Synthesis and biological evaluation of nandrolone–bodipy conjugates Michal Jurášek a,1 , Silvie Rimpelová b,1 , Vladimíra Pavlícˇková b , Tomáš Ruml b , Oldrˇich Lapcˇík a , Pavel B. Drašar a,⇑ a Department of Chemistry of Natural Compounds, Institute of Chemical Technology in Prague, 166 28 Prague, Czech Republic b Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague, 166 28 Prague, Czech Republic article info Article history: Received 15 June 2014 Received in revised form 19 September 2014 Accepted 1 October 2014 Available online xxxx Keywords: Androgens Nandrolone Fluorescent conjugates Cytotoxicity abstract Here, we report synthesis and biological evaluation of fluorescent nandrolone-3-carboxymethyloxime derivatives conjugated with green-emitting bodipy dye via PEG linkers. All the newly-synthesized com- pounds were evaluated for their effect on cell proliferation in vitro in MCF-7, LNCaP, PC-3 and HEK 293T model cell lines using WST-1 assay. By means of live-cell fluorescence microscopy, the intracellular localization of nandrolone–bodipy conjugates was revealed in endoplasmic reticulum. Moreover, we performed competitive localization study with nonfluorescent nandrolone, metandrolone, boldenone, trenbolone, and testosterone. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Norsteroids are xenobiotics possessing both androgenic and anabolic properties. Originally these substances were designed for the treatment of hematological and post-surgical conditions and substitutive supplementation [1]. Anabolic androgenic steroids (AAS) are widely used in medicine for treatment of various condi- tions, such as male hypogonadism, chronic wasting conditions, can- cer, burns, renal and hepatic failure, anemia, cachexia, and AIDS [2]. Nevertheless, there has been still increasing illicit misusage of these steroids by athletes and bodybuilders. Nandrolone (also known as 19-nortestosterone or 17b-hydroxy-19-nor-4-androsten-3-one) is one of the most abused androgenic anabolic steroids (AAS), especially its commercially available form, 17b-decanoate [3]. This performance-enhancing drug is banned in sports by International Olympic Committee [4]. In addition to the androgen receptor, the effects of nandrolone are associated with progesterone receptor and several other signaling pathways (see Fig. 1). Nandrolone naturally occurs in a tiny amount in the human body since it is one of metabolites of testosterone aromatization [5–8]. Generally, androgens and estrogens have opposing effects on the growth and development of malignant human breast tissues. Androgens, such as testosterone, dihydrotestosterone, and andro- stenedione, exert an inhibitory effect and estrogens, such as estra- diol and its derivatives, have mitogenic effect. Androgen receptors are present in 50–90% of breast cancers and their overexpression has been associated with better response to hormone therapy and longer survival of the patients [9]. Recently, Chimento et al. [10] reported that AAS are involved in progression of testicular cancer. Previous study dealing with the function of androgens in growth of MCF-7 cell line (a model of invasive breast ductal carcinoma) showed that androgens can inhibit cell proliferation in vitro [9]. Nandrolone as well as many other important steroids naturally bear oxo-group in C-3 position of the structure which might serve as convenient point for chemical modifications. In this work, we present syntheses of a series of green-emitting bodipy-labeled nandrolone-3-carboxymethyl-oxime (CMO) derivatives containing PEGylated linkers of different length. Bodipy is a small organic, lipophilic and membrane permeable dye often used for fluorescent lipid labeling [11,12]. Different spacing between nandrolone and bodipy was used in order to minimize the interference of the dye and the steroidal part of the molecule. We assessed the impact on cell proliferation of the newly synthesized derivatives in MCF-7, LNCaP, PC-3 and HEK 293T cells. Further, intracellular trafficking of the derivatives was performed using live-cell fluores- cence microscopy. 2. Results and discussion To our knowledge, information about the intracellular traffick- ing and localization of nandrolone is very scarce. Therefore, the aim of our study was to develop a functional fluorescent nandro- http://dx.doi.org/10.1016/j.steroids.2014.10.002 0039-128X/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Tel.: +420 220 443 698. E-mail address: Pavel.Drasar@vscht.cz (P.B. Drašar). 1 These authors contributed equally to this work. Steroids xxx (2014) xxx–xxx Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids Please cite this article in press as: Jurášek M et al. Synthesis and biological evaluation of nandrolone–bodipy conjugates. Steroids (2014), http://dx.doi.org/ 10.1016/j.steroids.2014.10.002