On the Heterogeneous Nature of Cisplatin-1-Methyluracil Complexes: Coexistence of Different Aggregation Modes and Partial Loss of NH3 Ligands as Likely Explanation.

The conversion of the 1 : 1-complex of Cisplatin with 1-methyluracil (1MeUH), cis-[Pt(NH3 )2 (1MeU-N3)Cl] (1 a) to the aqua species cis-[Pt(NH3 )2 (1MeU-N3)(OH2 )]+ (1 b), achieved by reaction of 1 a with AgNO3 in water, affords a mixture of compounds, the composition of which strongly depends on sample history. The complexity stems from variations in condensation patterns and partial loss of NH3 ligands. In dilute aqueous solution, 1 a, and dinuclear compounds cis-[(NH3 )2 (1MeU-N3)Pt(µ-OH)Pt(1MeU-N3)(NH3 )2 ]+ (3) as well as head-tail cis-[Pt2 (NH3 )4 (µ-1MeU-N3,O4)2 ]2+ (4) represent the major components. In addition, there are numerous other species present in minor quantities, which differ in metal nuclearity, stoichiometry, stereoisomerism, and Pt oxidation state, as revealed by a combination of 1 H NMR and ESI-MS spectroscopy. Their composition appears not to be the consequence of a unique and repeating coordination pattern of the 1MeU ligand in oligomers but rather the coexistence of distinctly different condensation patterns, which include µ-OH, µ-1MeU, and µ-NH2 bridging and combinations thereof. Consequently, the products obtained should, in total, be defined as a heterogeneous mixture rather than a mixture of oligomers of different sizes. In addition, a N2 complex, [Pt(NH3 )(1MeU)(N2 )]+ appears to be formed in gas phase during the ESI-MS experiment. In the presence of Na+ ions, multimers n of 1 a with n=2, 3, 4 are formed that represent analogues of non-metalated uracil quartets found in tetrastranded RNA.

Amide-based prodrugs of spermidine-bridged dinuclear platinum. Synthesis, DNA binding, and biological activity.

The chemistry and biology of acetyl-protected spermidine-bridged dinuclear platinum complexes [{ trans-PtCl(NH 3) 2] 2-mu-NH 2(CH 2) 3N(COR)(CH 2) 4NH 2]X 2 (R = H, X = Cl (1,1/t,t-spermidine, BBR3571); R = CH 3 , X = Cl ( 2); R = CH 2 Cl, X = ClO 4 ( 3); R = CF 3 , X = Cl ( 4)) are compared with their carbamate analogues. The compounds are potential prodrugs for the parent compound 1, a highly potent antitumor agent. At pH 6-8 hydrolysis of the blocking group with the release of the "parent" protonated species follows the order 4 > 3 >> 2. For 4, rate constants for the deprotection increase in this pH range. The DNA binding profile of 4 is similar to the Boc derivative, confirming the central influence of charge on DNA binding properties. The differences in cytotoxicity for the protected compounds in ovarian carcinoma cell lines sensitive and resistant to cisplatin cannot completely be explained by spontaneous release of 1,1/t,t-spermidine at physiological pH. Inherent cytotoxicity and cell line specificity may contribute to the observed behavior. The properties of the compounds present them also as possible "second-generation" analogues of the clinically relevant trinuclear complex [{ trans-PtCl(NH 3) 2} 2-mu- trans-Pt(NH 3) 2(NH 2(CH 2) 6NH 2) 2](NO 3) 4, ( 8, BBR3464).

Effects of geometric isomerism in dinuclear antitumor platinum complexes on their interactions with N-acetyl-L-methionine.

