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A fundamental problem obscuring the role of the ammine and primary amine groups in the activity of clinically used Pt anticancer drugs is the dynamic character of the adducts with DNA and DNA constituents. Dynamic motion is slower in analogues containing only secondary or tertiary amines, but such agents are not used clinically. Recently we found that enclosing the N center within a piperidine (pip) ring greatly reduces dynamic motion. In this work, we test the hypothesis that a diamine with only one pip ring, 2-aminomethylpiperidine (pipen), would slow dynamic motion enough for insightful study of adducts with one site (cis to the primary amine) closely reflecting the coordination environment of clinically used drugs. Racemic pipen was prepared and resolved by improved methods. PtCl(2)(pipen) synthesized with the pipen enantiomer having an R configuration of the asymmetric carbon (determined on the basis of the [alpha](D) sign) has the S stereochemistry at the N asymmetric center. In the adduct (S,R)-pipenPt(5'-GMP)(2), restricted rotation of the two nonequivalent N7-coordinated 5'-GMP's about the Pt-N7 bonds potentially could lead to two head-to-tail (LambdaHT and DeltaHT) and two head-to-head (HH(1) and HH(2)) atropisomers. However, 1D and 2D NOESY NMR data at pH approximately 3 indicated the dominance of the two HT atropisomers in a LambdaHT:DeltaHT ratio of 2:1. Deprotonation of the phosphate group (pH 7) further stabilized the LambdaHT form, and the CD signal had the shape characteristic of a LambdaHT form with a positive peak at approximately 280 nm. However, at pH 9.5, where the 5'-GMP N1H was largely deprotonated, the NMR spectrum and the approximately 280 nm CD peak both revealed that the LambdaHT form had decreased. When the pH was jumped down to 6.9, the NMR signals of the LambdaHT form and the approximately 280 nm CD peak increased with a half-time of approximately 3 min. Thus, the pip ring lengthens the atropisomerization time from seconds for ethylenediaminePt(5'-GMP)(2) to minutes for (S,R)-pipenPt(5'-GMP)(2). This pH jump experiment indicates that the signs of the CD signal are opposite for the LambdaHT and DeltaHT forms. Changes with pH in both the relative abundance and shifts of the H8 signals of the LambdaHT and DeltaHT forms correlated with an increase in hydrogen bonding by the phosphate group of the 5'-GMP cis to the primary amine. The hydrogen bonding changes the 5'-GMP base tilt and hence the H8 chemical shift. Such information is not obtainable on 5'-GMP adducts of clinically used anticancer drugs.

Chirality-controlling chelate (CCC) ligands are a class of chiral diamine ligands with one or two chiral secondary amine ligating groups. Analogues of platinum anticancer agents containing CCC ligands exhibit unusual steric and dynamic features. In this study NMR and CD methods were used to evaluate the influence of the N9 substituent in guanine derivatives (G) on conformer distribution in one class of (CCC)PtG(2)() complexes. We employed the CCC ligand, N,N'-dimethyl-2,3-diaminobutane [Me(2)()DAB with S,R,R,S or R,S,S,R configurations at the four asymmetric centers, N, C, C, and N]. For each Me(2)()DABPtG(2) complex, the presence of four G H8 signals demonstrated formation of all three possible atropisomers: DeltaHT (head-to-tail), LambdaHT, and HH (head-to-head). Different G ligands (5'-GMP, 3'-GMP, 1-MeGuo, Guo, or 9-EtG) were chosen to assess the effect of the N9 substituent on the relative stability and spectral properties of the atropisomers. The conformations of the atropisomers of Me(2)()DABPtG(2) were determined from CD spectra and from NOE cross-peaks (assigned via COSY spectra) between G H8 signals and those for the Me(2)()DAB protons. Regardless of the N9 substituent, the major form was HT. However, this form had the opposite chirality, LambdaHT and DeltaHT, and base tilt direction, left- and right-handed, respectively, for the S,R,R,S and R,S,S,R configurations of the Me(2)()DAB ligand. Thus, the chirality of the CCCligand, not hydrogen bonding, is the most important determinant of conformation. For each Me(2)()DABPtG(2) complex, the tilt direction of all three atropisomers is the same and, except for 5'-GMP, the order of abundance was major HT > minor HT > HH. For 5'-GMP, the HH atropisomer was three times as abundant as the minor HT species, suggesting that phosphate-NH(Me(2)()DAB) hydrogen bonds could be present since such bonding is possible only for the 5'-GMP derivatives. However, if such phosphate-NH hydrogen bonds exist, they are weak since the percentage of the major HT form of 5'-GMP complexes is similar and indeed can be smaller compared to this percentage for complexes with other G's. The CD spectra of all (S,R,R,S)-Me(2)()DABPtG(2) complexes were similar and opposite to those of all (R,S,S,R)-Me(2)()DABPtG(2) complexes, indicating the CD signature is characteristic of the dominant HT conformer, which has a chirality dictated by the chirality of the CCC ligand and not the N9 substituent.

