Prevention of HIV-1 infection by platinum triazines.

To identify and explore the activity of compounds which may act as anti-HIV virucidal agents, we have investigated platinum compounds, especially those containing N-donor aromatic ligands. After screening over 70 related agents, including N-donor aromatic ligands and metal precursors, we have identified a novel class of platinum(II) complexes with 2-pyridyl-1,2,4-triazine derivatives and Pt(II) formulations with these derivatives (ptt compounds) as having the highest anti-HIV activity. The maximum activity was observed when the agents were added immediately post-infection. The ptt agents did not block cell fusion activity of HIV-1 Env proteins in cells bearing CD4X4 or CD4R5 receptors, indicating a lack of interaction with the Env protein. The ptt compounds exhibit low toxicity for human epithelial cells, and are thus promising candidates for use as microbicides or antiviral agents against HIV.

A rare example of three abundant conformers in one retro model of the cisplatin-DNA d(GpG) intrastrand cross link. Unambiguous evidence that guanine O6 to carrier amine ligand hydrogen bonding is not important. possible effect of the Lippard base pair step adjacent to the lesion on carrier ligand hydrogen bonding in DNA adducts.

Guanine O6 to carrier ligand hydrogen bonding is a central feature of many hypotheses advanced to explain the anticancer activity of cis-type anticancer drugs, cis-PtA(2)X(2) (A(2) = diamine or two amines). Early structural evidence suggested that cis-Pt(NH(3))(2)(d(GpG)) (the cross-link model for the key cisplatin-DNA adduct) and other cis-PtA(2)(d(GpG)) adducts exist exclusively or mainly as the HH1 conformer with head-to-head (HH) bases. The dynamic motion of the d(GpG) in these adducts is too rapid to permit definitive characterization of both the conformation and the H-bonding. Hence, we use retro models having A(2) ligands designed to slow the motion. Here, we employ Me(2)ppz (N,N'-dimethylpiperazine), which lacks NH groups. Me(2)ppz is unique in having sp(3) N-methyl groups directly in the coordination plane, allowing the coexistence of multiple conformers but hindering dynamic motion in Me(2)ppzPt(d(GpG)) and Me(2)ppzPt(GpG) retro models. Dynamic processes are decreased enough in Me(2)ppzPt(d(GpG)) to permit HPLC separation of three abundant forms. After HPLC separation, the three re-equilibrate, proving that the three forms must be conformers and that Me(2)ppz has little influence on conformer distribution. This marks the first reported characterization of three abundant conformers for one cis-PtA(2)(d(GpG)) adduct. From NMR evidence, the Me(2)ppzPt(d(GpG)) HH1 conformer has uncanted bases. Another conformer, one of two recently discovered conformer types, has head-to-tail (HT) bases with Delta chirality. For this Delta HT1 form, several lines of evidence establish that the dinucleotide moieties have essentially identical structures in d(GpG) (and GpG) adducts of Me(2)ppzPt and other cis-PtA(2) complexes. For example, the shifts of the highly structure-sensitive G H8 NMR signals are almost identical for the Delta HT1 form of all adducts. In previous models, the stabilization of the Delta HT1 form could be attributed to G O6 H-bonding to A(2) NH groups. Such H-bonds are not possible for Me(2)ppz. The unambiguous conclusions are that G O6 H-bonding is weak and that neither canting nor H-bonding is essential in HH forms. These two features are present in almost all other small models but are essentially absent in the cross-link base pair (bp) step in duplexes. We conclude from our work that the forces favoring canting and H-bonding are weak, and we hypothesize that steric effects within the Lippard bp step adjacent to this cross-link bp step easily overcome these forces.

Synthesis and DNA binding of novel water-soluble cationic methylcobalt porphyrins.

Organocobalt derivatives of tetracationic water-soluble porphyrins are difficult to prepare via the typical reductive alkylation of the Co(II)(por) (porH(2) = porphyrin ligand). None have been reported. The problem may arise because the porphyrin core is made relatively electron poor by the positively charged peripheral groups. We have circumvented this problem by using the [Co(III)(NH(3))(5)CH(3)](2+) reagent, which inserts the Co(III)-CH(3) moiety directly into porH(2) in water under basic conditions. The method afforded two new [CH(3)Co(por)](4+) derivatives, [CH(3)CoTMpyP(4)](4+) and [CH(3)CoTMAP](4+), where [TMpyP(4)](4+) and [TMAP](4+) are the coordinated, NH-deprotonated forms of meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin and meso-tetrakis(N,N,N-trimethylaniliniumyl)porphyrin, respectively. The binding of the two new [CH(3)Co(por)](4+) cations to DNA and to the synthetic DNA polymers [poly(dA-dT)](2) and [poly(dG-dC)](2) was studied. Using published criteria by which changes in DNA viscosity and in the visible and CD spectra in the Soret region can be used to assess DNA binding, we conclude that both are outside binders. A large hypochromicity of the Soret bands of the [CH(3)Co(por)](4+) cations observed upon outside binding to DNA may indicate a high degree of self-stacking. The visible absorption and CD spectra of the [CH(3)Co(por)](4+) cations in the presence of 1:1 mixtures of [poly(dA-dT)](2) and [poly(dG-dC)](2) are nearly identical to those with [poly(dA-dT)](2) alone and are very different from those of [poly(dG-dC)](2) alone. Thus, both cations show a high preference for outside binding at AT-rich over GC-rich DNA sites. Upon binding of each of the [CH(3)Co(por)](4+) cations to all of the DNA polymers, the Soret bands exhibit blue shifts, whereas the Soret bands of the corresponding [(H(2)O)(2)Co(por)](5+) cations exhibit red shifts. The blue shifts strongly suggest that the [CH(3)Co(por)](4+) cations, particularly [CH(3)CoTMAP](4+), become five-coordinate forms to some extent on DNA binding; this result is the first good evidence for the presence at equilibrium of five-coordinate CH(3)Co(III)(N(4)) forms in water.

