2328 A bifunctional colchicinoid that binds to the androgen receptor Nima Sharifi,1,3 Ernest Hamel,2 Markus A. Lill,4 activity of this compound increases cytoplasmic AR levels Prabhakar Risbood,5 Charles T. Kane, Jr.,6 in prostate cancer cells. Finally, we found that this com- Md Tafazzal Hossain,6 Amanda Jones,7 pound has greater toxicity against androgen-independent James T. Dalton,7 and William L. Farrar1 prostate cancer cells than the combination of colchicine and nilutamide. Together, these data point to several 1Cancer Stem Cell Section, Laboratory of Cancer Prevention, ways of inhibiting AR function in CRPC. [Mol Cancer Ther National Cancer Institute at Frederick, Center for Cancer 2007;6(8):2328–36] Research, and 2Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, Introduction National Cancer Institute, Frederick, Maryland; 3Medical Oncology Branch, Center for Cancer Research, National Cancer Prostate cancer is the leading cause of nonskin malignancy Institute, Bethesda, Maryland; 4Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, in men (1). Growth and survival of prostate cancer is West Lafayette, Indiana; 5Drug Synthesis and Chemistry Branch, dependent on androgens and androgen receptor (AR) Division of Cancer Treatment and Diagnosis, National Cancer signaling. Therefore, advanced disease is generally first Institute, Rockville, Maryland; 6Starks Associates, Inc., Buffalo, treated with androgen deprivation therapy by medical or 7 New York; and Division of Pharmaceutics, College of Pharmacy, surgical castration (2). Metastatic disease almost always Ohio State University, Columbus, Ohio overcomes androgen deprivation and progresses as cas- trate-resistant prostate cancer (CRPC). A wealth of evi- Abstract dence suggests that CRPC is still reliant on AR for tumor Castrate-resistant prostate cancer (CRPC) continues to be cell survival and disease progression (3). In the castrate- dependent on the androgen receptor (AR) for disease resistant setting, AR is reactivated by a variety of progression. We have synthesized and evaluated a novel mechanisms that include but are not limited to AR gene compound that is a conjugate of colchicine and an AR amplification and other mechanisms of increasing AR antagonist (cyanonilutamide) designed to inhibit AR expression and ligand-independent activation by growth function in CRPC. A problem in multifunctional AR-binding factors and cytokines (4). Furthermore, a subset of compounds is steric hindrance of binding to the embedded androgen-responsive genes are reactivated in CRPC (5), hydrophobic AR ligand-binding pocket. Despite the bulky and prostate-specific antigen declines will often occur with side chain projecting off of the AR-binding moiety, this secondary hormonal therapies that also target AR (6). novel conjugate of colchicine and cyanonilutamide binds Together, this evidence suggests that AR is still a valid target in CRPC, and compounds that have novel AR- to AR with a Ki of 449 nmol/L. Structural modeling of this compound in the AR ligand-binding domain using a targeting mechanisms should provide new avenues for combination of rational docking, molecular dynamics, and prostate cancer therapy (7). steered molecular dynamics simulations reveals a basis for The rationale for the design and synthesis of a compound how this compound, which has a rigid alkyne linker, is able that has independent tubulin-binding and AR-binding to bind to AR. Surprisingly, we found that this compound moieties is 4-fold. First, many tubulin-binding drugs are also binds to tubulin and inhibits tubulin function to a used for cancer chemotherapy. In fact, the only form of greater degree than colchicine itself. The tubulin-inhibiting chemotherapy shown to prolong survival for metastatic prostate cancer patients is a tubulin-binding drug (8, 9). Addition of an AR-binding moiety to a therapeutic agent could selectively target AR-expressing prostate cancer cells, with minimal impact on cells that do not express AR. Received 3/7/07; revised 5/14/07; accepted 6/15/07. Second, the nuclear import of steroid hormone receptors is Grant support: Intramural Research Program of the NIH and the Center for Cancer Research, National Cancer Institute, and in part with Federal funds a microtubule-dependent process (10). The use of colchi- from the National Cancer Institute, NIH, under contract N02-CM-52209. cine, which only binds to the soluble tubulin heterodimer The costs of publication of this article were defrayed in part by the and disrupts tubulin polymerization, may thereby inhibit payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to the nuclear import of AR. Third, independent AR-binding indicate this fact. and tubulin-binding moieties in a single compound would