We synthesized a novel platinum drug, cis-[PtCl(NH3)2(N7-ACV)]+, in which ACV is the antiviral drug acyclovir [a deoxyriboguanosine analogue, 9-(2-hydroxyethoxymethyl)guanine]. This new compound exhibits antiviral efficacy in vitro and exhibits an antitumor activity profile different from that of cisplatin [Metal-Based Drugs 2:249-256 (1995)]. To contribute to understanding the mechanisms underlying biological activity of this new compound, we studied modifications of natural and synthetic DNAs in cell-free media by cis-[PtCl(NH3)2(N7-ACV)]+ by various biochemical and biophysical methods. The results indicated that the major DNA adduct of cis-[PtCl(NH3)2(N7-ACV)]+ was a stable monofunctional adduct at guanine residues. In contrast to DNA adducts of other monodentate and clinically ineffective platinum(II) compounds, the adducts of cis-[PtCl(NH3)2(N7-ACV)]+ terminated in vitro DNA and RNA synthesis. In addition, although DNA adducts of cis-[PtCl(NH3)2(N7-ACV)]+ and cisplatin were different, some properties of DNA modified by either compound were qualitatively similar. Such similarities were not noticed if DNA modifications by other ineffective monofunctional platinum(II) complexes were investigated. Thus, the DNA binding mode of monofunctional cis-[PtCl(NH3)2(N7-ACV)]+ was different from that of other monofunctional but ineffective platinum(II) complexes. It has been suggested that the unique capability of cis-[PtCl(NH3)2(N7-ACV)]+ to modify DNA may be relevant to a distinct antitumor efficiency of this novel drug in comparison with cisplatin. It also has been suggested that at least some aspects of DNA interactions of cis-[PtCl(NH3)2(ACV)]+ revealed in the current study could be exploited in the search for and development of new antiviral platinum complexes containing, as a part of the coordination sphere, antiviral nucleosides.

