Synthesis of 2- and 7- Substituted C19 Steroids Having a 1,4,6-Triene or 1,4-Diene Structure and Their Cytotoxic Effects on T47D and MDA-MB231 Breast Cancer Cells

2-Chloro-, 2-bromo- and 2-azido-1,4,6-androstatriene-3,17-diones were synthesized from 1α,2α-epoxy-4,6-androstadiene-3,17-dione (2) using HCl, HBr and NaN3, respectively. Compound 2 was also reacted with NaCN to give 2-cyano-1,4,6-androstatriene-3,17-dione (5) and 2β-cyano-1α-hydroxy-4,6-androstadiene-3,17-dione (6). 6α,7α-Epoxy-1,4-androstadiene-3,17-dione (8) was reacted with HCl, HBr and NaN3 to form the corresponding 7β-chloro-, 7β-bromo- and 7β-azido-6α-hydroxy-1,4-androstadiene-3,17-diones. The cytotoxic activity of these compounds towards T47D (estrogen-dependent) and MDA-MB231 (estrogen-independent) breast cancer cell lines was evaluated. The 6α-hydroxy-7β-substituted analogs were more active than the 2-substituted analogs on both cell lines. Compound 2 showed the highest selective activity against the T47D (IC507.1 μM) cell line and 5 showed good cytotoxic activity on MDA-MB231 (IC5018.5 μM) cell line, respectively. The 6α,7α-epoxy analog 8 also showed high cytotoxic activity on both cell lines (IC50 17.3 μM on T47D and IC50 26.9 μM on MDA-MB231).


Introduction
Breast cancer is the most commonly diagnosed cancer among women and continues to be a major cause of cancer related deaths. The medical treatments available for breast cancer include endocrine therapy, cytotoxic chemotherapy and adjuvant treatments. Estrogens are involved in numerous physiological processes, including the development and maintenance of the female sexual organs, the reproductive cycle, reproduction, and various neuroendocrine functions. On the other hand, estrogens enhance growth and proliferation of certain target cells, such as breast epithelial cells and estrogendependent mammary carcinoma cells. Tamoxifen is an estrogen receptor antagonist in breast tissue and has been the standard endocrine (antiestrogen) therapy for hormone-sensitive early breast cancer in post-menopausal women, however, prolonged treatment may cause endometrial cancer [2]. Estrone and estradiol are biosynthesized from 4-androstene-3,17-dione (4-AD) and testosterone by aromatase and consequently compounds (e.g., exemestane, formestane and anastrozole) that inhibit the aromatase enzyme find application in the treatment of advanced estrogen-dependent breast cancers [3,4]. Locally advanced or metastatic breast cancer is typically treated with chemotherapy [5]. Cyclophospamide, methotrexate and fluorouracil have all been used as chemotherapeutic drugs to treat breast cancer. Currently available cytotoxic drugs do not discriminate between cancer and normal cells undergoing rapid division, although some antiestrogen and aromatase inhibitors offer a greater breadth of endocrine therapy and lower toxicity than some other drugs.
We describe here the synthesis of some 2-substituted androstatriene-and 6α-hydroxy-7βsubstituted androstadiene-3,17-dione derivatives which were modified with unsaturation in the A and/or B rings. Chloro, bromo, cyano, and azido groups were selected as substituents at the C-2 and C-7 positions in the androstane skeleton. The in vitro cytotoxic activity of these substituted androstadiene and androstatriene derivatives against estrogen-dependent (T47D) and estrogen-independent (MDAMB231) breast cancer cells was studied.
When 8 was treated with sodium cyanide and ammonium chloride in ethanol/H 2 O (8:1) solution, the reaction did not proceed at room temperature, but when 8 was reacted under reflux conditions, 2αcyano-6α,7α-epoxy-4-androstene-3,17-dione (14) was obtained in 77% yield. The structure of 14 was determined by the presence of peaks at δ 6.29 ppm for only one double bond hydrogen (H-4) and at 3.17 (H-2) ppm and at

Cytotoxic effects
We assessed the cytotoxicity of breast cancer cells. The results are shown in  We assessed the cytotoxicity of synthesized compounds 1-5 and 7-14 on T47D are shown in Figures 3-6 and Table 1.

General
All non-aqueous reactions were performed under an atmosphere of dry nitrogen. The commercial reagents were purchased from Aldrich, Fluka, or Sigma. Solvents were purified and dried prior to use. Melting points were measured on Thomas-Hoover melting point apparatus and not corrected. 1 H-, 13 C-NMR, HSQC, HMQC and NOESY spectra were recorded on a Varian 400 MHz spectrometer in CDCl 3 and DMSO-d 6 . Chemical shifts (δ) are in parts per million (ppm) relative to tetramethylsilane, and coupling constants (J) are in Hertz. IR spectra were determined on a Jasco FT-IR 300E spectrometer as KBr pellets, unless indicated otherwise. GC/MS spectra were obtained on a Shimadzu QP 5050 and JEOL GC Mate 2 mass spectrometers. MPLC was run on a Yamazen YFLC-AI instrument. Analytical TLC was performed on pre-coated silica gel 60 F 254 plates (Merck). Solvent systems for TLC were ethyl acetate/n-hexane mixtures and 10% methanol in dichloromethane. Column chromatography was carried out on Merck silica gel 9385 (230-400 mesh), eluting with ethyl acetate/n-hexane mixtures.