Relationship of solution and protein-bound structures of DNA duplexes with the major intrastrand cross-link lesions formed on cisplatin binding to DNA.

DNA bases in the three-base-pair (3bp) region of duplexes with the two major lesions of cisplatin (cis-PtCl(2)(NH(3))(2)) with DNA, namely d(XGG) and d(XAG) ( = N7-platinated base), differ in their relative positions by as much as approximately 3.5 A in structures in the literature. Such large differences impede drug design and assessments of the effects of protein binding on DNA structure. One recent and several past structures based on NMR-restrained molecular dynamics (RMD) differ significantly from the reported X-ray structure of an HMG-bound XGG 16-mer DNA duplex (Ohndorf, U.-M.; Rould, M. A.; He, Q.; Pabo, C. O.; Lippard, S. J. Nature 1999, 399, 708). This 16-mer structure has several significant novel and unique features (e.g., a bp step with large positive shift and slide). Hypothesizing that novel structural features in the XGG or XAG region of duplexes elude discovery by NMR methods (especially because of the flexible nature of the 3bp region), we studied an oligomer with only G.C bp's in the XGGY site by NMR methods for the first time. This 9-mer gave a 5'-G N1H signal with a normal shift and intensity and showed clear NOE cross-peaks to C NHb and NHe. We assigned for the first time (13)C NMR signals of a duplex with a GG lesion. These data, by adding NMR-based criteria to those inherent in NOESY and COSY data, have more specifically defined the structural features that should be present in an acceptable model. In particular, our data indicated that the sugar of the X residue has an N pucker and that the GG cross-link should have a structure similar to the original X-ray structure of cis-Pt(NH(3))(2)(d(pGpG)) (Sherman S. E.; Gibson, D.; Wang, A. H.-J.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 7368). With these restrictions added to NOE restraints, an acceptable model was obtained only when we started our modeling with the 16-mer structural features. The new X-ray/NMR-based model accounted for the NOESY data better than NOE-based models, was very similar in structure to the 16-mer, and differed from solely NOE-based models. We conclude that all XGG and XAG (X = C or T) duplexes undoubtedly have structures similar to those of the 16-mer and our model. Thus, protein binding does not change greatly the structure of the 3bp region. The structure of this region can now be used in understanding structure-activity relationships needed in the design of new carrier ligands for improving Pt anticancer drug activity.

Assessment of the existence of hyper-long axial Co(II)-N bonds in cobinamide B(12) models by using electron paramagnetic resonance spectroscopy.

Protein control of cobalt-axial nitrogen ligand bond length has been proposed to modulate the reactivity of vitamin B(12) coenzyme during the catalytic cycle of B(12)-dependent enzymes. In particular, hyper-long Co-N bonds may favor homolytic cleavage of the trans-cobalt-carbon bond in the coenzyme. X-ray crystallographic studies point to hyper-long bonds in two B(12) holoenzymes; however, mixed redox and ligand states in the crystals thwart clear conclusions. Since EPR theory predicts an increase in Co(II) hyperfine splitting as donation from the axial N-donor ligand decreases, EPR spectroscopy could clarify the X-ray results. However, the theory is apparently undermined by the similar splitting reported for the 2-picoline (2-pic) and pyridine (py) adducts of Co(II) cobinamide (Co(II)Cbi(+)), adducts thought to have long and normal Co-N axial bond lengths, respectively. Cobinamides, with the B(12) 5,6-dimethylbenzimidazole loop removed, are excellent B(12) models. We studied Co(II)Cbi(+) adducts of unhindered 4-substituted pyridines (4-X-py's) in ethylene glycol to separate orbital size effects from Co-N axial distance effects on these splittings. The linear increase in splitting with the decrease in 4-X-py basicity found is consistent with the theoretically predicted increase in unpaired electron spin density as axial N lone pair donation to Co(II) decreases. No adduct (and hence no hyper-long Co(II)-N axial bond) was formed even by 8 M 2-pic, if the 2-pic was purified by a novel Co(III)-affinity distillation procedure designed to remove trace nitrogenous ligand impurities present in 2-pic distilled in the regular manner. Adducts formed by impurities in 2-pic and other hindered pyridines misled previous investigators into attributing results to adducts with long Co-N bonds. We find that many 2-substituted py's known to form adducts with simple synthetic Co models do not bind Co(II)Cbi(+). Thus, the equatorial corrin ring sterically impedes binding, making Co(II)Cbi(+) a highly selective binding agent for unhindered sp(2) N-donor ligands. Our results resolve the apparent conflict between EPR experiment and theory. The reported Co(II) hyperfine splitting of the enzyme-bound cofactor in five B(12) enzymes is similar to that of the relevant free cofactor. The most reasonable interpretation of this similarity is that the Co-N axial bond of the bound cofactor is not hyper-long in any of the five cases.