Complexes of the type [PtX(2)(Me(2)DAP)] (Me(2)DAP = 2,4-bis(methylamino)pentane) have been prepared and studied by (1)H NMR spectroscopy, molecular mechanics/dynamics (MMD) calculations, and X-ray crystallography. The coordinated Me(2)DAP ligand has four asymmetric centers. Complexes with an enantiomeric form of the ligand (e.g., RR-Me(2)DAP, RR being the configurations of the two asymmetric carbons) have three possible configurations, namely, SRRR, RRRR, and SRRS at N, C, C, and N, respectively. Indeed these three isomers were obtained in respective ratios of 9:1: approximately 0 for X = I(-) and of 7:3:1 for X = Cl(-). Complexes with the meso ligand (RS-Me(2)DAP) have four possible configurations, i.e., SRSR, RRSS, RRSR, and SRSS at N, C, C, and N, respectively (the latter two constitute an enantiomeric pair). Only the two symmetrical isomers were obtained in the ratios of 9:1 for X = I(-) and 1:1 for X = Cl(-). In addition, the preferred chelate ring conformations in solution (CDCl(3)) were determined to be the following: delta-chair (SRRR), lambda-skew (RRRR), fluxional chair (SRRS), fluxional skew (SRSR), and lambda-chair (RRSS). This information was used to assess the contributions of intra- and interligand interactions to determine the conformational stability. MMD calculations employing the AMBER force field [as modified by Yao et al. (Yao, S.; Plastaras, J. P.; Marzilli, L. G. Inorg. Chem. 1994, 33, 6061) and extended to include parameters for the Cl(-) ligands] provided minimum-energy structures for all five X = Cl(-) complexes. These structures agreed well with the experimentally determined solution conformations except for SRSR, which had a lowest energy delta-chair instead of skew conformation. X-ray structural studies of the SRSR species (X = Cl(-) and X = I(-)) confirmed the delta-chair conformation. The results suggest that an equatorial N-methyl group has nonbonded clashes with the cis chloride ligand; therefore, axial N-methyl groups are favored. However, in solution, the solvent and entropy (connected with fluxionality of conformers) are factors which, to some degree, can overcome the interligand steric interaction.

Recent findings that an analogue of clinically ineffective transplatin, trans-[PtCl2(E-iminoether)2], exhibits antitumor activity has helped reevaluation of the empirical structure-antitumor activity relationship generally accepted for platinum(II) complexes. According to this relationship, only the cis geometry of leaving ligands in the bifunctional platinum(II) complexes, should be therapeutically active. Global modifications of natural DNAs in cell-free media by trans-[PtCl2(E-iminoether)2] were studied through various molecular biophysical methods and compared with modifications by cis-[PtCl2(E-iminoether)2], transplatin, cisplatin, and monofunctional chlorodiethylenetriamineplatinum(II) chloride. Thus, the results of this study have extended our recent finding, indicating that the prevalent lesion occurring in double-helical DNA on its modification by trans-[PtCl2(E-iminoether)2] is a monofunctional adduct at guanine residues. The modification by trans-[PtCl2(E-iminoether)2] has been found to induce local distortions in DNA, which have a character differing fundamentally from those induced by both clinically ineffective or antitumor platinum complexes tested in this study. The different character of alterations induced in DNA by the adducts of trans-[PtCl2(E-iminoether)2] and transplatin has been suggested to be relevant to the unexpected observation that the new complex with leaving chloride groups in trans position exhibits antitumor efficacy. In addition, the results support the idea that platinum drugs that bind to DNA in a manner fundamentally different from that of cisplatin can exhibit altered biological properties, including differing spectra and intensities of antitumor activity.

The B-->Z transition of DNA modified by platinum(II) complexes has attracted considerable interest because of a possible relationship with the molecular mechanism of antitumor activity of these metal-based compounds. Until recently it was generally accepted that the cis geometry of leaving groups in the bifunctional platinum(II) complexes should be therapeutically active. This paradigm had to be abandoned recently due to the finding that several analogues of clinically ineffective trans-diamminedichloroplatinum(II) (transplatin) exhibit antitumor activity. One of these therapeutically active trans compounds is trans-dichlorobisiminoetherplatinum(II) (trans-EE) [iminoether = E-HN = C(OMe)Me]. In view of the fact that DNA is the main pharmacological target for platinum(II) complexes and that these complexes attack preferentially guanine residues in DNA, it is of interest to examine the effect of iminoether platinum(II) complexes on the conformation of double-stranded poly(dG-dC) and its synthetic analogues. As these polymers can undergo the B-->Z transition, we have investigated in detail the effect of trans-EE and its cis isomer (cis-EE) on this conformational transformation using different techniques. They include circular dichroism spectroscopy, Raman spectroscopy and immunochemical assay employing specific antibodies against Z-DNA. It has been shown that cis-EE somewhat facilitates the B-->Z transition induced by increasing NaCl concentration and radically lowers its cooperativity. The polymers modified by cis-EE at low or high salt concentrations have been found to adopt distorted conformations which belong to the B and Z families respectively. Thus, cis-EE affects the B-->Z transition in DNA in a way similar to antitumor cis-diamminedichloroplatinum(II) (cisplatin). On the other hand, trans-EE was found to affect B-->Z transition (its cooperativity or the NaCl concentration corresponding to the midpoint of the salt-induced transition) only slightly. This behavior of trans-EE was, however, fundamentally different from that of clinically ineffective transplatin, which hinders B-->Z transition and lowers its cooperativity. The different effects of trans-EE on the B-->Z transition in comparison with the effects of cisplatin, transplatin and monofunctional chlorodiethylenetriamineplatinum(II) chloride are consistent with a unique DNA binding mode of this new antitumor trans compound, which might be associated with its unexpected biological efficacy.