In this study, the reactions of N-acetyl-L-methionine (AcMet) with [{trans-PtCl(NH(3))(2)}(2)-mu-H(2)N(CH(2))(6)NH(2)](NO(3))(2) (BBR3005: 1,1/t,t 1) and its cis analog [{cis-PtCl(NH(3))(2)}(2)-mu-{H(2)N(CH(2))(6)NH(2)}]Cl(2) (1,1/c,c 2) were analyzed to determine the rate and reaction profile of chloride substitution by methionine sulfur. The reactions were studied in PBS buffer at 37 degrees C by a combination of multinuclear ((195)Pt, {(1)H-(15)N} HSQC) magnetic resonance (NMR) spectroscopy and electrospray ionization time of flight mass spectrometry (ESITOFMS). The diamine linker of the 1,1/t,t trans complex was released as a result of the trans influence of the coordinated sulfur atom, producing trans-[PtCl(AcMet)(NH(3))(2)](+) (III) and trans-[Pt(AcMet)(2)(NH(3))(2)](2+) (IV). In contrast the cis geometry of the dinuclear compound maintained the diamine bridge intact and a number of novel dinuclear platinum compounds obtained by stepwise substitution of sulfur on both platinum centers were identified. These include (charges omitted for clarity): [{cis-PtCl(NH(3))(2)}-mu-NH(2)(CH(2))(6)NH(2)-{cis-Pt(AcMet)(NH(3))(2)}] (V); [{cis-Pt(AcMet)(NH(3))(2)}(2)-mu-NH(2)(CH(2))(6)NH(2)] (VI); [{cis-PtCl(NH(3))(2)}-mu-NH(2)(CH(2))(6)NH(2)-{PtCl(AcMet)NH(3)] (VII); [{PtCl(AcMet)(NH(3))}(2)-mu-NH(2)(CH(2))(6)NH(2)] (VIII); [{trans-Pt(AcMet)(2)(NH(3))}-mu-NH(2)(CH(2))(6)NH(2)-{PtCl(AcMet)(NH(3))] (IX) and the fully substituted [{trans-Pt(AcMet)(2)(NH(3))}(2)-mu-{NH(2)(CH(2))(6)NH(2)] (X). For both compounds the reactions with methionine were slower than those with glutathione (Inorg Chem 2003, 42:5498-5506). Further, the 1,1/c,c geometry resulted in slower reaction than the trans isomer, because of steric hindrance of the bridge, as observed previously in reactions with DNA and model nucleotides.

A surprisingly stable macrochelate formed from the reaction of cis dinuclear platinum antitumor compounds with reduced glutathione.

The structurally unique macrochelate [{Pt(en)}2-mu-{H2N(CH2)6 NH2}-mu-(SG)] (I) is the principal product of the reaction of the dinuclear compound [{PtCl(en)}2-mu-{H2N(CH2)6 NH2}]Cl2 (1) with reduced glutathione (GSH) in a stoichiometric 1:1 ratio in phosphate buffered saline (PBS) (pH 7.35). The macrochelate is formed through simultaneous bridging of the hexanediamine linker and glutathione thiolate. This represents a novel structure for glutathione adducts of platinum. At higher (1:4) ratios of Pt complex to GSH, an interesting interchange between bridged Pt-(SG)-Pt and terminal Pt-SG species is observed with the diamine linker still remaining intact in all cases. The integrity of I is further evident when reaction ratios are increased to 1:4 (Pt complex/GSH), and additional minor products are identified as [{Pt(en)SG}2-mu-{NH2(CH2)6 NH2}] (II), which transforms to [{Pt{NH2(CH2)2 NH2}(SG)}2-mu-{H2N(CH2)6 NH2}-mu-(SG)] (III), where the chelate ring is broken to produce a dangling monodentate ethylenediamine. The chemical shifts of the Pt-NH2 linker in all compounds are explained by consideration of the enhanced rigidity of the macrochelate (I) leading to shielding in comparison to the "open" monodentate structures (II, III). The remarkable stability of I is discussed in terms of possible biological implications.

Synthesis and DNA conformational changes of non-covalent polynuclear platinum complexes.

Polynuclear platinum compounds demonstrate many novel phenomena in their interactions with DNA and proteins as well as novel anti-cancer activities. Previous studies indicated that the high positive charge and the non-coordinated "central linker" of the polynuclear compounds could have major contributions to these features. Therefore, a series of non-covalent polynuclear platinum complexes, [[Pt(NH(3))(3)](2)-mu-Y](n+) (Y=polyamine linker or [trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]) was synthesized and the DNA interactions of these platinum complexes were investigated. The conformational changes induced by these compounds in polymer DNA were studied by circular dichroism and the reversibility of the transition was tested by subsequent titration with the DNA intercalating agent ethidium bromide (EtBr). Fluorescent quenching was also used to assess the ability of EtBr to intercalate into A and Z-DNA induced by the compounds. The non-covalent polynuclear platinum complexes induced both B-->A and B-->Z conformational changes in polymer DNA. These conformational changes were partially irreversible. The platinum compound with the spermidine linker, [[Pt(NH(3))(3)](2)-mu-spermidine-N(1),N(8)]Cl(5).2H(2)O, is more efficient in inducing the conformational changes of DNA and it is less reversible than complexes with other linkers. The melting point study showed that the non-covalent polynuclear platinum complexes stabilized the duplex DNA and the higher the electrical charge of the complexes the greater the stabilization observed.

