Synthesis, Crystal Structure and Interaction With DNA of N,N′-(Butane-1,4-Diyl)Bis(Guanidinium) Tetrachloroplatinate (II)

The design, synthesis, crystal structure and interaction with DNA of the N,N′-(butane-1,4-diyl)bis(guanidinium) tetrachloroplatinate(ll) are described. Crystal data: a = 8.152(1), b = 8.889(4), c = 10.700(3) Å , α = 81.59(3), β = 87.99(5), γ = 78.48(6)°, V = 752(1) Å3, Z = 2 , space group P-1. The structure was refined to R = 0.039 and Rw = 0.046 from 1853 reflections (I > 3σ(I)). This compound, named PtC4Gua, does not exhibit a center of symmetry and the center linker chain C(2) - C(3) - C(4) - C(5) is in gauche conformation. The cation is bisprotonated with the H+ attached to the imine group of each terminal guanidinium function. The presence of the platinum moiety reinforces the binding of the butane(bis)guanidinium structure with double stranded DNA as judged from thermal denaturation studies and DNA unwinding experiments.


Introduction
Platinum complexes, like cis-diamminedichloroplatinum (11) (cisplatin) and carboplatin, are effective antitumor agents used for the treatment of genitourinary and head and neck cancers [1]. Very recently, oxaliplatin (1R,2R-diaminocyclohexanedicarboxylatoplatinum(ll)) has been recommended in metastatic colon rectal cancer treatment with neither nephrotoxicity nor cross-resistance to cisplatin [2]. The biological effects are attributed to the formation of adducts with DNA. Cisplatininduced bifunctional intrastrand cross-links between neighbouring purine base residues induce marked conformational kinks in DNA. These lesions are considered relatively difficult to repair [3].
The severe side effects of cisplatin and the development of resistance have encouraged the design of alternative platinum compounds with a broader spectrum of activity, in particular against cisplatin-resistant cell lines. Over the last ten years, a large number of drugs containing the basic cis-[PtX2(amine)2] motif were synthesized [3]. Dimers formed by linking the monofunctional platinum species [Pt(dien)CI] + by a tetramethylene linker exhibit a greatly enhanced ability for interstrand cross-linking compared to cisplatin itself [4]. The bis(platinum) complex shown in Figure represents a promising antitumor agent with a pronounced cytotoxic effect toward cell lines resistant to cisplatin [5]. Recently, a novel bifunctional triplatinum(ll) complex, BBR 3464, was shown to exhibit comparable or superior efficacity to cisplatin and is now entering phase clinical trials [6]. Its structure can be described as two trans [PtCI(NH)] units linked by a central NH(CH)eNH-trans Pt(NHa) NH(CH)eNH_ diamine chain.
Several non-classical platinum complexes that do not contain the aforementioned basic motif have also been developed. Among them, the compound [rhodamine-123] PtCI4 was the first tetrachloroplatinate (11) endowed with potent anticancer activity [7]. In addition, an (ethidium) PtCI4 conjugate showed reduced toxicity to mice compared to ethidium but equal trypanocidal activity [8]. These considerations prompted us to synthesize the bis(guanidinium) tetrachloroplatinate(ll) salt, hereafter named PtC4Gua, which incorporates a (butane-1,4-diyl)bis(guanidinium) cation and PtCI4 as counteranion.