Proton magnetic spectroscopy (1H-MRS) investigation was performed on CSF samples of patients with neurological inflammatory diseases including 52 cases of multiple sclerosis (MS). 12 acute idiopathic polyneuropathies, 20 acute meningitides (10 viral and 10 bacterial). Spectra were compared with those acquired in 18 neurological controls. High CSF lactate levels were found in MS patients during clinical exacerbation of relapsing-remitting course (p = 0.036 vs neurological controls). In MS patients with MRI evidence of Gd-enhanced plaques CSF lactate was higher than in patients with MRI inactive plaques (p = 0.017). CSF lactate positivity correlated with number of CSF mononuclear cells in MS patients with clinical activity (p = 0.05) as well as in MS patients with MRI enhancement (p = 0.003). A comparative 1H-MRS investigation in vivo on localized demyelinating areas confirmed an elevated lactate signal in Gd-enhanced (61%) more frequently than in unenhanced (22%) plaques (p = 0.03). MS patients with high lactate signal in active plaques showed high lactate levels in CSF. Increased CSF lactate was found also in patients with acute meningitis and idiopathic polyneuropathy. These data suggest that changes in lactate levels may depend on anaerobic glycolytic metabolism in activated leukocytes during inflammatory diseases. A decrease of CSF formulate levels was found in MS patients during active and inactive clinical phase (p = 0.037, p=0.05 vs neurological controls respectively). Formate changes might be related to a disorder of choline-glycine cycle in MS. 1H-MRS in vivo showed significant increase of choline in acute plaques, whereas a decrease of N-acetyl aspartate was found in chronic plaques; these metabolites are undetectable in CSF. CSF glucose levels were lower in bacterial than in viral meningitis (p = 0.014) and in neurological controls (p = 0.05). These observations suggest that 1H-MRS may be able to detect CSF metabolic impairment in neurological inflammatory diseases. In MS some CSF findings reflect metabolic changes occurring in brain demyelinating areas, and they could be useful foe evaluation of disease activity in different stages of disease evolution.