Long range 1,4 and 1,6-interstrand cross-links formed by a trinuclear platinum complex. Minor groove preassociation affects kinetics and mechanism of cross-link formation as well as adduct structure.

Reported here is a comparison of the kinetics of the stepwise formation of 1,4- and 1,6-GG interstrand cross-links by the trinuclear platinum anticancer compound (15)N-[[trans-PtCl(NH(3))(2)](2)[mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+), (1,0,1/t,t,t (1) or BBR3464). The reactions of (15)N-1 with the self-complementary 12-mer duplexes 5'-[d(ATATGTACATAT)(2)] (I) and 5'-[d(TATGTATACATA)(2)] (II) have been studied at 298 K, pH 5.3 by [(1)H,(15)N] HSQC 2D NMR spectroscopy. The kinetic profiles for the two reactions are similar. For both sequences initial electrostatic interactions with the DNA are observed for 1 and the monoaqua monochloro species (2) and changes in the chemical shifts of certain DNA (1)H resonances are consistent with binding of the central charged [PtN(4)] linker unit in the minor groove. The pseudo first-order rate constants for the aquation of 1 to 2 in the presence of duplex I (3.94 +/- 0.03 x 10(-5) s(-1)), or II(4.17 +/- 0.03 x 10(-5) s(-1)) are ca. 40% of the value obtained for aquation of 1 under similar conditions in the absence of DNA. Monofunctional binding to the guanine N7 of the duplex occurs with rate constants of 0.25 +/- 0.02 M(-1) s(-1) (I) and 0.34 +/- 0.02 M(-1) s(-1) (II), respectively. Closure to form the 1,4- or 1,6-interstrand cross-links (5) was treated as direct from 3 with similar rate constants of 4.21 +/- 0.06 x 10(-5) s(-1) (I) and 4.32 +/- 0.04 x 10(-5) s(-1) (II), respectively. Whereas there is only one predominant conformer of the 1,6 cross-link, evidence from both the (1)H and [(1)H,(15)N] NMR spectra show formation of two distinct conformers of the 1,4 cross-link, which are not interconvertible. Closure to give the major conformer occurs 2.5-fold faster than for the minor conformer. The differences are attributed to the initial preassociation of the central linker of 1 in the minor groove and subsequently during formation of both the monofunctional and bifunctional adducts. For duplex I, molecular models indicate two distinct pathways for the terminal [PtN(3)Cl] groups to approach and bind the guanine N7 in the major groove with the central linker anchored in the minor groove. To achieve platination of the guanine residues in duplex II the central linker remains in the minor groove but 1 must diffuse off the DNA for covalent binding to occur. Clear evidence for movement of the linker group is seen at the monofunctional binding step from changes of chemical shifts of certain CH(2) linker protons as well as the Pt-NH(3) and Pt-NH(2) groups. Consideration of the (1)H and (15)N shifts of peaks in the Pt-NH(2) region show that for both the 1,4 and 1,6 interstrand cross-links there is a gradual and irreversible transformation from an initially formed conformer(s) to product conformer(s) in which the amine protons of the two bound [PtN(3)] groups exist in a number of different environments. The behavior is similar to that observed for the 1,4-interstrand cross-link of the dinuclear 1,1/t,t compound. The potential significance of preassociation in determining kinetics of formation and structure of the adducts is discussed. The conformational flexibility of the cross-links is discussed in relation to their biological processing, especially protein recognition and repair, which are critical determinants of the cytotoxicity of these unique DNA-binding agents.

A comparison of DNA binding profiles of dinuclear platinum compounds with polyamine linkers and the trinuclear platinum phase II clinical agent BBR3464.

The DNA binding profiles of three bis Pt(II) polyamine-linked compounds, [[ trans-PtCl(NH(3))(2)](2)[mu-spermine- N(1), N(12)]](4+), [[ trans-PtCl(NH(3))(2)](2)[mu-spermidine- N(1), N(8)]](3+), and [[ trans-PtCl(NH(3))(2)](2)[mu-BOC-spermidine]](2+), were compared with that of a novel trinuclear phase II clinical agent, [[ trans-PtCl(NH(3))(2)](2)[mu- trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+). All of the compounds bind preferentially in a bifunctional manner, according to unwinding of supercoiled DNA circles. The kinetics of binding of these compounds corresponds to their relative charge (2+ to 4+). The preference for the formation of interstrand crosslinks, however, does not follow a charge-based pattern. By studying the results of DNA polymerase extension products on a DNA template modified by the compounds, and by incorporating the complementary method of RNA transcription mapping, it was possible to determine the nucleotide bases that are preferred sites of binding. The stop sites due to platinum adducts were determined, and some preliminary observations concerning the range and type of crosslinks were established. It can be concluded that dinuclear Pt compounds are similar to their trinuclear counterpart, and that charge differences do not contribute solely to the variances between the compounds.

