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 k...

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.

Charge delocalization (6+ to 8+) in "noncovalent" linear trinuclear platinum complexes produces compounds with cytotoxicity in some cases equivalent to cisplatin. The cellular uptake of a novel 8+ compound is greater than that of neutral cisplatin as well as other multinuclear Pt compounds.

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.

BBR3464 is a novel trinuclear platinum drug currently in Phase II clinical trials. Polyamine-bridged dinuclear platinum compounds as represented by [[trans-Pt(NH(3))(2)Cl](2)-mu-spermidine-N(1),N(8)]Cl(3) (1) are highly interesting second-generation analogues of BBR3464 because the hydrogen-bonding and electrostatic contributions of the central platinum-amine group in BBR3464 are replicated by the free, noncoordinated "central" quaternary nitrogens of the linear polyamine linker while the presence of two separate Pt-Cl bonds maintains the bifunctional binding mode on the DNA adducts. Preclinical investigations confirm the potency of these species with cytotoxicity in the nanomolar range. This remarkable potency results in a relatively narrow therapeutic index. To enhance the therapeutic index of these drugs, we investigated the potential for "pro-drug" delivery of less toxic and better tolerated derivatives such as the compounds [[trans-Pt(NH(3))(2)Cl](2)-mu-N(4)-R-spermidine-N(1),N(8)]Cl(2) where N(4)-R represents BOC (tert-butyl), CBz (benzyl), and Fmoc (fluorenylmethyl) carbamate blocking groups, 2-4, respectively. The bulky Fmoc derivative showed evidence for conformational isomers by (1)H NMR spectroscopy due to the inequivalence of the two n-propyl and n-butyl side chains of the spermidine moiety. The rate constants for hydrolysis and release of 1 were calculated. Release of cytotoxic 1 at physiologically relevant pH followed the order 4 > 2 > 3. The calculated values for 4 (pH 5, 6.0(+/-3.9) x 10(-10) s(-1); pH 6, 6.5(+/-0.2) x 10(-9) s(-1); pH 7, 6.0(+/-0.2) x 10(-8) s(-1); pH 8, 1.6(+/-0.1) x 10(-7) s(-1)) show a more pronounced pH dependence compared to 2 (pH 5, 4.6(+/-0.1) x 10(-8) s(-1); pH 6, 4.2(+/-0.1) x 10(-8) s(-1); pH 7, 3.2(+/-0.1) x 10(-8) s(-1)). Preliminary biological assays of cellular uptake and cytotoxicity confirm the utility of the pro-drug concept. While blocked-polyamine compounds such as 2-4 are, in general, 2-3 orders of magnitude less cytotoxic than 1, there is significant cell type variability. Specifically, the Fmoc derivative 4 showed significantly enhanced cytotoxicity warranting further study of the pro-drug concept for greater selectivity and/or oral delivery.

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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.

The chemistry and biology of acetyl-protected spermidine-bridged dinuclear platinum complexes [{trans-PtCl(NH 3 ) 2 ] 2 -μ-NH 2 (CH 2 ) 3 N(COR)(CH 2 ) 4 NH 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 -μ-trans-Pt(NH 3 ) 2 (NH 2 (CH 2 ) 6 NH 2 ) 2 ](NO 3 ) 4 , (8, BBR3464).