The effect of the phenanthroline substituents on the structure and reactivity of platinum(II) and platinum(IV) complexes has been investigated. The X-ray crystal structures of the compounds [PtI(2)(4,7-Ph(2)phen)].CHCl(3) (1dz.CHCl(3)), [PtI(4)(4,7-Ph(2)phen)].CHCl(3) (2dz.CHCl(3)), [PtI(2)(2,9-Me(2)-4,7-Ph(2)phen)] (1fz), and [PtI(4)(2,9-Me(2)-4,7-Ph(2)phen)].I(2) (2fz.I(2)) have shown that complexes 1fz and 2fz, containing ortho-substituted phenanthrolines, exhibit a remarkable displacement of the equatorial iodine atoms from the N-Pt-N' plane (average 0.477(2) and 0.199(2) Å, respectively), a bending of the phenanthroline [angle between outer rings of 19.9(7) and 14.2(7) degrees, respectively] and a rotation of the N-C-C'-N' plane with respect to the N-Pt-N' plane [32.3(10) and 26.5(9) degrees, respectively]. Comparison between the structures of 1fz and 2fz, both having the phenanthroline with methyl substituents in the ortho position, indicates that, in the latter case, because of the presence of the two axial iodine ligands, the displacements of the ligands from the equatorial plane are smaller and find a compensation in a narrowing of the I(1)-Pt-I(1') angle (5 degrees ) and a lengthening of the Pt-N bonds (0.07 Å). The electrochemical behavior of the four-coordinate platinum(II) complexes shows that compounds possessing regular planar geometry have access to the one-electron reduced species, whereas those with distorted coordination geometry are irreversibly reduced by collapsing of the complex geometry. This is in sharp contrast with the behavior of related nickel complexes for which the pseudo-tetrahedral coordination imposed by bulky 2,9-substituents of phenanthroline stabilizes the nickel(I) species. Spectroscopic results allow us to assign a significant Pt(I) character to [1d](-) monoanions. The electrogenerated, plus one electron, complexes are not indefinitely stable and, because of conjugation with the phen ligand, progressively restore the Pt(II) oxidation state by transferring the electron to the peripheral organic ligand. The latter process can involve multiple electron additions in the macroelectrolysis time scale. The related platinum(IV) complexes [PtX(4)(L)] undergo irreversible two-electron reduction accompanied by fast release of the axial ligands and formation of the corresponding platinum(II) species.

The antitumor drugs 1-nitro-9-[(2-(dialkylamino)ethyl)amino]acridines (alkyl = Me, A(1); Et, A(2)) with platinum(II) give tridentate coordination compounds in which the two nitrogens of the ethylenediamine side chain and the C(8) carbon atom of the acridine ring system act as donor atoms. An excess of triphenylphosphine displaces the residual chloride coordinated to platinum but leaves unaltered the tridentate acridine ligand. The structures of [Pt(A(1)-H)Cl], 1, and [Pt(A(1)-H)(PPh(3))]Cl, 3, have been solved by single-crystal X-ray diffraction. 1.CHCl(3) crystallizes in the orthorhombic system, space group P2(1)2(1)2(1) (no. 19), with a = 8.715(1) Å, b = 11.045(2) Å, c = 22.609(4) Å, Z = 4, R = 0.0559, and R(w) = 0.0561 for 1502 reflections with F > 3sigma(F). 3.(1)/(2)CH(2)Cl(2) crystallizes in the monoclinic system, space group P2(1)/c (no. 14), with a = 13.418(3) Å, b = 14.053(3) Å, c = 18.918(4) Å, beta = 97.21(3) degrees, Z = 4, R = 0.0591, and R(w) = 0.0611 for 3608 reflections with F > 4sigma(F). In both complexes the acridine ligand adopts an imino-type configuration with the proton of the exocyclic 9-amino group shifted on N(10). Because of a severe steric interaction between the nitro group in the 1-position and the chelate diamine chain in the 9-position, the acridine moiety is folded about the C(9)-N(10) vector with an average angle between outer rings of 12 degrees. Moreover, the acridine aromatic moiety and the platinum coordination planes are twisted, forming a dihedral angle of ca. 20 degrees. The steric repulsion between the nitro and the diamine groups appears to provide the driving force to metalation. The H(10) proton has a great tendency to be engaged in H-bonding as shown by X-ray and solution studies. The formation of a H-bond with a rather poor acceptor such as the chloride ion can cause a downfield shift of the H-resonance as large as 6 ppm.

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.

