Kinetically Favored Platination of Adenine in the G-Rich Human Telomeric Repeat

The interactions of PT-ACRAMTU, a cytotoxic platinum−acridine conjugate, with the human telomeric G-quadruplex have been studied using in-line high-performance liquid chromatography−mass spectrometry and footprinting assays. The conjugate reacts significantly faster with quadruplex DNA (t1/2 = 1.2 h) than with double-stranded DNA, and A-N7, and not G-N7, is the kinetically preferred target, an unprecedented reactivity feature in platinum−DNA interactions. Unlike the clinical platinum drug cisplatin, which targets the human telomeric sequence nonspecifically, the platinum−intercalator technology has the potential to produce telomere-specific anticancer agents via a mechanism that kinetically discriminates between G and A in the two DNA secondary structures

Modulation of Nucleotide Binding of <i>trans</i> Platinum(II) Complexes by Planar Ligands. A Combined Proton NMR and Molecular Mechanics Study

Nonclassical trans platinum complexes containing planar nitrogen bases show biological activity different from that of trans-diamminedichloroplatinum(II) (trans-DDP). In search of the mechanism of action of such compounds, a comparative study on the nucleobase chemistry of trans-DDP and trans-[PtCl2(NH3)(quinoline)] (trans-QUIN) was performed using 1D and 2D NMR spectroscopy and molecular modeling techniques. The two simple monofunctional adducts trans-[PtCl(9-ethylguanine-N7)(NH3)L]NO3 (L = NH3, 1; L = quinoline, 2) were synthesized by employing the AgNO3/DMF method. Reactions of these species with 5‘-guanosine monophosphate (5‘-GMP) and 5‘-cytidine monophosphate (5‘-CMP) were used to simulate potential second binding steps on DNA. Guanine-N7 proved to be the kinetically preferred binding site for both 1 and 2. Reactions with 2 proceeded significantly slower than those with 1 under the same conditions. These differences in reactivity are attributed to an altered hydrolytic behavior of 2 due to steric influences of quinoline upon associative substitution reactions. This is supported by interligand NOEs observed in the 2D NOESY spectrum of 2 and by AMBER-based geometries for different conformers of 2. Signal splittings observed in the 1H NMR spectra of 2 and the bifunctional adducts trans-[Pt(9-EtGua-N7)(5‘-GMP-N7)(NH3)L] (4) and trans-[Pt(9-EtGua-N7)2(NH3)L]2+ (6) (L = quinoline) indicate hindered rotation about the Pt−N (guanine and quinoline) bonds. Temperature-dependent NMR spectra and molecular mechanics results are in agreement with frozen rotamers in solution at room temperature where unfavorable repulsive interligand interactions result in different head-to-head and head-to-tail orientations of the bases. For the different rotamers of 4, a high barrier of interconversion of 87 kJ mol-1 was estimated from NMR data. The consequences of these kinetic and geometric effects with respect to target DNA are discussed

Inversion of the Cis Geometry Requirement for Cytotoxicity in Structurally Novel Platinum(II) Complexes Containing the Bidentate N,O-Donor Pyridin-2-yl-acetate

Water soluble platinum(II) complexes have been synthesized that contain the N,O-chelate pyridin-2-yl acetate (PyAc) as a novel structural motif in platinum antitumor complexes. The trans-platinum complex trans-[PtCl(PyAc-N,O)(NH3)] (2) (N-donors are trans) and its isomer cis-[PtCl(PyAc-N,O)(NH3)] (4) (N trans to Cl) were prepared from trans-[PtCl2((NH3)(PyAcH)]·H2O (1·H2O) and cis-[PtCl2(NH3)(PyAcMe) (3), respectively, employing the bidentate ligand as its methylester (PyAcMe). 2 and 4 are readily formed from the respective dichloro species, even at low pH and in the presence of extra chloride, indicating a high thermodynamic stability of the PyAc chelate ring. 1·H2O and 2−4 were characterized by 1H NMR and IR spectroscopy and elemental analyses. The solid-state structure of 2 was determined:  triclinic, P1̄ (no. 2), with a = 8.170(2) Å, b = 9.274(3) Å, c = 7.374(2) Å, α = 108.68(2)°, β = 113.27(2)°, γ = 74.40(2)°, V = 479.7(6) Å3, Z = 2. The six-membered metallacyclus in 2 adopts a “boat” form, allowing a strainless coordination of platinum. The most promising cytotoxic properties in the above series of compounds have been established for 2 (and 1, which rapidly transforms into 2 at 37 °C and neutral pH). Preliminary ID50 values were 0.88 and 1.26 μM, respectively, in cisplatin-sensitive L1210 leukemia. Both compounds proved to be cross-resistant to the clinical drug. Reactions of 2 and 4 with 5‘-guanosine monophosphate (5‘-GMP) under physiological conditions gave the monofunctional adducts trans- and cis-[Pt(5‘-GMP-N7)(PyAc-N,O)(NH3)] (I and II). Chelate-bound carboxylate was not replaced by guanine-N7 when an excess of nucleotide was applied (NMR). In an analogous reaction, 2 reacts with the oligonucleotide d(TCGT) [5‘-T(1)-C(2)-G(3)-T(4)-3‘] to give the adduct d(TCGT)-N7(3)-Pt(PyAc-O,N)(NH3) (III), which was characterized by a combination of total correlation spectroscopy, double-quantum-filtered correlation spectroscopy, nuclear Overhauser effect spectrometry, and rotating-frame Overhauser enhancement spectroscopy experiments. Binding of the [Pt(PyAc-N,O)(NH3)]+ fragment to N7 of G(3) causes an increase of N-type character of the T(4) and G(3) deoxyribose residues relative to the unplatinated sequence, while those of T(1) and C(2) remain S-type. An internucleotide nuclear Overhauser effect between H6(4) and H2‘(3) indicates stacking between guanine and the 3‘-thymine base. The most striking feature proved to be the pronounced upfield shift and broadening of the 1H NMR signals assigned to the base protons H5 and H6 in III. Magnetization transfer between H5(2) and H3 of pyridine suggests that this effect is caused by base−base interactions involving the planar ligand on platinum, which must be situated on the 5‘ face of guanine. Possible implications for the DNA binding and cytotoxic effect of the compounds are discussed

Cellular Recognition and Repair of Monofunctional–Intercalative Platinum–DNA Adducts

The cellular recognition and processing of monofunctional–intercalative DNA adducts formed by [PtCl­(en)­(L)]­(NO₃)₂ (<b>P1-A1</b>; en = ethane-1,2-diamine; L = <i>N</i>-[2-(acridin-9-ylamino)­ethyl]-<i>N</i>-methylpropionamidine, acridinium cation), a cytotoxic hybrid agent with potent anticancer activity, was studied. Excision of these adducts and subsequent DNA repair synthesis were monitored in plasmids modified with platinum using incubations with mammalian cell-free extract. On the basis of the levels of [α-³²P]-dCTP incorporation, <b>P1-A1</b>–DNA adducts were rapidly repaired with a rate approximately 8 times faster (<i>t</i>_1/2 ≈ 18 min at 30 °C) than the adducts (cross-links) formed by the drug cisplatin. Cellular responses to <b>P1-A1</b> and cisplatin were also studied in NCI-H460 lung cancer cells using immunocytochemistry in conjunction with confocal fluorescence microscopy. At the same dose, <b>P1-A1</b>, but not cisplatin, elicited a distinct requirement for DNA double-strand break repair and stalled replication fork repair, which caused nuclear fluorescent staining related to high levels of MUS81, a specialized repair endonuclease, and phosphorylated histone protein γ-H2AX. The results confirm previous observations in yeast-based chemical genomics assays. γ-H2AX fluorescence is observed as a large number of discrete foci signaling DNA double-strand breaks, pan-nuclear preapoptotic staining, and unique circularly shaped staining around the nucleoli and nuclear rim. DNA cleavage assays indicate that <b>P1-A1</b> does not act as a typical topoisomerase poison, suggesting the high level of DNA double-strand breaks in cells is more likely a result of topoisomerase-independent replication fork collapse. Overall, the cellular response to platinum–acridines shares striking similarities with that reported for DNA adduct-forming derivatives of the drug doxorubicin. The results of this study are discussed in light of the cellular mechanism of action of platinum–acridines and their ability to overcome resistance to cisplatin

Unexpected Reactivity of the 9-Aminoacridine Chromophore in Guanidylation Reactions

The 9-aminoacridine chromophore is an important building block of DNA-targeted chemotherapeutic agents. The success of 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea as a carrier group in cytotoxic platinum−intercalator conjugates prompted us to explore the synthesis of an analogous guanidine-functionalized acridine. In a successful effort to generate such a derivative, various methods of guanidylation were employed, which demonstrate that the acridine C9−N9 linkage is highly susceptible to electrophilic and nucleophilic attack. The newly established reactivities provide efficient pathways to novel cyclic and spirocyclic acridine derivatives

Unexpected Reactivity of the 9-Aminoacridine Chromophore in Guanidylation Reactions

The 9-aminoacridine chromophore is an important building block of DNA-targeted chemotherapeutic agents. The success of 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea as a carrier group in cytotoxic platinum−intercalator conjugates prompted us to explore the synthesis of an analogous guanidine-functionalized acridine. In a successful effort to generate such a derivative, various methods of guanidylation were employed, which demonstrate that the acridine C9−N9 linkage is highly susceptible to electrophilic and nucleophilic attack. The newly established reactivities provide efficient pathways to novel cyclic and spirocyclic acridine derivatives

Replacement of a Thiourea-S with an Amidine-NH Donor Group in a Platinum−Acridine Antitumor Compound Reduces the Metal’s Reactivity with Cysteine Sulfur

The reactivity of two DNA-targeted platinum−acridine conjugates with cysteine sulfur was studied. The conjugate containing an amidine-NH donor group cis to the chloride leaving group showed considerably reduced reactivity with N-acetylcysteine compared to the prototypical derivative containing a thiourea-S linkage. The opposite scenario has been observed previously in reactions with nucleobase nitrogen. Possible consequences of the unique target-selective tuning of the substitution chemistry for the pharmacodynamic properties and biological activity of these agents are discussed

Solution Structural Study of a DNA Duplex Containing the Guanine-N7 Adduct Formed by a Cytotoxic Platinum−Acridine Hybrid Agent^†^,^‡

[PtCl(en)(ACRAMTU-S)](NO3)2 (PT-ACRAMTU; en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) is a dual metalating/intercalating DNA binding drug conjugate that shows cytotoxicity at micromolar to nanomolar concentrations in a wide range of solid tumor cell lines. In ∼80% of its adducts, PT-ACRAMTU binds to guanine-N7 in the major groove, selectively at 5‘-CG sites [Budiman, M. E. et al. (2004) Biochemistry 43, 8560−8567]. Here, we report the synthesis, physical characterization, and NMR solution structure of a site-specifically modified octamer containing this adduct, 5‘-CCTCG*TCC-3‘/3‘-GGAGCAGG-5‘, where the asterisk indicates the [Pt(en)ACRAMTU)]3+ fragment. The structure was determined by a combination of high-resolution 2-D NMR spectroscopy and restrained molecular dynamics/molecular mechanics (rMD/MM) calculations using 179 NOE distance restraints and refined to an r6 weighted residual (Rx) of 9.2 × 10-2 using the complete relaxation matrix approach. An average structure was calculated from the final ensemble of 19 rMD geometries showing pairwise root-mean-square deviations of <1.05 Å. The dual binding increases the thermal stability of the octamer compared to the unmodified duplex (ΔTm = 13.2°). The modified sequence shows structural features reminiscent of both B- and A-type DNA. Watson−Crick hydrogen bonding is intact at and beyond the adduct site. Platinum is bound to the N7 position of G5 in the major groove, and ACRAMTU intercalates into the central 5‘-C4G5/C12G13 base-pair step on the 5‘-face of the platinated nucleobase. The chromophore's long axis is aligned with the long axes of the adjacent base pairs, maximizing intermolecular π−π stacking interactions. PT-ACRAMTU lengthens (rise, 6.62 Å) and unwinds (twist, 15.4°) the duplex at the central base-pair step but does not cause helical bending. No C3‘-endo deoxyribose pucker and no significant roll are observed at the site of intercalation/platination, which clearly distinguishes the PT-ACRAMTU-induced damage from the 1,2-intrastrand cross-link formed by cisplatin. Overall, the DNA perturbations produced by PT-ACRAMTU do not appear to mimic those caused by the major cisplatin lesion. Instead, intriguing structural similarities are observed for PT-ACRAMTU's monoadduct and the N7 adducts of dual major-groove alkylating/intercalating antitumor agents, such as the pluramycins

