary objective in the present study was to further delineate differences in the interactions of AMPZ and cisplatin with natural, high-molecular-mass DNA using a combination of biochemical and molecular biophysics techniques. The results demonstrate for the first time that little conformational distortions induced by AMPZ in highly polymeric DNA with a random nucleotide sequence represent a structural motif recognizable by DNA repair systems less efficiently than distortions induced by cisplatin. Thus, DNA adducts of azolato-bridged dinuclear Pt(II) complexes can escape repair mechanisms more easily than those of cisplatin, which may potentiate antitumor effects of these new metallodrugs in cancer cells.

Second- and higher-order structural changes of DNA induced by antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes with different types of 5-substituent.

Here, we used circular dichroism (CD) and fluorescence microscopy (FM) to examine the interactions of a series of antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes, [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)](n+) (R=CH3 (1), C6H5 (2), CH2COOCH2CH3 (3), CH2COO(-) (4), n=2 (1-3) or 1 (4)), which are derivatives of [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N2,N3)](2+) (5-H-Y), with DNA to elucidate the influence of these interactions on the secondary or higher-order structure of DNA and reveal the mechanism of action. The CD study showed that three derivatives, 1-3, with a double-positive charge altered the secondary structures of calf thymus DNA but that 4, the only complex with a single positive charge, induced almost no change, implying that the B- to C-form conformational change is influenced by ionic attraction. Unexpectedly, single-molecule observations with FM revealed that 4 changed the higher-order structure of T4 DNA into the compact-globule state most efficiently, at the lowest concentration, which was nearly equal to that of 5-H-Y. These contradictory results suggest that secondary structural changes are not necessarily linked to higher-order ones, and that the non-coordinative interaction could be divided into two distinct interactions: (1) ionic attraction and (2) hydrogen bonding and/or van der Waals contact. The relationship between diffusion-controlled non-coordinative DNA interactions and cytotoxicities is also discussed.

Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex

Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug–DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers.