Potent Chlorambucil-Platinum(IV) Prodrugs

Abstract

The DNA-alkylating derivative chlorambucil was coordinated in the axial position to atypical cytotoxic, heterocyclic, and non-DNA coordinating platinum(IV) complexes of type, [Pt^IV(H_L)(A_L)(OH)₂](NO₃)₂ (where H_L is 1,10-phenanthroline, 5-methyl-1,10-phenanthroline or 5,6-dimethyl-1,10-phenanthroline, A_L is 1S,2S-diaminocyclohexane). The resultant platinum(IV)-chlorambucil prodrugs, PCLB, 5CLB, and 56CLB, were characterized using high-performance liquid chromatography, nuclear magnetic resonance, ultraviolet-visible, circular dichroism spectroscopy, and electrospray ionization mass spectrometry. The prodrugs displayed remarkable antitumor potential across multiple human cancer cell lines compared to chlorambucil, cisplatin, oxaliplatin, and carboplatin, as well as their platinum(II) precursors, PHENSS, 5MESS, and 56MESS. Notably, 56CLB was exceptionally potent in HT29 colon, Du145 prostate, MCF10A breast, MIA pancreas, H460 lung, A2780, and ADDP ovarian cell lines, with GI₅₀ values ranging between 2.7 and 21 nM. Moreover, significant production of reactive oxygen species was detected in HT29 cells after treatment with PCLB, 5CLB, and 56CLB up to 72 h compared to chlorambucil and the platinum(II) and (IV) precursors.

Keywords: 56MESS; 5MESS; DNA; PHENSS; ROS; chlorambucil; cytotoxicity; platinum(II); platinum(IV).

Figure 1

Chemical structures of platinum(II) chemotherapeutics.

Figure 2

An illustration of the clinically used platinum(II) drugs and the likely molecular interactions before it reaches the primary target, the DNA.

Figure 3

Platinum(II) complexes undergo oxidation to generate their platinum(IV) derivatives, subsequently platinum(IV) complexes undergo reduction to generate their platinum(II) congeners, as well as release the bioactive or non-bioactive axial ligands. L = ligand. A_L = axial ligand.

Figure 4

Structures of CLB, unconventional non-covalent DNA-binding platinum(II) complexes, PHENSS, 5MESS, and 56MESS, together with the studied platinum(IV)-CLB prodrugs, PCLB, 5CLB, and 56CLB.

Figure 5

Synthetic pathway to create the studied platinum(IV)-CLB prodrugs. Counter-ions of the platinum complexes are omitted for clarity.

Figure 6

Proton labeling system of the platinum(IV)-CLB complexes.

Figure 7

¹H-NMR spectrum of PCLB in D₂O obtained at 298 K, with proton assignment. Inset: structure of PCLB with proton labeling system.

Figure 8

Stacked ¹H-NMR spectra of PHENSS, PHENSS(IV)(OH)₂ and PCLB in D₂O obtained at 298 K, with arrows highlighting the movement of chemical resonances and change in multiplicity. Inset: structures of PHENSS, PHENSS(IV)(OH)₂ and PCLB with proton labeling systems.

Figure 9

Expanded ¹H-¹⁹⁵Pt-HMQC spectrum of PCLB, highlighting the coupling between H2, H9, H3, and H8 protons with platinum. Region: 400 ppm. Inset: structure of PCLB with proton labeling system and arrows that indicate coupling.

Figure 10

Stacked UV spectra of PCLB, 5CLB, and 56CLB obtained at 298 K, showing UV absorptions at different wavelengths. Inset: color-coded structures of PCLB, 5CLB, and 56CLB.

Figure 11

1D-¹⁹⁵Pt-NMR spectra of PCLB, 5CLB, and 56CLB in 10 mM PBS (~7.4 pH) within the regions of −2800 and 400 ppm at 37 °C. The red box encloses the platinum resonances recorded for the complexes within the region of 400 ppm. Inset: structures of PCLB, 5CLB, and 56CLB.

Figure 12

Zoomed ¹H-NMR spectra of PCLB with PBS and AsA in D₂O at 310.15 K, in different time intervals, highlighting the movement of resonances of the phenanthroline protons and the aromatic protons of the CLB ligand as indicated by the red arrows. T represents time in min.

Figure 13

1D-¹⁹⁵Pt-NMR spectra of PCLB with PBS and AsA in D₂O at 310.15 K, within the regions of 400 and −2800 ppm, highlighting the complete reduction in the complex after 1 h. Inset: structures of PCLB and its platinum(II) scaffold, PHENSS.

Figure 14

ROS production upon treatment with platinum(IV)-CLB prodrugs, platinum(II) and (IV) scaffolds, CLB, and cisplatin in HT29 colon cells at 24, 48, and 72 h. **** indicates p < 0.0001 compared with control. *** indicates p < 0.001 compared with control. ** indicates p < 0.01, compared with control. * indicates p < 0.05 compared with control. Data points denote mean ± SEM. n = 3 from three independent experiments where samples were run in triplicate.