Biodistribution and renal excretion of isomers of the cationic tracer, (99m)Tc diaminocyclohexane (DACH): biodistribution of cationic renal tracers.

The buildup of organic anions in the plasma in the uremic state can competitively inhibit the tubular extraction of para-aminohippurate or (131)I ortho-iodohippurate (OIH) and lead to spuriously low measurements of effective renal plasma flow (ERPF). This problem can be circumvented by the use of cationic tracers. The cationic renal tracer, (99m)Tc labeled diaminocyclohexane ((99m)Tc DACH), has a clearance of 80% of OIH in mice but its clearance in humans is relatively low, only 30% of OIH. The (99m)Tc DACH isomer(s) used in prior studies, however, was not clearly defined and may have consisted of a single isomer or a combination of isomers. Since the anionic isomers of some (99m)Tc renal tracers have been shown to have widely different clearances, the biodistribution and urine excretion of the (99m)Tc cis-, trans-S,S, trans-R,R and +/-trans-DACH isomers were compared in Sprague-Dawley rats at 10 minutes and 60 minutes postinjection to determine if one of the (99m)Tc DACH isomers may be a significantly better renal tracer than the others. The red cell binding of (99m)Tc +/- trans-DACH was also determined. All of the isomers showed a high degree of specificity for the kidney with minimal secretion into the gastrointestinal tract. Urine excretion of the 4 tracers, however, was only 38-48% that of OIH at 10 minutes and 66-84% that of OIH at 60 minutes. Red cell binding was 6.9%. Cationic renal tracers have the potential to provide a more accurate measurement of ERPF than anionic tracers. Based on the animal data, however, it is unlikely that any of the (99m)Tc DACH isomers will have a substantially higher clearance in humans than the form of (99m)Tc DACH originally tested. Development of alternative cationic renal tracers is warranted.

Imprinting structural information from a GpG ligand into the configuration of a chiral diamine ligand through second-sphere communication in platinum(II) complexes.

Cisplatin forms the cis-Pt(NH3)2(d(GpG)) cross-link with DNA. We have recently created novel d(GpG) conformations by using "retro models" (complexes having bulky carrier ligands designed to slow d(GpG) dynamic motion). Our results define four conformer classes: HH1, HH2, delta HT1, and delta HT2, with a head-to-head or head-to-tail base orientation and a phosphodiester backbone with a normal (1) or opposite (2) propagation direction. Moreover, each G residue can be syn or anti, and the base canting can be left-handed (L) or right-handed (R). Thus, 32 variants of cis-Pt(NH3)2(d(GpG)) are conceivable, but the adduct is too dynamic to study. Thus far, by using retro models, we have obtained evidence for five variants with d(GpG) but only four with GpG. We therefore selected Me2DAPPt(GpG) complexes for study by 1H and 31P NMR spectroscopy, CD spectroscopy, and molecular mechanics and dynamics (MMD) calculations. Coordinated Me2DAP (N,N'-dimethyl-2,4-diaminopentane) has N, C, C, N chiral centers designated, for example, as R,R,R,R. This ligand has greater flexibility and more readily inverted N centers than ligands used previously in GpG retro models. One goal was to determine whether the GpG ligand can control the configuration of a carrier ligand. (R,R,R,R)-Me2DAPPt(GpG) forms the anti, anti HH1 R variant almost exclusively. Equal populations of the two possible linkage isomers of (S,R,R,R)-Me2DAPPt(GpG) are formed, both favoring the anti, anti HH1 R, variant; however, the isomer with the 5'-G cis to the S nitrogen has sharper signals, suggesting that interligand interactions are more favorable. Indeed, this linkage isomer was the major product of isomerization when (R,R,R,R)-Me2DAPPt(GpG) was kept at pH approximately 9.5 to allow N center equilibration. Steric clashes between the Me2DAP C-Me groups and the G O6 atoms found by MMD calculations appear to disfavor the HH1 conformer of (S,S,S,S)-Me2DAPPt(GpG) and (S,S,S,R)-Me2DAPPt(GpG) complexes. These two complexes have a significant population of the anti, syn delta HT1 conformer, as indicated by broad 1H NMR signals and by 31P NMR and CD data. Equilibration of (S,S,S,R)-Me2DAPPt(GpG) at pH 9.5 leads to a mixture of (S,S,S,S)-Me2DAPPt(GpG) and at least one isomer of (S,S,S,R)-Me2DAPPt(GpG). Thus, second-sphere communication (hydrogen bonding and steric interligand interactions) influences both GpG conformation and Me2DAP configuration.

Cisplatin-DNA cross-link retro models with a chirality-neutral carrier ligand: evidence for the importance of "second-sphere communication".