The biological activity of cis and trans complexes of formula [PtCl2(HN = C(OMe)Me)2] has been investigated. The iminoether ligands can have either E or Z configuration about the C = N double bond, therefore EE, EZ and ZZ isomers are obtainable. Substitution of iminoether with EE configuration for amine leads to unexpectedly high antitumor activity for the complex with trans geometry which turns out to be more active than the cis congener in the P388 leukaemia system. The same trans-EE complex shows an activity comparable to that of cisplatin in reducing the primary tumour mass and lung metastases in mice bearing Lewis lung carcinoma, thus representing a trans platinum complex active on both limphoproliferative and solid metastasizing murine tumours. Also the cytotoxicity, the inhibition of DNA synthesis and the mutagenic activity, which are greater for the cis- with respect to the trans-isomer in the amine complexes, are instead greater for the trans- than for the cis- isomer in the case of iminoether compounds. Binding to calf thymus DNA is slower for iminoether complexes than it is for amine complexes, however after 24 h reaction time the level of binding is similar for both types of complexes. Trans-EE, like trans-DDP, does not give the DNA conformational alterations (terbium fluorescence) typical of antitumour-active cis- platinum compounds, but, under strictly analogous experimental conditions, shows a greatly reduced DNA interstrand cross-linking ability (heat denaturation/renaturation assay) with respect to either trans-DDP or cis-EE and cis-DDP. The data in hand point to a new trans platinum antitumour complex with a mechanism of action different from that of cis-DDP and classical analogues.

A platinum(II) complex with the antiviral drug acyclovir was synthesized and its antiviral and anticancer properties were investigated in comparison to those of acyclovir and cisplatin. The platinum-acyclovir complex maintained the antiviral activity of the parent drug acyclovir, though showing a minor efficacy on a molar basis (ID(50) = 7.85 and 1.02 muMu for platinum-acyclovir and cisplatin, respectively). As anticancer agent, the platinum-acyclovir complex was markedly less potent than cisplatin on a mole-equivalent basis, but it was as effective as cisplatin when equitoxic dosages were administered in vivo to P388 leukaemia-bearing mice (%T/C = 209 and 211 for platinum-acyclovir and cisplatin, respectively). The platinum-acyclovir complex was also active against a cisplatin-resistant subline of the P388 leukaemia (%T/C = 140), thus suggesting a different mechanism of action. The DNA interaction properties (sequence specificity and interstrand cross-linking ability) of platinum-acyclovir were also investigated in comparison to those of cisplatin and [Pt(dien)Cl](+), an antitumour-inactive platinum-triamine compound. The results of this study point to a potential new drug endowed, at the same time, with antiviral and anticancer activity and characterized by DNA interaction properties different from those of cisplatin.

The stereochemical factors that influence the tendencies for sequence specific binding of platinum antitumour drugs to DNA are examined. The NHs of the platinum-amine moiety can form hydrogen bonds to the O6 of guanine or to a phosphate oxygen of DNA. Modelling the stereochemistry of the NH atoms can lead to compounds with a strong preference for forming one type of adduct with DNA.

Platinum complexes with N,N'-bis(1-hydroxybut-2-yl)ethylenediamine, [PtCl2(ethambutol)] were prepared and the biological activity of three isomers [with (-), (+) and (+/-) ethambutol, respectively] investigated. All species interact with the Bam HI and Ava I recognition sequences showing a binding preference for GC rich sequences of DNA. The complex which showed the greatest interaction with adjacent guanines, [PtCl2[+/-)ethambutol)] was also found to be the most mutagenic of the three. On the other hand, only [PtCl2[+)ethambutol)] had a considerable antitumour activity against both P388 leukaemia and Lewis lung carcinoma, and this was not correlated either with restriction enzyme blocking activity or with mutagenicity.

The ability of enantiomeric platinum complexes to block the action of selected restriction enzymes has been investigated. The complexes [PtCl2(DAC)], [PtCl2(DAB)] and [PtCl2(DAP)] (DAC = 1,2-diamminocyclohexane; DAB = 2,3-diamminobutane; DAP = 1,2-diamminopropane) exhibit a guanine-cytosine preference in accord with previous results on cis-[PtCl2(NH3)2] (cis-DDP). The extent of inhibition, however, is significantly different for the different isomers; the R,R form is more active than the others at short incubation time, as the time becomes longer, the differences among isomers level off. It also appears that cis-DDP is more active than [PtCl2(DAC)] in blocking the Cfo I enzyme, though it shows a preference for the G-X-G sequences.