Modification of Platinum(II) Antitumor Complexes with Sulfur Ligands. 1. Synthesis, Structure, and Spectroscopic Properties of Cationic Complexes of the Types [PtCl(diamine)(L)]NO₃ and [{PtCl(diamine)}₂(L-L)](NO₃)₂ (L = Monofunctional Thiourea Derivative; L-L = Bifunctional Thiourea Derivative)

A new class of mono- and dinuclear platinum(II) complexes is described that derives from the cisplatin analogues [Pt(en)Cl2] and [Pt(dach)Cl2] (en = 1,2-ethanediamine, dach = racemic trans-1,2-cyclohexanediamine). The selective substitution of one chloro ligand in these species by 1,1,3,3-tetramethylthiourea (tmtu), which requires abstraction of chloride with silver salt in DMF, gives [PtCl(en)(tmtu)]NO3 (1) and [PtCl(dach)(tmtu)]NO3 (2). Similarly, reactions employing the novel bifunctional thiourea derivatives C2H4(NMeCSNMe2)2 (3) and C6H12(NMeCSNMe2)2 (4) yield the dinuclear complexes [{Pt(en)Cl}2(μ-3-S,S‘)](NO3)2 (5) and [{Pt(en)Cl}2(μ-4-S,S‘)](NO3)2·0.5EtOH (6), respectively. The compounds were characterized by 1H, 13C, and 195Pt NMR spectroscopy, elemental analyses, and IR data. 195Pt chemical shifts in the −2895 to −2929 ppm region confirm the mixed-donor [PtN2ClS] coordination for 1, 2, 5, and 6 and thiourea-S coordination in all cases. The single-crystal X-ray structures of 2−4 have been determined. 2:  monoclinic, space group P21/n, a = 10.804 Å, b = 16.221 Å, c = 21.789 Å, β = 102.16(1)°, Z = 8. 3:  monoclinic, space group P21/n, a = 12.787(2) Å, b = 6.250(1) Å, c = 17.777(3) Å, β = 98.21(1)°, Z = 4. 4:  monoclinic, space group P21/n, a = 11.097(3) Å, b = 13.717 Å, c = 11.925 Å, β = 97.61(2)°, Z = 4. The Pt−S distance in 2 (2.285(2) Å, mean) is in accordance with the magnitude of shielding found for the 195Pt core and suggests weak π-acceptor properties of tmtu. The bifunctional thiourea derivatives 3 and 4 adopt highly elongated conformations in the solid state where the sulfur atoms and the n-(CH2)n (n = 2, 6) linkers are Z-oriented. Force field calculations on 3 and 4 imply that the Z-form should be the preferred conformer for the thiourea groups in solution. 1H NMR spectra indicate a dynamic equilibrium of different rotamers due to low barriers of rotation within the thiourea moieties in free and coordinated 3 and 4. It is suggested that the steric and electronic effects of the peralkylated thiourea derivatives in 1, 2, 5, and 6 may modulate the affinity of the complexes for biomolecules

Unexpected Reactivity of the 9-Aminoacridine Chromophore in Guanidylation Reactions

The 9-aminoacridine chromophore is an important building block of DNA-targeted chemotherapeutic agents. The success of 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea as a carrier group in cytotoxic platinum−intercalator conjugates prompted us to explore the synthesis of an analogous guanidine-functionalized acridine. In a successful effort to generate such a derivative, various methods of guanidylation were employed, which demonstrate that the acridine C9−N9 linkage is highly susceptible to electrophilic and nucleophilic attack. The newly established reactivities provide efficient pathways to novel cyclic and spirocyclic acridine derivatives