We employ retro models, cis-PtA2G2 (A2 = a diamine, G = guanine derivative), to assess the cross-linked head-to-head (HH) form of the cisplatin-DNA d(GpG) adduct widely postulated to be responsible for the anticancer activity. Retro models are designed to have minimal dynamic motion to overcome problems recognized in models derived from cisplatin [A2 = (NH3)2]; the latter models are difficult to understand due to rapid rotation of G bases about the Pt-N7 bond in solution and the dominance of the head-to-tail (HT) form in the solid. Observation of an HH form is unusual for cis-PtA2G2 models. Recently, we found the first HH forms for a cis-PtA2G2 model with A2 lacking NH groups in a study of new Me2ppzPtG2 models. (Me2ppz, N,N'-dimethylpiperazine, has inplane bulk which reduces dynamic motion by clashing with the G O6 as the base rotates into the coordination plane from the ground state position approximately perpendicular to this plane G = 5'-GMP and 3'-GMP.) The finding of an HH form (albeit in a mixture with HT forms) with both G H8 signals unusually downfield encouraged us to study additional Me2ppzPtG2 analogues in order to explain the unusual spectral features and to identify factors that influence the relative stability of HT and HH forms. Molecular modeling techniques suggest HH structures with the H8's close to the deshielding region of the z axis of the magnetically anisotropic Pt atom, explaining the atypical shift pattern. When G = 1-Me-5'-GMP, we obtained NMR evidence that the HH rotamer has a high abundance (34%) and that the three rotamers have nearly equal abundance. These findings and the observation that the relative HT distributions varied little or not at all as a function of pH when G = Guo, 1-MeGuo, or 1-Me-5'-GMP are consistent with two of our earlier proposals concerning phosphate groups in HT forms of cis-PtA2(GMP)2 complexes. We proposed that a G phosphate group can form hydrogen bonds with the cis G N1H ("second-sphere" communication) and (for 5'-phosphate) A2 NH groups. The new results with 1-Me-5'-GMP led us to propose a new role for a 5'-phosphate group; it can also favor the HH form by counteracting the natural preference for the G bases to adopt an HT orientation. Finally, the HH form was also sufficiently abundant to allow observation of a distinct 195Pt NMR signal (downfield of the resonance observed for the HT forms) for several complexes. This is the first report of an HH 195Pt NMR signal for cis-PtA2G2 complexes.

Nature of cysteine-based Re(V)=O(N2S2) radiopharmaceuticals at physiological pH ascertained by investigation of a new complex with a meso N2S2 ligand having carboxyl groups anti to the oxo group.

X-ray structural characterization of a new isomer of ReO(TMECH3) revealed that it is anti-ReO(DL-TMECH3) (1). (DL-TMECH6 is meso-tetramethyl-ethylene-dicysteine prepared from racemic penicillamine (penH4), the subscript on H indicating the number of dissociable protons; anti denotes the geometric isomer having both carboxyl groups anti to the oxo ligand.) In 1, one carboxyl is deprotonated and coordinated trans to the oxo ligand, and the other is protonated and dangling. The 1H NMR spectrum (assigned by 2D methods) of 1 at pH 4 in aqueous solution revealed that the structure of 1 is the same as in the solid state except for deprotonation of the dangling carboxyl group, affording the monoanion. All chelate ring protons and methyl groups are inequivalent and give sharp signals. As the pH was raised above 7, the 1D 1H NMR signals of the monoanion broadened. Broadening was severe for the methyl and ethylene signals of the tridentate half of the monoanion, and these signals were replaced by new signals for the dianion. The changes suggested a rate process that was intermediate on the NMR time scale, such as CO2- ligation/deligation. With increasing pH the dianion signals sharpened up to pH approximately 8 and then broadened up to pH approximately 10. Finally, the spectrum at pH 10.8 showed only half the number of signals. Each signal was at the midpoint shift between two corresponding signals observed at lower pH, indicating a time averaging between the halves of the DL-TMEC ligand, but no change in protonation state. Two Re=O stretching bands (923 and 933 cm-1) with a constant intensity ratio of approximately 1 were observed for the dianion. These results can be explained if the dianion exists detectably only as a NH-deprotonated/carboxyl-deligated form having two conformers. The conformers differ in the N lone pair (NLp) orientation (either endo or exo with respect to the oxo ligand) and thus have slightly different Re=O stretching frequencies. Although they can be detected by resonance Raman spectroscopy, the conformers are indistinguishable by NMR spectroscopy because NLp inversion (and hence conformer interconversion) is very fast. Interchange of the NH and NLp sites affects the NMR spectra. At pH 8.3 the signals of the dianion are sharpest because interchange is slowest. Below and above pH 8.3, the signals are broader because acid and base catalysis, respectively, increase the rate of interchange between the NH and NLp sites.

Re(V) complexes with an open-chain quadridentate ligand containing two amine and two amido donors. Synthesis, characterization, and solution equilibria of Re2O3(dioxo-tetH4)2 and [ReO(H2O)(dioxo-tetH4)]Cl (dioxo-tetH6 = 1,4,8,11-tetraazaundecane-5,7-dione).

