Influence of the Number of Axial Bexarotene Ligands on the Cytotoxicity of Pt(IV) Analogs of Oxaliplatin

We present the synthesis and cytotoxic potencies of new Pt(IV) complexes with bexarotene, an anticancer drug that induces cell differentiation and apoptosis via selective activation of retinoid X receptors. In these complexes bexarotene is positioned as an axial ligand. The complex of one bexarotene ligand attached to Pt(IV) oxaliplatin moiety was potent whereas its counterpart carrying two bexarotene ligands was inactive.


Introduction
The discovery of anticancer properties of platinum based complexes became a significant breakthrough in cancer treatment [1]. Currently cisplatin, carboplatin, and oxaliplatin remain major drugs for the first line treatment (alone and in combination) for a variety of malignancies including head and neck, testicular, breast, and ovarian tumors [2][3][4][5]. However, despite the success of platinum containing drugs, the intrinsic or acquired resistance, general toxicity, and other severe side effects are clinically unfavorable [4][5][6]. To overcome these problems novel strategies for the search of active antitumor compounds are being developed. Octahedral Pt(IV) complexes are of interest because of their kinetic inertness, low general toxicity, and possibility for oral administration [7,8]. Satraplatin was the first Pt(IV) compound to enter phase III clinical trials as an oral drug for treatment of hormone-refractory prostate cancer. Unfortunately, this compound showed no convincing benefit for overall patient survival and was not approved by the FDA. Still, clinical trials of satraplatin in combination with different organic drugs continued [9,10].
Combinations of two drugs in one molecule are extensively used in modern drug discovery and allow for control of activity, selectivity, and pharmacokinetics. The synthetic advantage of Pt(IV) complexes is the suitability for chemical modifications of axial positions which makes introduction of new active compounds relatively easy. Based on the proposed mechanism of action for Pt(IV) complexes, that is, activation by reduction, the release of the axial ligands can be useful for drug targeting and delivery to cancer cells [11]. This approach yielded a variety of promising complexes containing axial ligands such as folic acid [12], estradiol [13], short peptides [14], inhibitors of glutathione-S-transferase [15], pyruvate dehydrogenase kinase [16][17][18], histone deacetylase [19][20][21], cyclooxygenase [22][23][24], mitochondria associated hexokinase [25], or p53 activators [26].
Retinoids are biologically active analogs of vitamin A, which play an essential role in cell proliferation, differentiation, and apoptosis. Bexarotene, a selective agonist of retinoid X receptors, is used to treat cutaneous T-cell lymphoma by inducing cell differentiation and apoptosis and inhibiting metastasis [27][28][29]. Recently we have shown that introduction of bexarotene into Ru(II)-arene compounds resulted in highly cytotoxic agents [30]. Here, we report the synthesis, chemical characterization, and antiproliferative activity of Pt(IV) complexes with covalently attached axial ligand bexarotene.
Complexes 3 and 4 were characterized by 1 H, 13 C, 15 N, 195 Pt 1D, and 2D NMR spectroscopy, ESI mass spectrometry, and elemental analysis. In the 13 C{ 1 H} spectra we observed a shift of the carboxylic group that confirms the attachment of bexarotene to the platinum center. The nature of the axial ligand had only a minor influence on the 1 H and 13 C resonances in the oxaliplatin moiety [25,36]. In ESI mass spectra the most abundant peaks were assigned to the [M+Na + ] + ion in the positive ion mode or to the [M−H + ] − ion in the negative ion mode for complex 4, respectively; additionally minor peaks can be assigned to proton and potassium adducts. For all mass spectra the experimental isotopic patterns were in a good agreement with calculated isotopic distribution (Figure 1). 195 Pt NMR spectroscopy is a known method for monitoring the coordination sphere of the Pt(IV) center [34,37]. For complexes 3 and 4, the resonance at 3228 ppm and 3233 ppm, respectively, indicates a Pt(IV)N 2 O 4 coordination sphere ( Figure 2). As reported earlier, the nature of carboxylates in the axial position has no dramatic influence on the resonance in 195 Pt spectra [25,36,37].
The cytotoxicity of complexes 3 and 4, bexarotene, and cisplatin was evaluated in the human MCF7 breast cancer cell line and its doxorubicin/cisplatin resistant subline MCF7D, as well as against colon carcinoma SW480 cells, non-small cell lung carcinoma A549, and immortalized human nonmalignant keratinocyte HaCat cell line using the colorimetric MTT-test after 72 h of incubation (  Complex 3 with two axial bexarotene ligands did not affect the viability of SW480, MCF7, MCF7D, and HaCat cells at concentrations < 100 M. Also, the cytotoxic effect against non-small cell lung carcinoma cell line A549 (IC 50 = 83 ± 16 M) was minor. In contrast, complex 4 with one bexarotene ligand showed a considerably higher cytotoxicity than cisplatin against SW480, HaCat, MCF7, MCF7D, and A549 cells. Complex 4 was notably more active than parent bexarotene and exhibited high sensitivity against breast cancer cells: the IC 50 value in the MCF7 cell line was in the submicromolar range, providing a promising basis for further investigation (Table 1). Such a specificity (complex 4 is active, but 3 is not) is unexpected but rarely reported in the literature for Pt(IV) complexes with different ligands [37,38], although it is not a general rule. Recently we presented a similar design with lonidamine as biologically active component and no such specificity was observed [25].

Conclusions
A novel platinum(IV) complex featuring the oxaliplatin core and one axial bexarotene ligand exhibited high cytotoxicity against a panel of tumor cell lines. This complex is more active than cisplatin and preferential sensitivity of a breast cancer cell line to 4 compared to nonmalignant cells was found. Remarkably, complex 3 with two bexarotene ligands showed no activity in the tested cell lines. These results reveal a good Oxaliplatin 0.9 ± 0.3 [34] 11.5 ± 3.9 [34] 43.8 [35] -- * Data are mean ± standard deviation from 3 independent experiments; each drug concentration was tested in triplicate. -: no data. potential for the use of bexarotene as ligand in the search for new metal-based anticancer compounds.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.