Dichloro[N-[(η⁶-phenyl)methyl]-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8tetrahydronaphthalen-2-yl)vinyl)benzamide](1,3,5-triaza-7-phosphatricyclo [3.3.1.1^3,7]decane-κP⁷)ruthenium

Abstract

Bexarotene-tethered RuII(arene) compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) were prepared as an analog of RAPTA antitumor complexes in order to evaluate their in vitro antiproliferative activity against human cancer cell lines.

1. Introduction

Ruthenium-based antitumor drugs are the most widely studied after platinum-based complexes [1,2,3] and are first-in-class approved by FDA as an orphan drug against gastric cancer BOLD 100 (Figure 1) [4]. OrganorutheniumII(arene) compounds with PTA co-ligands, termed RAPTA (Figure 1), have previously shown excellent tumor-inhibiting properties [5].

Tethering the known metal-based drugs with the biologically active molecule is a promising approach in medicinal chemistry to improve cytotoxicity and selectivity [6,7,8]. Recently we have utilized the antitumor drug bexarotene, a selective agonist of retinoid X receptors (RXRs) and an FDA-approved drug, to treat cutaneous T cell lymphoma, improving the antitumor activity of Ru(II), Ru(III), and Pt(IV) complexes [9,10,11]. In this paper, we have followed the same approach and prepared RAPTA analogs with bexarotene moiety attached to the arene part of the complex. The synthesized compound was fully characterized using NMR spectroscopy and mass-spectrometry with electrospray ionization (ESI-MS). The antiproliferative activity was investigated against human cancer cell lines.

2. Results

The RAPTA analog with the bexarotene moiety 2 (Figure 2) was prepared by the reaction of the PTA ligand with Ru-dimer 1 in CH₂Cl₂ at room temperature (Scheme 1). A pure compound was isolated by precipitation out of the reduced-in-volume reaction mixture by the petroleum ether. The compound was characterized by ¹H, ¹³C{¹H}, and ³¹P{¹H} NMR spectroscopy, ESI-MS, and elemental analysis (full spectra available in Supplementary Materials). ³¹P{¹H} NMR spectroscopy was used to monitor the formation of the complex. These spectra included a shift from ca. −100 to −30 ppm for the PTA ligand upon coordination. The ¹H NMR spectrum had no significant shift in the proton signals for both the arene fragment and the CH₂ groups of the PTA ligand compared to the initial ruthenium dimer. ESI-mass spectra were recorded without the addition of any acid or base in both the positive and negative modes, hence, the ions corresponding to complex 2 appeared mainly due to the loss or gain of the chloride ([M − Cl]⁺ and [M + Cl]⁻ ions).

For the obtained complex 2, the antiproliferative activity was studied by the MTT-assays on a series of human cancer cell lines (lung carcinoma A549, colon carcinoma HCT116, breast adenocarcinoma MCF7, colon adenocarcinoma SW480) and is presented in

Table 1. The 50% inhibitory concentrations of complex 2, bexarotene and cisplatin. Values are means ± SDs obtained by the MTT-assay (exposure time: 72 h).

Compounds	A549	HCT116	MCF7	SW480
2	31 ± 4	32 ± 7	27 ± 2	23.4 ± 0.6
cisplatin	8.8 ± 0.9	12 ± 2	13 ± 1	6.2 ± 0.6
Bexarotene	50 ± 2	51 ± 2	75 ± 1	69 ± 1

. The complex exhibits activity in the range of low micromolar concentrations, exceeding the activity of the parent drug bexarotene Ru-dimer 1 and greatly exceeding the activity of RAPTA-type complexes.

3. Materials and Methods

3.1. General

All commercial reagents were used without further purification. All solvents were purified and degassed before use. ¹H NMR, ¹³C NMR, and ³¹P spectroscopy were performed at 298 K on Bruker Avance 600. ¹H and ¹³C NMR spectra were calibrated against the residual solvent: CDCl₃. ³¹P spectra were calibrated by external reference (85% H₃PO₄ in H₂O, δ = 0 ppm). The splitting of the proton resonances in the reported ¹H spectra is defined as s = singlet, d = doublet, t = triplet, and m = multiplet. Mass-spectra were recorded using TSQ Endura (Thermo Scientific, Waltham, MA, USA) mass-spectrometer with an electrospray ionization source (ESI). The methanol solution of each compound was introduced through a syringe pump directly into the ion source at 5–10 μL/min. Mass spectra were acquired in both positive and negative modes. The system was controlled by the Xcalibur software, which was also used for data collection and data processing. The ion transfer tube temperature was set to 275 °C, and the vaporizer temperature to 40 °C. The Sheath and Aux gases were 6 and 5 units, while the spray voltage was 3.4 and 2.5 kV for both positive and negative modes correspondingly. The spectra were recorded during 30 s in the m/z range 150–1400.