We are interested in identifying mononuclear cationic [M(V)=O]3+ (M = Tc, Re) complexes for radiopharmaceutical applications. The open-chain ligand, 1,4,8,11-tetraazaundecane-5,7-dione-(dioxo-tetH6) with two amine and two amide donors, was selected for investigation since the literature led us to expect that a five-coordinate [Re(V)=O(dioxo-tetH4)]+ cation would dominate. Instead, the neutral mu-oxo bridged dinuclear complex, Re2O3(dioxo-tetH4)2 (1), and a salt of the six-coordinate mononuclear cation, [ReO(H2O)(dioxo-tetH4)]+ (2), were isolated; the structure of each was determined by X-ray crystallography. The cation (2) is unusual because it has a trans-oxo/aqua core. Such aqua compounds are rarely isolated, and the Re-OH2 distance is relatively short (2.185 A). The cation has two pKa values, 4.1 and 8.7, determined with visible spectroscopy. Since the Re-OH2 bond is short, the coordinated water is likely to be acidic. Thus the two pKa's are assigned to the stepwise deprotonation of the water ligand to give a trans-oxo/hydroxo neutral form and a trans-dioxo anion. Although 1 was the first product isolated following ligand exchange of ReOCl3(Me2S)(OPPh3) with dioxo-tetH6 under neutral conditions, it probably formed from the hydroxo mononuclear complex. Under concentrated conditions (approximately 300 mM) the dinuclear complex deposited from solution, but the 1H NMR spectra of 2 (approximately 20 mM) were consistent with the presence of only monomeric forms in D2O, pH 3-12. 1H NMR experiments demonstrated that in DMSO-d6 2 converts to 1 upon addition of base, consistent with the proposal that two units of the hydroxo monomer condense to give the dinuclear form. In addition, all spectra of pure 1 dissolved in DMSO-d6 included extra low intensity signals that were characteristic of the monomer. Thus, although 1 is favored over the neutral monomer in DMSO-d6, the two complexes exist as a mixture of equilibrating forms. Our results do not support the previous findings for the Re(V) complex with a macrocyclic diamine-diamide ligand related to dioxo-tetH6. The data indicate that the ability of an amido group to donate electron density to a Re(V) center is moderately greater than the donating ability of a neutral amine group.

Effect of protein binding on renal extraction of 131I-OIH and 99mTc-labeled tubular agents.

UNLABELLED: The clearance of 99mTc-mercaptoacetyltriglycine (MAG3) is less than the clearances of o-131I-iodohippurate (OIH) and 99mTc-labeled DD- and LL-ethylenedicysteine (EC). This difference could be associated with the lower affinity of MAG3 for the tubular transport receptor, but MAG3 is more highly protein bound than OIH and the EC isomers; protein binding could also be an important factor governing tubular extraction. To separate the effects of protein binding from tubular receptor affinity, the extraction fractions (EFs) of MAG3, OIH, and the DD, LL, and DL isomers of 99mTc-EC were measured in an isolated perfused rat kidney model using a protein-free perfusate and perfusates containing bovine serum albumin. METHODS: The right kidney was removed from the rat and perfused with modified Krebs-Henseleit buffers containing 7.5 or 2.5 g/dL bovine serum albumin or a protein-free perfusate. OIH was coinjected into the renal artery with each of the 99mTc-tracers. Protein binding was measured in each of the perfusates, and the venous outflow was collected to determine the EF. RESULTS: The protein binding of MAG3 in the albumin perfusates ranged from 87% to 95%, significantly higher than the 20%-34% range of protein binding observed with the three EC complexes (P < 0.05). In the 2.5 g/dL albumin perfusate, the EF of MAG3 was 44%, significantly less than the 57%-77% EF of the three EC complexes; in the 7.5 g/dL perfusate, the MAG3 EF fell to 18% versus 39%-45% for the EC complexes (P < 0.05). However, in the protein-free perfusate, the EF of MAG3 was 64%, equal to or higher than the 46%-62% EF of the three EC complexes. CONCLUSION: Protein binding modulates the tubular extraction of renal tracers. Protein binding and receptor affinity must be considered in the design of future renal radiopharmaceuticals as well as radiopharmaceuticals targeting other receptors.

Understanding the orientation and dynamic motion of planar heterocyclic N-donor ligands by exploiting the symmetry properties of mixed-ligand mu-oxorhenium(V) dinuclear complexes.

Factors influencing the orientation and dynamic motions of planar N-donor heterocyclic ligands (L) are of interest since such features have broad relevance in metallobiochemistry [Marzilli, L. G.; Marzilli, P. A.; Alessio, E. Pure Appl. Chem. 1998, 70, 961-968]. We found that mu-oxorhenium(V) dinuclear complexes [ReOCl2LsLt]-O-[ReOCl2LsLt] bearing either symmetrical (L = py = pyridine; 3,5-lut = 3,5-lutidine) or lopsided (L = Me3-Bzm = 1,5,6-trimethylbenzimidazole) cis L ligands are particularly useful for studying these factors. NMR data showed that terminal (Lt) and stacked (Ls) ligands were exchanged by approximately 180 degrees rotation about the Re-O-Re bond system. Such exchange occurred, however, between degenerate chiral conformers. Here we report a combined X-ray structural and solution NMR investigation of the AA + CC (racemic) and AC (meso) forms of two mixed-ligand mu-oxorhenium dimers that bear one lopsided and one symmetrical ligand on each Re atom, namely, Re2O3-Cl4(py)2(Me3Bzm)2 (1rac and 1meso) and Re2O3Cl4(3,5-lut)2(Me3Bzm)2 (2rac and 2meso). The presence of two different cis L ligands in 1 and 2 breaks the local symmetry at each Re atom, so that, in the racemic dimers, the exchange of terminal and stacked ligands leads to nondegenerate conformers. Overall, NMR data showed that the unsymmetrical dimers 1 and 2 undergo two dynamic processes contemporaneously, namely, 180 degrees rotation about the Re-N(py or 3,5-lut) bond and coupled rotation about the Re-O-Re/Re-N bonds. Both processes reach the slow exchange limit below -80 degrees C. Rotation of py in 1 occurs faster than that of 3,5-lut in 2; this difference is attributed to the higher steric demands of 3,5-lut compared to py. For both dimers NMR data provided compelling evidence of the preferred conformers in solution, including ligand orientations. The low-T solution structure of 1meso and 2meso is chiral, the same as that found in the solid state for 2meso, where the Me3Bzm on one Re atom is stacked with the 3,5-lut on the other Re atom. The remaining Me3Bzm and 3,5-lut, one on each Re atom, are both terminal. In solution the coupled Re-O-Re/Re-N rotations interconvert the two halves of each meso dimer to yield the same overall stable chiral conformation. For the racemic dimers, however, this process does not interconvert one enantiomer into the other, but instead interconverts two rotamers, R1 and R2, each of which is chiral. We found that, in the case of both 1rac and 2rac, the conformer with stacking symmetrical ligands (R1) is roughly 1 order of magnitude more stable than that with stacking Me3Bzm ligands (R2). Moreover, the solution conformation of R1 is the same as that found in the solid state of 1rac. Solution- and solid-state data indicate that the key interaction favoring the observed conformations is very likely the electrostatic attraction between the delta+ H2 atoms on the Me3Bzm ligands and the negative O and Cl groups in the core of the dimers. Finally, for both meso and racemic dimers we were also able to elucidate the preferred pathways of the coupled dynamic motions and establish that, very likely, the two halves of the dimers swing back and forth by approximately 130 degrees through the anti eclipsed form.

