Arylisothiocyanato selective androgen receptor modulators (SARMs) for prostate cancer

https://doi.org/10.1016/j.bmc.2006.06.019Get rights and content

Abstract

A new series of androgen receptor targeted agents (ARTA) was prepared and tested in androgen-dependent and -independent prostate cancer cell lines. These agents were bicalutamide analogs with isothiocyanato substituted B-rings. Also, the linker sulfone of R-bicalutamide was maintained or replaced with several alternative linkages including ether, amine, N-methylamine, thioether, and methylene (in this case the product was a racemic mixture) functional groups at the X-position. To expand the structure–activity relationship (SAR) of these arylisothiocyanato AR ligands, B-ring halogenated arylisothiocyanato ligands were also prepared and tested. The arylisothiocyanato AR ligands showed strong binding affinities to AR ranging from 0.6 to 54 nM. Among them, thioether and ether linkages demonstrated high binding affinities (0.6 and 4.6 nM, respectively) and selective cell growth inhibition (approximately 3- to 6-fold) for LNCaP, an androgen-dependent prostate cancer cell line, when compared to the androgen independent prostate cell lines (DU145, PC-3, and PPC-1) and a bladder cell line (TSU-Pr1). However, the ligands were inactive (IC50>100 mM) in a normal monkey kidney cell line (CV-1) that was used as the control for non-specific toxicity.

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/R23, 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.

References and notes (30)

  • J.D. McConnell

    Urol. Clin. North Am.

    (1991)
  • A. Mukherjee et al.

    Biochem. Pharmacol.

    (1999)
  • J. Chen et al.

    Endocrinology

    (2005)
  • G. Leclerc et al.

    Eur. J. Med. Chem.

    (1983)
  • D.J. Hwang et al.

    Bioorg. Med. Chem.

    (2001)
  • F.E. Romesberg et al.

    J. Am. Chem. Soc.

    (1998)
    G. Bartoli et al.

    J. Org. Chem.

    (1986)
  • H. Tucker et al.

    J. Med. Chem.

    (1988)
    D.A. Evans et al.

    J. Org. Chem.

    (1974)
    T.R. Hoye et al.

    J. Org. Chem.

    (1979)
  • C.A. Marhefka et al.

    J. Med. Chem.

    (2001)
  • N. Haga et al.

    J. Am. Chem. Soc.

    (1992)
    N. Hodgson

    J. Chem. Soc.

    (1941)
  • R.T. Greenlee et al.

    CA-Cancer J. Clin.

    (2001)
  • O.O. Clement et al.

    J. Med. Chem.

    (2003)
  • B.S. Carter et al.

    Prostate

    (1990)
  • C.E. Bohl et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (2005)
    V.K. Reddy et al.

    J. Med. Chem.

    (2001)
    A. Valasinas et al.

    J. Med. Chem.

    (2001)
    B. Frydman et al.

    J. Med. Chem.

    (2004)
    H.T. Le et al.

    J. Biol. Chem.

    (2003)
    H. Ohtsu et al.

    J. Med. Chem.

    (2002)
    P.M. Matias et al.

    J. Med. Chem.

    (2002)
    S.F. Brandy et al.

    J. Med. Chem.

    (2002)
  • W. Kassouf et al.

    J. Urol.

    (2003)
    M. Dukes et al.

    Steroids

    (2000)
  • F.H. Schroder et al.

    Eur. Urol.

    (2004)
  • Cited by (31)

    • Targeting the N-terminal domain of the androgen receptor: The effective approach in therapy of CRPC

      2023, European Journal of Medicinal Chemistry
      Citation Excerpt :

      Duane D. Miller et al. have done much work on selective AR degraders (SARDs) compounds [82–85]. Their first generation of SARDs were metabolically labile secondary and tertiary amines (I) designed by structural modification of the traditional AR antagonist compound 6 [86] and the tissue-selective AR agonist compound 7 (Fig. 4A) [87]. Class I was exemplified by compound 8 (3, (S)–N-(4-cyano-3-(trifluoromethyl) phenyl)-3-((6- cyano-[1,1′-biphenyl]-3-yl) (methyl)amino)-2-hydroxy-2- methylpropanamide) (Fig. 4A) [83].

    • Design and synthesis of novel bicalutamide and enzalutamide derivatives as antiproliferative agents for the treatment of prostate cancer

      2016, European Journal of Medicinal Chemistry
      Citation Excerpt :

      The antiproliferative results are reported in Tables 1 and 2 (absolute IC50 in μM). The overall activity of the four reference compounds (3, 5, 45a, 44e) is consistent with previous reported data for some of these specific cell lines and it represents a confirmation of the reliability of the test [20–22]. 70% of our new derivatives performed better than bicalutamide, either as a racemic mixture or pure R-isomer (3, 45a), significantly improving its antiproliferative activity up to 50-fold (overall antiproliferative activity in the four cell lines reported as geometric mean).

    • Preliminary investigations into triazole derived androgen receptor antagonists

      2014, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      A common structural feature of non-steroidal AR antagonists is the extremely electron-deficient N-phenyl amide moiety. The aryl portion commonly bears a trifluoromethyl group (CF3) in addition to a strongly deactivating group at the para-position, relative to the amide.3–8 Typical examples of para-substitution are the nitro group (NO2) in the case of 1 and 2, or the nitrile (CN) of 3 and MDV3100.

    View all citing articles on Scopus
    View full text