Synthesis and Cytotoxic Evaluation of Steroidal Copper (Cu (II)) Complexes

Using estrone and pregnenolone as starting materials, some steroidal copper complexes were synthesized by the condensation of steroidal ketones with thiosemicarbazide or diazanyl pyridine and then complexation of steroidal thiosemicarbazones or steroidal diazanyl pyridines with Cu (II). The complexes were characterized by IR, NMR, and HRMS. The synthesized compounds were screened for their cytotoxicity against HeLa, Bel-7404, and 293T cell lines in vitro. The results show that all steroidal copper (II) complexes display obvious antiproliferative activity against the tested cancer cells. The IC50 values of complexes 5 and 12 against Bel-7404 (human liver carcinoma) are 5.0 and 7.0 μM.


Introduction
Metal-based antitumor drugs play a relevant role in antiblastic chemotherapy [1,2]. Cisplatin is regarded as one of the most effective drugs [3][4][5][6][7][8], even if severe toxicities and drug resistance phenomena limit its clinical use [9]. Therefore, in recent years, there has been a rapid expansion in research and development of novel metal-based anticancer drugs in order to improve clinical effectiveness, reduce general toxicity, and broaden the spectrum of activity [10][11][12].
Copper (Cu) is a transition metal that can exist in oxidised and reduced states. This allows it to participate in redox and catalytic chemistry, making it a suitable cofactor for a diverse range of enzymes and molecules. Cu deficiency or toxicity is implicated in a variety of pathological conditions.
Steroid hormones play an important role in the biochemistry of many cancers; a number of steroidal complexes connected to a metal pharmacophore had been designed and synthesized by many research groups, and their physiological activities were evaluated [13,14].
Thiosemicarbazones have received considerable attention since the discovery of their cytotoxic activity against cancer cells and bacteriostatic effects [15]. As the disruption of copper homeostasis is a pathological feature of cancer cells, copper complexes had been investigated for their potential applications as anticancer drugs [16]. Cu complexes of thiosemicarbazone (TSC) compounds had been explored as antimalarial, antifungal, antinociceptive, and antibacterial agents [17][18][19]. Cu complexes of bis(thiosemicarbazones) (CuII(btsc)s) had also been investigated as metallodrugs and diagnostic agents [20]. More recently, Adsule et al. [13] investigated the bioactivity of some new steroidal thiosemicarbazones Cu (II) metal complexes and discovered that some compounds had better antineoplastic activity.
In the present study, some steroidal copper complexes were synthesized by the condensation of steroidal ketones with thiosemicarbazide or diazanyl pyridine and then complexation of steroidal thiosemicarbazones or steroidal diazanyl pyridines with Cu (II). The synthesized compounds were screened for their cytotoxicity against HeLa, Bel-7404, and 293T cell lines in vitro.

Materials.
The sterols were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. All chemicals and solvents are of analytical grade from commercial sources. All solvents were used without further purification unless otherwise specified.

Instrumentation and Methods.
Melting points were determined on an X4 apparatus (Beijing Tech Instrument Co. Ltd., Beijing, China) and were uncorrected. The 1 H and 13 C NMR spectra were recorded in CDCl 3 on a Bruker AV-600 spectrometer at working frequencies of 600 and 150 MHz and a Bruker AV-300 spectrometer at working frequencies of 300 and 75 MHz, respectively. Chemical shifts are expressed in parts per million ( ) values and coupling constants (J) in Hertz. Infrared spectra were measured with a Thermo Scientific Nicolet IS-10 Spectrophotometer (Thermo Scientific, USA). HREIMS was measured on an Agilent 6210 TOFMS instrument (Agilent Technologies, USA). The cell proliferation assay was undertaken by a MTT method using 96-well plates on a MLLTISKAN MK3 analysis spectrometer (Thermo Scientific, Shanghai, China).

General Procedure for Preparation of Steroidal
Thiosemicarbazones. Steroidal ketone (0.38 mmol) was dissolved in 40 mL 95% ethanol. After the solution was heated to 65 ∘ C, a few drops of glacial acetic acid were added to adjust pH to 3-5, and thiosemicarbazide (1.70 mmol) was added. The mixture was stirred at 60-70 ∘ C for 6 h (the progress of the reaction was monitored by TLC, ethyl acetate : petroleum ether = 1 : 2). Then, the reaction was terminated and majority of solvent was evaporated under reduced pressure. Suitable amount of water was added to the reaction mixture, and the product was extracted with CH 2 Cl 2 . The combined extract was washed with saturated NaHCO 3 solution, water, and saturated brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting residue was separated by the column chromatography using a mixture of ethyl acetate : petroleum ether (1 : 2) to give target products.

General Procedure for Preparation of Steroidal Diazanyl
Pyridine. A mixture of steroidal ketone (1 mmol) and diazanyl pyridine (1 mmol) in 95% ethanol (30 mL) was stirred at 70-80 ∘ C for 6 h. After completion of the reaction, the majority of solvent was evaporated and some water was added to this solution. The mixture was extracted with CH 3 COOC 2 H 5 and the extract was washed with saturated brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting residue was chromatographed on a column of silica gel with mixture of petroleum ether/ethyl acetate (1 : 1) to give steroidal diazanyl pyridine. (9,9 ). 3 -Hydroxypregnenolone-20-(2 -diazanyl)pyridine (10,10

General Procedure for Preparation of Copper Complexes.
Steroidal ligand (0.1 mmol) and 0.1 mmol CuCl 2 ⋅2H 2 O were added to 8 mL of methanol. The mixture was stirred for 5 hour at 70 ∘ C. The reaction was terminated when large precipitant emerged. The resulting suspension was filtered, washed with ethyl acetate and water, and dried in a desiccator over phosphorus pentoxide to give target products.

Cytotoxicity Assay.
The antiproliferative activity of all Cu(II) metal complexes and steroidal thiosemicarbazones on Bel-7404 (human liver carcinoma), HeLa (human cervical carcinoma), and HEK-293T (normal kidney epithelial) cell lines was determined by using the MTT method and cisplatin as a positive control. The detailed procedure had been reported in our previous work [22].   Table 1.

Results and Discussion
From the data shown in Table 1, all steroidal copper (Cu (II)) complexes show an obvious antiproliferative activity  Comparing compound 7 with compound 8, we can observe that after 3-hydroxyl group of 7 was converted into 3acetoxy group (compound 8), the antiproliferative activity of the compound was remarkably decreased and the cytotoxicity to normal cells 293T was increased. The result shows that 3-hydroxyl of the compound to the antiproliferative activity plays an important role.
Unfortunately, these steroidal copper (Cu (II)) complexes to normal kidney epithelial cells (293T) show similar cytotoxicity except for compound 5 which exhibits a smaller activity to 293T cells compared to cisplatin (27 M versus 10.3 M).

Conclusion
In conclusion, using estrone and pregnenolone as starting materials, through different chemical methods, some steroidal copper (II) complexes were synthesized and characterized by IR, NMR, and HRMS. Their antiproliferative activities were assayed by MTT method. The results show that all steroidal copper (II) complexes display obvious antiproliferative activity against the tested cancer cells, and compounds

Conflicts of Interest
The authors declare that they have no conflicts of interest.