Influence of carrier ligand NH hydrogen bonding to the O6 and phosphate group of guanine nucleotides in platinum complexes with a single guanine ligand.

Coordinated N,N',N"-trimethyldiethylenetriamine (Me3dien) has several possible configurations: two have mirror symmetry (R,S configurations at the terminal nitrogens) and the terminal N-Me's anti or syn with respect to the central N-Me (anti-(R,S) and syn-(R,S) isomers, respectively), and two are nonsymmetrical (R,R and S,S configurations at terminal nitrogens, rac denotes a 1:1 mixture of the two isomers). For each configuration, two Me3dienPtG atropisomers can be formed (anti or syn orientation of central N-Me and G 06, G = guanine derivative), and these can be observed since the terminal N-Me's decrease the rate of G rotation about the Pt-N7 bond. In symmetrical syn-(R,S)-Me3dienPtG derivatives with G = 9-EtG and 3'-GMP, the anti rotamer, which can form O6-NH H-bonds, was slightly favored over the syn rotamer but never more than 2:1. This anti rotamer is also favored by lower steric repulsion between the terminal N-Me's and G O6; thus, the contribution of O6-NH H-bonding to the stability of the anti rotamer could be rather small. With G = 5'-GMP, an O6-NH H-bond in the anti rotamer and a phosphate-NH H-bond in the syn rotamer can form. Only the syn rotamer was detected in solution, indicating that NH H-bonds to 5'-phosphate are far more important than to O6, particularly since steric factors favor the anti rotamer. Interconversion between rotamers was faster for syn-(R,S)- than for rac-Me3dien derivatives. This appears to be determined by a smaller steric impediment to G rotation of two "quasi equatorial" N-Me's, both on one side of the platinum coordination plane (syn-(R,S) isomer), than one "quasi equatorial" and one "quasi axial" N-Me on either side of the coordination plane (rac isomer).

Cisplatin--DNA cross-link models with an unusual type of chirality-neutral chelate amine carrier ligand, N,N dimethylpiperazine (Me2ppz): Me2ppzPt(guanosine monophosphate)2 adducts that exhibit novel properties.

Most simple cis-PtA2G2 complexes that model the G-G cross-link DNA lesions caused by the clinically used anticancer drug cis-PtCl2(NH3)2 undergo large fluxional motions at a rapid rate (A2 = two amines or a diamine; G = guanine derivative). The carrier amine ligands in active compounds have NH groups, but the fundamental role of the NH groups has been obscured by the dynamic motion. To assess carrier ligand effects, we examine retro models, cis-PtA2G2 complexes, in which dynamic motion has been reduced by the incorporation of steric bulk into the carrier ligands. In this study we introduce a new approach employing the chirality-neutral chelate (CNC) ligand, Me2ppz (N,N'-dimethylpiperazine). Because they lie in the Pt coordination plane, the methyl groups of Me2ppz do not clash with the 06 of the base of G ligands in the ground state, but such clashes sterically hinder dynamic motion. NMR spectroscopy provided conclusive evidence that Me2ppzPt(GMP)2 complexes (GMP = 5'- and 3'-GMP) exist as a slowly interconverting mixture of two dominant head-to-tail (HT) conformers and a head-to-head (HH) conformer. Since the absence of carrier ligand chirality precluded using NMR methods to determine the absolute conformation of the two HT conformers, we used our recently developed CD pH jump method to establish chirality. The most abundant HT Me2ppzPt(5'-GMP)2 form had A chirality. Previously this chirality was shown to be favored by phosphate-cis G NIH hydrogen-bonding interligand interactions; such interactions also favor the HT conformers over the HH conformer. For typical carrier ligands, G O6 and phosphate interactions with the carrier ligand NH groups also favor the HT forms. These latter interactions are absent in Me2ppzPt(GMP)2 complexes, but the HT forms are still dominant. Nevertheless, we do find the first evidence for an HH form of a simple cis-PtA2G2 model with A2 lacking any NH groups. In previous studies, the absence of the HH conformer in cis-PtA2G2 complexes lacking carrier NH groups may be due to the presence of out-of-plane carrier ligand bulk. Such bulk forces both G O6-G O6 and G O6-carrier ligand clashes, thereby disfavoring the HH form. The major DNA cross-link adduct has the HH conformation. Thus, for anticancer activity, the small bulk of the NH group may be more important than the H-bonding interaction.

