Arylisothiocyanato selective androgen receptor modulators (SARMs) for prostate cancer
Graphical abstract
Introduction
Prostate cancer (CaP) is a major health hazard for men living in United States1 and developed countries. Approximately 90% of prostate tumors are androgen-dependent.2 Androgens, primarily testosterone (T) and dihydrotestosterone (DHT), bind to the androgen receptor (AR) and support the development and maintenance of normal prostate tissue but more importantly can have a pathologic role in prostate cancer patients by promoting the growth of the tumor.3 Antiandrogen therapy to block these pathological effects of endogenous androgens has been the golden standard for unrespectable prostate cancer (disease spread beyond prostate). However, despite the high initial response rate to hormonal therapy, almost all patients relapse and become hormone refractory.4 Our effort is directed at finding potent non-steroidal AR inhibitors that are effective even in hormone refractory prostate cancer.5 Until now, the blockage of the pharmacological effects of androgens by non-steroidal AR ligands was achieved through the use of nilutamide, flutamide, and bicalutamide in advanced CaP patients.6, 7, 8
All current AR antagonists are reversible ligands that interact with the AR via non-covalent bonds such as hydrophobic, electrostatic, and hydrogen bond interactions. Unlike a reversible ligand, an irreversible ligand can bind to the receptor via a covalent bond that permanently attaches the ligand to the receptor. Our laboratory has a long interest in the discovery of novel anticancer drugs that act via the AR. Recently, we reported that an isothiocyanate substituent at the para-position of the B-ring of thioether and sulfonyl-linked derivatives of bicalutamide bound tightly and may form a covalent bond to the AR.9 In this study, we expand our previous efforts10, 11 regarding non-steroidal AR ligands to include bicalutamide analogs with the potential to bind irreversibly to the AR. In pursuit of this objective, chiral arylisothiocyanato derivatives were synthesized and tested in the prostate cancer cell lines LNCaP, DU145, PC-3, PPC-1, and a bladder cancer cell line (TSU-Pr1) as well as a normal monkey kidney cell line (CV-1). In this report, our previous ligands S-23 and R-35 (Table 1)10, 11 were tested on CaP cell lines as selective androgen receptor modulators (SARMs) and diversified at the X-position and halogenated on the B-ring. This work was carried out to find suitable ligands, which are selective AR-dependent CaP inhibitors with strong AR binding affinity and no cytotoxicity on normal cell lines, and to address the key issues in enhancing AR binding affinities of bicalutamide derivatives and their use in prostate cancer therapy. First, we focused on which linker was most suitable for AR potential prostate cancer therapy. With this objective, we prepared and tested several analogs with ether, methylene, thioether, sulfone, amino, and methylamino groups as outlined in general structure S/R-1 (Fig. 1). The synthetic methods for these linker variants are shown in Scheme 1, Scheme 2, Scheme 3, Scheme 4, Scheme 5. Our second goal was the determination of the pharmacological effects of halogenation at the 2- or 3-position of the 4-isothiocyanato substituted B-ring derivatives as outlined in general structure S-2 (Fig. 1). Kim et al.12 reported the para B-ring substituent is a major structural determinant of in vivo disposition and activity of non-steroidal AR agonists (a.k.a. selective androgen receptor modulators or SARMs). SARMs are a new set of non-steroidal AR ligands with potential therapeutic activity similar to testosterone. They may be used orally with androgenic and anabolic activity and have fewer side effects than testosterone. Chen et al.13 reported that halogenation at the 2- or 3-position of the B-ring increased their binding affinities to AR. To further probe the SAR of 2- or 3-halogenated SARMs, we tested the AR binding affinity and cancer cell growth inhibition of the 2- or 3-halogenated isothiocyanato ligands. Our last goal was the characterization of SAR A-ring para-position regarding AR affinity and cell growth inhibition. We compared the NO2 or CN group on the A-ring of our analogs. The in vitro prostate cancer directed cell growth inhibitory properties of the chiral and racemic AR ligands in Figure 1 were characterized. The AR binding affinity and the concentration of the ligand that inhibited cell growth by 50% (IC50) were determined by radioligand competitive binding assay and sulforhodamine B (SRB) assay, respectively. The AR binding affinities and cell growth inhibitory effect of these compounds (S and/or R)-23 to (S and/or R)-37 with diverse substituents on the aromatic A and B rings are reported.
Section snippets
Chemistry
A total of 17 arylisothiocyanato derivatives (S and/or R)-23 to (S and/or R)-37 (Table 1) with several linkages (X = O, CH2, NH, NCH3, S, and SO2) were prepared as discussed in Scheme 1, Scheme 2, Scheme 3, Scheme 4, out of which 14 novel SARMs were newly synthesized. The target arylisothiocyanato derivatives were mainly synthesized by two known methods (Leclerc14 and Marhefka10). Scheme 1 describes the synthesis of ether-linked analogs. The protected arylamines (S-9 to S-14) were converted to
Results and discussion
A novel series of arylisothiocyanato 1,3-disubstituted-2-hydroxy-2-methylpropionamide AR ligands were prepared (Table 1) with several variations of the linkers (O, CH2, NH, N–CH3, S, and SO2; S/R–23, S-24, 31, S-32, S-33, R-34, S/R-35, R-36, and R-37) and modifications in position and degree of halogenation (2-, or 3-positions with F/Cl substitution on B-ring; S-25 to S-30). Irreversibly binding ligands have been proven to be very useful pharmacological tools in a number of systems.23 The
Cell culture
Four prostate cancer cell lines (LNCaP, DU145, PC-3, and PPC-1), a bladder cancer cell line (TSU-Pr1), and a normal monkey kidney cell line (CV-1) were obtained from ATCC. Prostate cancer cells and bladder cancer cell were grown in RPMI-1640 medium and CV-1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM l-glutamine supplemented with 10% fetal bovine serum (FBS) and maintained in a 5% CO2/95% air humidified atmosphere at 37 °C, respectively.
Assay for cell growth inhibition (sulforhodamine B assay)
Viable cells were
Acknowledgments
This work was supported by grants from National Institutes of Health (NIH-5R01DK065227-02 and -03) and GTx-Inc.
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