New Oxaliplatin-Pyrophosphato Analogs with Improved In Vitro Cytotoxicity

Abstract

Two new Pt(II)-pyrophosphato complexes containing the carrier ligands cis-1,3-diaminocyclohexane (cis-1,3-DACH) and trans-1,2-diamine-4-cyclohexene (1,2-DACHEX), variants of the 1R,2R-diaminocyclohexane ligand present in the clinically used Pt-drug oxaliplatin, have been synthesized with the aim of developing new potential antitumor drugs with high bone tropism. The complexes are more stable at physiological pH than in acid conditions, with Na₂[Pt(pyrophosphato)(cis-1,3-DACH)] (1) slightly more stable than [Pt(dihydrogenpyrophosphato)(1,2-DACHEX)] (2). The greater reactivity at acidic pH ensures a greater efficacy at the tumor site. Preliminary NMR studies indicate that 1 and 2 react slowly with 5'-GMP (used as a model of nucleic acids), releasing the pyrophosphate ligand and affording the bis 5'-GMP adduct. In vitro cytotoxicity assays performed against a panel of four human cancer cell lines have shown that both compounds are more active than oxaliplatin. Flow cytometry studies on HCT116 cells showed that the pyrophosphato compounds with the non-classical 1,3- and 1,4-diaminocyclohexane ligands (1 and 4) are the most capable to induce cells' death by apoptosis and necrosis.

Keywords: antitumor drugs; bone tumors; cisplatin; oxaliplatin; phosphaplatins; pyrophosphate.

Figure 1

Pt(II)-pyrophosphato complexes.

Scheme 1

(a) Water, excess KI (8-fold), 5 min, room temperature, and then cis-1,3-diamino-cyclohexane (cis-1,3-DACH), 40 °C, 2.5 h. (b) Water, Ag₂CO₃, 40 °C, 2 h. (c) Water, pH = 8, sodium pyrophosphate decahydrate at 55 °C for 3 h, and then pH = 6 at room temperature for 30 min.

Figure 2

(a) COSY (300 MHz), (b) [¹H-¹³C] HSQC (¹³C, 75.5 MHz), (c) ³¹P{¹H} (121.5 MHz), and (d) ¹⁹⁵Pt{¹H} (64.5 MHz) NMR spectra of 1 dissolved in D₂O.

Figure 3

(a) COSY (300 MHz), (b) [¹H-¹³C] HSQC (75.5 MHz, ¹³C), (c) ³¹P{¹H} (121.5 MHz), and (d) ¹⁹⁵Pt{¹H} (64.5 MHz) NMR spectra of 2 in D₂O (pH* = 9).

Figure 4

(a) Scheme of the reaction mechanism for the formation of complex 2. (b) ³¹P NMR spectra of the reaction mixture in H₂O/D₂O (90:10) recorded at different time intervals.

Figure 5

Plot of ³¹P chemical shift vs. pH* for 1 (a) and 2 (b).

Scheme 2

Protonation/deprotonation steps for 1 (a) and 2 (b) with corresponding pK_a1 and pK_a2.

Figure 6

³¹P NMR (121.5 MHz) spectra at different times of 1 (a) and 2 (b) in physiological conditions (D₂O, HEPES buffer 50 mM, pH* = 7.4, 120 mM NaCl, 37 °C).

Figure 7

³¹P NMR (121.5 MHz) spectra at different times of 1 (a) and 2 (b) in acidic conditions (D₂O, HEPES buffer 50 mM, pH* = 6.5, 120 mM NaCl, 37 °C).

Figure 8

³¹P NMR spectra at different time intervals of 1 (5 mM) in the presence of 5’-GMP (12.5 mM) at pH* = 7.4 (D₂O, 50 mM HEPES buffer, 120 mM NaCl, 37 °C).

Figure 9

(a) ³¹P NMR spectra (121.5 MHz) at different time intervals of 2 (5 mM) in the presence of 5’-GMP (12.5 mM) at pH* = 7.4 (D₂O, 50 mM HEPES buffer, 120 mM NaCl, 37 °C). (b) ³¹P NMR spectrum (202.3 MHz) of 2 (5 mM) in the presence of 5’-GMP (12.5 mM) at pH* = 7.4 (D₂O, 50 mM HEPES buffer, 120 mM NaCl, 37 °C) recorded after 624 h.

Figure 10

¹H NMR spectra at different times of 2 (5 mM) in the presence of 5’-GMP (12.5 mM) at pH* = 7.4 (D₂O, 50 mM HEPES buffer, 120 mM NaCl, 37 °C). Diamonds indicate free 5’-GMP, circles indicate coordinated 5’-GMP, stars indicate starting complex 2, and triangles indicate the bis-adduct [Pt(5’-GMP)₂(1,2-DACHEX)].

Figure 11

In vitro cytotoxicity of compounds 1, 2, 3, 4, oxaliplatin (OXP), and cisplatin (CDDP) towards HCT-116, PC3, OV2008, and MDAMB231 cell lines.

Figure 12

FCM dot plots of Annexin V/PI-positive HCT116 cells after 24 and 48 h incubation with compounds 1–4, oxaliplatin (OXP), and cisplatin (CDDP) at their IC₅₀ concentrations (reported in Supplementary Table S1). The lower left quadrants of each panel show the viable cells. The upper left quadrants contain the necrotic cells. The upper right quadrants contain the late apoptotic cells. The lower right quadrants contain the early apoptotic cells. Each quadrant includes the corresponding % of cells measured by flow cytometry.