Stepwise assembly of unsymmetrical supramolecular arrays containing porphyrins and coordination compounds.

The stepwise coordination of meso-4'-pyridyl/phenyl porphyrins (4'-PyPs) to different metal centers proved to be an efficient synthetic approach leading to unsymmetrical arrays containing porphyrins and coordination compounds. The first step of this process, treatment of 4'-PyPs with a less than stoichiometric amount of cis,fac-RuCl2(Me2-SO)3(CO) (1), leads to the selective coordination of [cis,cis,cis-RuCl2(Me2SO)2(CO)] fragments ([Ru]) to some of the peripheral 4'-N sites of the 4'-PyPs. Column separation afforded four partially ruthenated 4'-PyPs in pure form: 4'-cis-DPyP[Ru] (2), 4'-trans-DPyP[Ru] (3), (4'-TPyP)[Ru] (4), and (4'-TPyP)[Ru]3 (5). These compounds, which have residual unbound peripheral 4'-N(py) sites (either one or three), were allowed to react with other metal centers that may belong either to a metalloporphyrin or to a coordination compound. When building blocks 2-5 were treated with [Ru(TPP)(CO)(EtOH)] (TPP = meso-tetraphenylporphyrin) in chloroform at room temperature, axial coordination of Ru(TPP)(CO) units ((Ru)) to the available 4'-N(py) sites readily occurred, generating the following arrays containing both perpendicular porphyrins and coordination compounds: (Ru)-(mu-4'-cis-DPyP)[Ru], (Ru)(mu-4'-trans-DPyP)[Ru], (Ru)3(mu-4'-TPyP)[Ru], and (Ru)(mu-4'-TPyP)[Ru]3. Furthermore, building blocks 2, 3, and 5 were treated with a series of coordination compounds capable of binding two pyridylporphyrins either cis to each other (trans-RuCl2(Me2SO)4 and trans,cis,cis-RuCl2(Me2SO)2(CO)2) or trans to each other (trans-PdCl2(C6H5CN)2). Homo- (Ru) and heterobimetallic (Ru-Pd) arrays with as many as seven metal atoms (six Ru and one Pd) and two 4'-PyPs were obtained as follows: trans,cis,cis-RuCl2(Me2SO)2(4'-cis-DPyP[Ru])2, trans,cis,cis-RuCl2(Me2SO)2(4'-trans-DPyP[Ru])2, trans,cis,cis-RuCl2(CO)2(4'-cis-DPyP[Ru])2, and trans-PdCl2(4'-TPyP[Ru]3)2. All the products were thoroughly characterized by 1H NMR spectroscopy. Since the [Ru] fragment is chiral, diastereomers are formed when two or more [Ru] units are bound to a porphyrin. We found that when two 4'-cis-DPyP[Ru] (2) units are coordinated cis to each other on the same metal center, the mutual anisotropic effect of the cis porphyrins differentiates the sulfoxide methyl resonances for the two forms. These and other results indicate that the pyridyl units react independently of the presence or absence of a substituent on the other py rings. Thus, the synthetic strategy should be a general method for linking diverse metal centers through pyridylporphyrins.

Preparation and crystal structure of a rhenium analogue of the cationic renal agent, tc-99m diaminocyclohexane.

We report here a chemical study on a Re analogue of one of the few cationic Tc-99m tracers previously investigated as an agent for effective renal plasma flow (ERPF) measurement. Cationic Tc-99m tracers have the potential for overcoming problems associated with common anionic Tc-99m tracers in patients who have developed a uremic state. The Tc-99m-DACH tracer, prepared from 1,2-diaminocyclohexane (1,2-DACH), is the only cationic renal agent tested in humans and has seven possible isomers. The complex isolated from the reaction of the racemic mixture, (+/-)-trans-1,2-DACH, and ReIO(2)(PPh(3))(2) after conversion to the BPh(4) (-) salt was found by X-ray crystallography to be the meso isomer, trans-[ReO(2) (trans-R,R-1,2-DACH)(trans-S,S-l,2-DACH)][BPh(4)].MeOH.2H(2)O (1). The structural parameters for 1 are normal. The complex is highly symmetrical, suggesting that the analogous meso Tc-99m-DACH agent is also symmetrical. Studies of other Tc-99m-DACH agents that were made from cis-1,2-DACH or individual trans-1,2-DACH enantiomers show that the biodistribution is not very dependent on the starting 1,2-DACH ligand stereochemistry; these agents must be less symmetrical than the meso Tc-99m-DACH agent analogue of 1. Thus, the overall charge and lipophilicity (similar for all Tc-99m-DACH isomers) exert a greater influence on biodistribution than the specific structural features of the different Tc-99m-DACH isomers.

The influence of stereoisomerism on the pharmacokinetics of Tc radiopharmaceuticals.