This cation does not exhibit an inversion center half away along the C(2)-C(3) bond and the central linker chain C(1 C(2) C(3) C(4) exhibits a gauche conformation, as shown by the torsion angle value of -64.14. In contrast, in the crystal structure of S,S'-(1,8-octanediyl)bis(thiouronium)tetrachloroplatinate(ll), the cation exhibits a centre of symmetry and packs in a mixed trans(t) and gauche(g) conformation, with a tgtttttgt sequence [9]. However, in the S,S'-(1,4-butanediyl)bis(thiouronium) tetrachloroplatinate (ll),the cation exhibits an extended trans configuration [10]. Moreover, in the title compound, the cation is bisprotonated with the H attached to the imine group of each terminal guanidinium function and with the three equivalent C-N bonds in the range from 1.31(1) to 1.34(1) A. The two guanidinium moieties are planar with the C(11) and C(21) out-of-plane displacements of-0.01 and 0.02 ,&, respectively and the dihedral angle is 20.4 between them. In the [PtCI4] counteranion, the platinum atom exhibits a quasi-ideal square planar coordination, the distances being in the range 2.301(3)-2.308(2) A and the angles 89.01-90.9(1). There is an extensive hydrogen-bonding network involving the four chloride atoms of each tetrachloroplatinate(ll) anion and hydrogen atoms in the different guanidinium moieties (Table 2). Ci2 CI4 iv Figure 2 Perspective view of PtC4Gua generated by CAMERON using 50% probability ellipsoids for the non-hydrogen atoms. The crystallographic labelling scheme is shown. The hydrogen bonds are shown by dotted lines. SynTnetycode i" x, l+y, z ii -x, l-y, 1-z ;iii" 1-x,-1-y, -z ;iv" l+x,-l+y, z.
The ability of the platinated compound PtC4Gua and its non-platinated analogue C4Gua to alter the thermal denaturation profile of double stranded DNA was used as a first indication of their propensity to bind to DNA. The zTm values are collated in Table 3. A much larger increase in the Tm of nucleic acids is observed with the platinated compound than with the Pt-free bisguanidine.
The stabilization of the DNA double helix by binding of the drug increases with increasing molar ratio of drug to DNA-phosphate (D/P). We can conclude that the bisguanidinium chain promotes the interaction with DNA and that the [PtCI4] anion contributes to reinforce the interaction with DNA.
(Butane-l, 4-Diyl)BIS(Guanadinium) Tetrachloroplatinate(II)  [11]. The local duplex unwinding is believed to be a major determinant in the recognition of DNA damages by repair enzymes and therefore it may be essential to the biological activity of the drug. Electrophoresis in native agarose gel was used to determine the unwinding  The refined cell constants and other relevant crystal data are presented in Table 4, together with details of the intensity measurements. The crystal was mounted, using glass fibers, on an ENRAF-NONIUS CAD4 diffractometer equipped with a graphite monochromator. The lattice parameters were refined using 25 reflections. The data were collected using the o-2e scan technique and with Mo K(z radiation 0.71073 ). During the data collection, three intensity control reflections were monitored every two hours, showing no loss of intensity. The data were corrected for Lorentz and polarisation effects. The structure was solved by a combination of direct methods using SIR procedure [13] and heavy-atom techniques and refined by full-matrix least-squares method based on F, using CRYSTALS [14].  Table 3. Criteria for a satisfactory complete analysis were ratios of rms shift to standard deviation less than 0.1 and no significant features in final difference maps. Details of data collection and refinement are given in Table 4. Calculations were performed with a PC CRYSTALS package program. The drawings of the molecules were generated using CAMERON [16]. The atomic scattering factors were taken from International Tables for X-ray Crystallography [17].Fractional atomic coordinates and equivalent isotropic thermal parameters were shown in table 5. We measured the change of the absorbance at 260 nm as a function of the temperature for both calf thymus DNA (42% AT base pairs) and the synthetic polynucleotide poly(dA-dT) in the absence and presence of the test drugs. The variation of the Tm of helix-to-coil transition of the two nucleic acids were determined in the presence of 10 and 20lLtM drug using 20 l.tM DNA.
Tm measurements were performed in BP buffer pH 7.1 (6 mM NaHPO4, 2 mM NaH_PO4 using 20 #M DNA at 260 nm with a heating rate of lC/min. Each drug concentration was tested in duplicate. Tm for DNA alone 42.5 and 58.4 C for poly(dA-dT) and calf thymus DNA, respectively. 3.4 Unwinding of supercoiled pUC19 plasmid DNA by PtC4Gua. The DNA (0.5 t.tg) was incubated with C4Gua or PtC,Gua at the indicated concentration (taM) for 3h at 37C before loading the samples on a 1% agarose gel. Control lanes Ct refer to the plasmid DNA incubated without drug. After 2h electrophoresis, the gel was stained with ethidium bromide (ll.tg/ml) then destained in water prior to being photographed under UV light.