Requests for reprints: Nima Sharifi or William L. Farrar, Room 21-81, potentially result in concomitant AR and tubulin binding, Cancer Stem Cell Section, Laboratory of Cancer Prevention, National thereby anchoring AR to tubulin. Thus, the bound AR Cancer Institute at Frederick, Building 560, Frederick, MD 21702. Phone: 301-451-4982; Fax: 301-846-7042. E-mail: [email protected] would remain in the cytoplasm. However, a difficulty is or [email protected] preservation of binding to the hydrophobic AR ligand- Copyright C 2007 American Association for Cancer Research. binding domain, in which androgen ligands are completely doi:10.1158/1535-7163.MCT-07-0163 buried (11). Adding a linker or a bulky moiety to an AR Mol Cancer Ther 2007;6(8). August 2007 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 2329 ligand could therefore easily lead to loss of AR-binding TLC was done on Merck 60 F254 silica gel glass plates. activity. Fourth, if AR ligand-binding activity can be Elemental analyses were done by Atlantic Microlabs. conserved with a compound that has a linker that extends Melting points are uncorrected. Unless stated, all solvents outside the ligand-binding domain, this could disrupt and reagents were purchased from commercial sources and binding to steroid receptor coactivators that are required used without further purification. for AR function (12). Benzonitrile, 4-[3-(4-Hydroxy-2-Butynyl)-4,4-Dimethyl- Colchicine, thiocolchicine, and combretastatin A-4 all 2,5-Dioxo-1-Imidazolidinyl]-2-(Trifluoromethyl)- (2). To bind to a common site on tubulin referred to as the 1 (ref. 15; 3.90 g, 13.1 mmol) in dimethylformamide colchicine site (13). Studies of the structure-activity rela- (DMF; 60.0 mL) was added cesium carbonate (3.85 g, 11.8 tionship of colchicine suggest that the C7 acetamido group mmol) and a solution of 4-chloro-2-butyn-1-ol (3.60 g, 34.4 can be modified while preserving tubulin-binding activity mmol) in DMF (5.0 mL). After 3 h at room temperature, (14). We therefore chose this site for one end of the linker. the mixture was filtered. The filtrate was diluted with Cogan and Koch (15) used a rigid alkyne linker with a EtOAc (500 mL), washed with water (1.2 L), then dried N cleavable salicylamide -Mannich base in doxorubicin- (MgSO4) and concentrated. The white solid was dried targeting for prostate cancer. Although we used the alkyne under reduced pressure to give 2 (2.85 g, 59%). 1HNMR N y J moiety, we excluded the salicylamide -Mannich base and (500 MHz, DMSO-d6): 8.32 (d, 1H, = 8.4 Hz); 8.20 (d, 1H, selected a noncleavable linker to allow for the possibility of J = 1.6 Hz); 8.06–8.04 (dd, 1H, J = 1.7, 8.4 Hz); 5.16–5.14 concomitant AR and tubulin binding. We constructed a (t, 1H, J = 5.9 Hz); 4.30 (s, 2H); 4.08–4.07 (m, 2H); 1.54 linker with sufficient length to extend the colchicine moiety (s, 6H). Mass spectroscopy: electrospray (positive ion): m/z outside the AR ligand-binding domain. Cyanonilutamide is calculated for C17H14F3N3O3 = 365; found: (relative + structurally similar to nilutamide and has a slightly higher intensity) 383 [(M + NH4) , 100%]. HPLC: retention time affinity for the AR than does nilutamide (15). 13.879 min with 100% peak area [Luna C18, 4.6  250 mm, A These considerations led us to synthesize acetamide, 2-[4- 5 . Mobile phase: A = 0.05 mol/L H3PO4 buffer (pH, 3); [3-[(4-cyano-3-trifluoro-methyl)phenyl]-5,5-dimethyl-2,4- B = acetonitrile + 1% water. Linear gradient = 95% A; 70% dioxoimidazol-idin-1-yl]-but-2-ynyloxy]-N-[(7S)-5,6,7,9-tet- A; 100% B; 95% A]. rahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen-7- Acetic Acid, 2-[4-[3-[(4-Cyano-3-Trifluoromethyl)- yl], and will hereafter refer to this compound as CCN, for Phenyl]-5,5-Dimethyl-2,4-Dioxoimidazolidin-1-yl]But-2- colchicine-cyanonilutamide. Here, we describe the proper- ynyloxy]-Methyl Ester (3). A mixture of 2 (1.80 g, 4.93 ties of CCN and the structural basis for how it binds AR. mmol) and thallium ethoxide (349 AL, 4.93 mmol) in CCN binds to tubulin and inhibits tubulin assembly with CH3CN (17.0 mL) was stirred for 1 h at ambient greater potency than colchicine and has activity that is temperature and then concentrated to dryness. The powder comparable to the more potent thiocolchicine (13). Despite was dissolved in DMF (17.0 mL), and methyl bromoacetate the hydrophobic binding pocket of the AR ligand-binding (3.8 g, 24.8 mmol) was added. The mixture was heated at domain and the relatively bulky nature of colchicine, CCN 60jC for 3 h and then cooled to room temperature, and still retains AR-binding activity. We determined the water (50 mL) was added. This was extracted with diethyl  structural basis of binding to the AR ligand-binding ether (4 100 mL), dried (MgSO4), and concentrated.