The influence of stereoisomerism on the pharmacokinetics of Tc mono-oxo complexes is reviewed. Tc(V) monooxo complexes formed with N/S ligands have four donor groups from the ligands in an equatorial plane; the oxo ligand coordinates in an axial position. Stereoisomerism in Tc (V) mono-oxo complexes can be centered within the ligand (carbon atom in the chelate ring or ligating nitrogen of amine donors) or at the Tc. The metal center becomes chiral when an equatorial ligand has a head and a tail (i.e., the two ends of the ligand differ). All types of stereocenters can produce significantly different pharmacokinetic profiles for individual isomers. Thus, biological evaluation of separated stereoisomers is necessary to identify the optimal sterochemical configuration, particularly for radiopharmaceuticals targeted to receptor molecules with low specificity. Because of interspecies variation, there is ultimately no substitute for human testing. Although it is possible that the increase in non-specific binding of agents incorporating L-vs D-amino acids may more than offset any increased receptor binding, much more information is needed. Stereochemical factors can also lead to unpredictable differences in coordination geometry and thermodynamic preference of a single isomer; thus chemical characterization of stereoisomers continues to be an important component of radiopharmaceutical development.

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-t rimethylammoniumethyl-amine)phenyl]porphy rin.

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine )phenyl]porphyrin (TthetaF4TAP) has been synthesized. The binding interactions of TthetaF4TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TthetaF4TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TthetaF4TAP binds to calf thymus DNA, [poly(dA-dT)]2, and [poly(dG-dC)]2 in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TthetaF4TAP/CT DNA resembled that of TthetaF4TAP/[poly(dA-dT)]2, suggesting that TthetaF4TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TthetaF4TAP binds preferentially to [poly(dA-dT)]2 over [poly (dG-dC)]2. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TthetaF4TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TthetaF4TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation.

Comparison of technetium-99m-ll-EC isomers in rats and humans.

UNLABELLED: Technetium-99m-L,L-ethylenedicysteine (99mTc-LL-EC) is a new renal imaging agent with pharmacokinetic properties reported to be slightly superior to those of 99mTc-mercaptoacetyltriglycine (99mTc-MAG3); however, to better define the potential of the enantiomer 99mTc-DD-EC and the diastereomer 99mTc-DL-EC as renal imaging agents, we compared the three EC stereoisomers with 131I-orthoiodohippurate (OIH) in a series of rats and humans. METHODS: Each 99mTc-EC stereoisomer was coinjected with OIH in six Sprague-Dawley rats for measurements of clearance and extraction fraction. Each stereoisomer was also coinjected with OIH in three human volunteers followed by sequential imaging, plasma clearance measurements and timed urine collections. RESULTS: Technetium-99m-DD-EC had the highest clearance and extraction efficiency in rats (p < or = 0.02). In humans, image quality was good with all three agents. The clearance ratio (EC/OIH) was 82% +/- 8% for 99mTc-DD-EC compared to 70% +/- 3% and 40% +/- 5% for 99mTc-LL-EC and 99mTc-DL-EC, respectively. Technetium-99m-DD and 99mTc-LL-EC were excreted more rapidly than 99mTc-DL-EC. CONCLUSION: Technetium-99m-DD-EC has excellent imaging properties and the data suggest that its clearance may approach that of OIH more closely than any other 99mTc renal agent. A potential limitation is the fact that both 99mTc-DD and LL-EC exist in dianionic (80%) and monoanionic (20%) forms at physiological pH and it is unlikely that these two forms have the same clearance or protein binding affinity.

Similar conformations of hairpins with TTT and TTTT sequences: NMR and molecular modeling evidence for T.T base pairs in the TTTT hairpin.

The conformations of the d[G(1)C(2)G(3)C(4)-T(a)T(b)T(c)T(d)-G(5)C(6)G(7)C(8)] (T4) and d[G(1)C(2)G(3)C(4)-T(a)T(b)T(c)-G(5)C(6)G(7)C(8)] (T3) DNA hairpins have been studied. The 1H and 31P signals of the two hairpins have been nearly completely assigned by means of two-dimensional NMR spectroscopy in D2O (NOESY (two-dimensional nuclear Overhauser effect and exchange spectroscopy) at mixing times of 5, 50, 100, 300 and 500 ms, double-quantum-filtered correlation spectroscopy (DQF-COSY) and 1H-31P reverse chemical shift correlation (RCSC), and one-dimensional NOE spectra in 90% H2O. Conformational analysis using distance geometry (DG), molecular mechanics (MM) and molecular dynamics (MD) gave model conformations, which were evaluated by comparison of experimental and simulated 2D NOESY spectra. For the T4 sequence in T4, both NMR data and modeling indicated a T(a).T(d) wobble base pair. Although two types of T(a).T(d) base pairs are possible, the one with T(a)NH-T(d)O4 and T(a)O2-T(d)NH H-bonds was calculated to be more stable. Because the T(a).T(d) base pair of T4 extends the stem, there are only two residues (T(b) and T(c) in the loop. Although there are three residues in the T3 loop, the T(c) base projects into the solvent. The resulting conformational models have very similar loop folding patterns (FP): the bases of the two adjacent residues that begin the loop [T(b)T(c) of T4 and T(a)T(b) or T3] have a minor groove/major groove orientation with the first residue each having a trans alpha torsion angle; and the phosphodiester group that links the residues at the 3' end of the loop and the 5' top of the stem [T(c)pT(d) of T4 and T(c)pG(5) of T3] has a gauche+, gauche+ zeta,alpha conformation with a trans gamma angle for the second residue in both. These or similar features appear to be present in most of the few other hairpins studied previously by conformational methods. Thus, we believe that the conformations of the loops in T3 and T4 hairpins have greater similarities than previously recognized.