Dichloro[N-[(η⁶-phenyl)methyl]-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8tetrahydronaphtha-len-2-yl)vinyl)benzamide](1,3,5-triaza-7-phosphatricyclo[3.3.1.1^3,7]decane-κP⁷)ruthenium.

A solution of 1,3,5-triaza-7-phosphaadamantane (26 mg, 0.16 mmol) in CH₂Cl₂ (1 mL) was added to a solution of ruthenium dimer 1 (100 mg, 0.08 mmol) in CH₂Cl₂ (14 mL). The reaction mixture was stirred for 2 h at room temperature. The solvent was evaporated under the vacuum to a minimum volume, the product was precipitated with petroleum ether, separated by centrifugation, and the resulting dark orange powder was dried in a vacuum.

Yield 107 mg (85%, T_dec = 230–235 °C)

¹H NMR (600.13 МHz, CDCl₃): δ(ppm) 8.08 (t, 1H, J = 5.4 Hz, NH), 7.88 (d, 2H, J = 8.0 Hz, H16; H18), 7.37 (d, 2H, J = 8.0 Hz, H15; H19), 7.12 (s, 1H, H6), 7.07 (s, 1H, H3), 5.80 (s, 1H, H13), 5.70–5.64 (m, 4H, H27; H28; H30; H31), 5.30 (s, 1H, H13), 5.14 (t, 1H, J = 4.8 Hz, H29),4.65 (d, 2H, J = 5.7 Hz, H13), 4.51 (s, 6Н, H4_PTA; H5_PTA; H6_PTA), 4.32 (s, 6Н, H1_PTA; H2_PTA; H3_PTA), 1.94 (s, 3H, H14), 1.70 (s, 4H, H8; H9), 1.29 (d, 12H, J = 17.2 Hz, H23; H24; H25; H26).

¹³C NMR (150.92 МHz, CDCl₃): δ (ppm) 166.7 (СО), 148.6 (С11), 144.2 (С12), 143.8 (С2), 141.8 (С1), 137.5 (С5), 132.2 (С4), 131.3 (С17), 127.5 (С3;С6), 127.0 (С16; С18), 126.4 (С15; С19), 116.0 (С13), 100.0 (С_arene), 86.3 (С_arene), 85.0 (С_arene), 85.0 (С_arene), 72.8 (J = 6.0 Hz, С4_PTA; C5_PTA; C6_PTA), 52.5 (J = 16.6 Hz, С1_PTA; C2_PTA; C3_PTA), 40.7 (С22), 34.7 (С8; C9), 33.5 (С7/C10), 33.4 (С7/C10), 31.5 (С23/C24/C25/C26), 31.4 (С23/C24/C25/C26), 19.5 (С14).

³¹P NMR (242.94 МHz, CDCl₃): δ (ppm) −30.2.

Elemental analysis calculated for C₃₇H₄₇Cl₂N₄OPRu: C 57.96, H 6.18, N 7.31, found: C 58.42, H 6.07, N 7.18.

ESI-MS: m/z: 731 [M − Cl]⁺, 801 [M + Cl]⁻.

3.2. Cells and In Vitro Antiproliferative Assays

The human HCT116 colorectal carcinoma, SW480 colon adenocarcinoma, A549 non-small cell lung carcinoma, and MCF7 breast adenocarcinoma cell lines were obtained from the European collection of authenticated cell cultures (ECACC; Salisbury, UK) All cells were grown in a DMEM medium (Gibco™, Dublin, Irland) supplemented with 10% fetal bovine serum (Gibco™, Brazil). The cells were cultured in an incubator at 37 °C in a humidified 5% CO₂ atmosphere and were sub-cultured 2 times a week. The effect of the investigated compounds on cell proliferation was evaluated using a common MTT assay. The cells were seeded in 96-well tissue culture plates («ТРР», Trasadingen, Switzerland) at a 1 × 10⁴ cells/well in 100 µL of the medium. After overnight incubation at 37 °C, the cells were treated with the tested compounds in the concentration range of 0 to 200 µM. Cisplatin was used as a standard. After 72 h of treatment, the solution was removed, and a freshly diluted MTT solution (100 µL, 0.5 mg/mL in cell medium) was added to the wells, and the plates were further incubated for 50 min. Subsequently, the medium was removed, and the formazan product was dissolved in 100 μL of DMSO. The number of living cells in each well was evaluated by measuring the absorbance at 570 nm using the «Zenith 200 rt» microplate reader (Biochrom, Cambridge, UK).

4. Conclusions

The synthesis and characterization of bexarotene—tethered RuII (arene) compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) as an analog of the RAPTA complex has been reported here for the first time. The antiproliferative activity of the compound 2 showed the potential applications of this compound as an antitumor agent.