Kinetic and equilibria studies of the aquation of the trinuclear platinum phase II anticancer agent [(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](4+) (BBR3464).

The hydrolysis profile of the bifunctional trinuclear phase II clinical agent [(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](4+) (BBR3464, 1) has been examined using [(1)H,(15)N] heteronuclear single quantum coherence (HSQC) 2D NMR spectroscopy. Reported are estimates of the rate and equilibrium constants for the first and second aquation steps, together with the acid dissociation constant (pK(a1) approximately equal to pK(a2) approximately equal to pK(a3)). The equilibrium constants for the aquation determined by NMR at 298 and 310 K (I = 0.1 M, pH 5.3) are similar, pK(1) = pK(2) = 3.35 +/- 0.04 and 3.42 +/- 0.04, respectively. At lower ionic strength (I = 0.015 M, pH 5.3) the values at 288, 293, and 298 K are pK(1) = pK(2) = 3.63 +/- 0.05. This indicates that the equilibrium is not strongly ionic strength or temperature dependent. The aquation and anation rate constants for the two-step aquation model at 298 K in 0.1 M NaClO(4) (pH 5.3) are k(1) = (7.1 +/- 0.2) x 10(-5) s(-1), k(-1) = 0.158 +/- 0.013 M(-1) s(-1), k(2) = (7.1 +/- 1.5) x 10(-5) s(-1), and k(-2) = 0.16 +/- 0.05 M(-1) s(-1). The rate constants in both directions increase 2-fold with an increase in temperature of 5 K, and rate constants increase with a decrease in solution ionic strength. A pK(a) value of 5.62 plus minus 0.04 was determined for the diaqua species [(trans-Pt(NH(3))(2)(OH(2)))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)-NH(2))(2))](6+) (3). The speciation profile of 1 under physiological conditions is explored and suggests that the dichloro form predominates. The aquation of 1 in 15 mM phosphate was also examined. No slowing of the initial aquation was observed, but reversible reaction between aquated species and phosphate does occur.

Synthetic Ways to Tris(nucleobase) Complexes Derived from cis-Diammineplatinum(II) and a Platinum(II) Complex Containing Four Different Ligands, Three of Which Are Nucleobases.

Reactions of cis-[PtCl(L-N7)(NH(3))(2)]Cl (L = 9-ethylguanine, 9-EtGH, or 9-methyladenine, 9-MeA) with excess KI leads in slightly acidic solution to trans-PtI(2)(L)(NH(3)). Partial or complete substitution of the iodo ligands by aqua, chloro, or nucleobase ligands can be achieved and gives a series of neutral or cationic complexes. Among others, tris(nucleobase) complexes containing three different nucleobases (9-EtGH, 9-MeA, and 1-methylcytosine, 1-MeC), [Pt(1-MeC)(9-EtGH)(9-MeA)(NH(3))](2+) with SP-4-3 and SP-4-4 configuration numbers have been prepared and the deprotonated form SP-4-4-[Pt(1-MeC-N3)(9-EtG-N7)(9-MeA-N7)(NH(3))]NO(3).2H(2)O (7b) has been X-ray structurally characterized: monoclinic system, space group C2/c, a = 23.817(5) Å, b = 10.992(2) Å, c = 21.647(4) Å, beta = 112.07(3) degrees, V = 5251.9 Å(3), Z = 8. Moreover, the X-ray crystal structure analyses of trans-PtCl(2)(9-EtGH-N7)(NH(3)).0.5H(2)O (4) and trans-[Pt(1-MeC-N3)(2)(9-EtG-N7)(NH(3))]NO(3).3H(2)O (6b) are reported. 4: triclinic system, space group P&onemacr; (No. 2), a = 11.557(3) Å, b = 12.742(2) Å, c = 8.880(2) Å, alpha = 96.95(1) degrees, beta = 94.91(2) degrees, gamma = 88.32(2) degrees, V = 1293.1(5) Å(3), Z = 4. 6b: triclinic system, space group P&onemacr; (No. 2), a = 10.957(2) Å, b = 11.022(2) Å, c = 12.916(2) Å, alpha = 81.18(3) degrees, beta = 89.93(3) degrees, gamma = 64.22(3) degrees, V = 1384.2(5) Å(3), Z = 2. Relative orientations of the nucleobases in these compounds appear to be dictated by intranucleobase H bond formation.