NovelTriﬂuoromethylatedEnobosarmAnalogues with Potent ... · Enobosarm (also referred to as Ostarine, GTx-024 or S-22) is a nonsteroidal SARM with a favorable safety and pharmacoki-netic

Small Molecule Therapeutics

Novel Trifluoromethylated EnobosarmAnalogueswith Potent Antiandrogenic Activity In Vitro andTissue Selectivity In VivoD. Alwyn Dart1,2, Sahar Kandil3, Serena Tommasini-Ghelfi2,Gilberto Serrano de Almeida2, Charlotte L. Bevan2,Wenguo Jiang1,and Andrew D.Westwell3

Abstract

Prostate cancer often develops antiandrogen resistance,possibly via androgen receptor (AR) mutations, whichchange antagonists to agonists. Novel therapies withincreased anticancer activity, while overcoming current drugresistance are urgently needed. Enobosarm has anaboliceffects on muscle and bone while having no effect on theprostate. Here, we describe the activity of novel chemicallymodified enobosarm analogues. The rational addition ofbistrifluoromethyl groups into ring B of enobosarm, pro-foundly modified their activity, pharmacokinetic and tissuedistribution profiles. These chemical structural modifica-tions resulted in an improved AR binding affinity—byincreasing the molecular occupational volume near helix12 of AR. In vitro, the analogues SK33 and SK51 showed verypotent antiandrogenic activity, monitored using LNCaP/ARLuciferase cells where growth, PSA and luciferase activity

were used as AR activity measurements. These compoundswere 10-fold more potent than bicalutamide and 100-foldmore potent than enobosarm within the LNCaP model.These compounds were also active in LNCaP/BicR cells withacquired bicalutamide resistance. In vivo, using the ARLucreporter mice, these drugs showed potent AR inhibitoryactivity in the prostate and other ARexpressing tissues,e.g., testes, seminal vesicles, and brain. These compoundsdo not inhibit AR activity in the skeletal muscle, and spleen,thus indicating a selective tissue inhibitory profile. Thesecompounds were also active in vivo in the Pb-Pten deletionmodel. SK33 and SK51 have significantly different andenhanced activity profiles compared with enobosarm andare ideal candidates for further development for prostatecancer therapy with potentially fewer side effects. Mol CancerTher; 17(9); 1846–58. �2018 AACR.

IntroductionProstate cancer is the most commonly diagnosed male cancer

in the Western world (1). Tumor growth is initially androgendependent—driven by the androgen receptor (AR). The ARsignaling pathway remains a key driver of prostate cancer,implicated throughout the different stages of the disease, andas such represents an obvious therapeutic target in prostatecancer therapy (2, 3). AR is a member of the family of steroidhormone receptors, which are ligand-dependent transcriptionalregulators for diverse sets of genes involved in both reproduc-tive and anabolic actions (4–6). The prototypical model for ARinvolves ligand binding that induces conformational changesin the ligand-binding domain (LBD), revealing a coactivator

dimerization surface composed of helices 3, 5, and 12, as theLBD site is adjacent to helix 12 (H12).

Currently, the mainstays of prostate cancer treatment areandrogen ablation (to castrate levels) and/or antiandrogentreatment, which block AR signaling (2, 3). The major anti-androgens in clinical use are bicalutamide and enzalutamide(and historically flutamide, hydroxyflutamide, and niluta-mide; Fig. 1A). However, these compounds bind to AR withlow affinity and can induce escape mechanisms. Furthermore,under AR amplification or mutation conditions some of thesecompounds exhibit agonist activity and fail to inhibit AR. Thisresistance may arise from, among other mechanisms, muta-tions to several key residues in the LBD (AR-LBD; refs. 7, 8).Common mutations in AR found in prostate cancer patientsinclude T877A, W741L, and W741C. Bicalutamide (Fig. 1A)can act as an agonist in ARW741L and ARW741C while hydro-xyflutamide is an agonist in ART877A (7, 8). Gain-of-functionmutations are common in prostate cancers and are correlatedwith disease progression, the lack of therapeutic responseand poor clinical outcomes. Additionally, AR mutationsmay be selected for during antiandrogen therapy—reviewedin ref. 9 and 10.

Additionally, the use of antiandrogens in patients may elicitmoderate side effects, e.g., muscle wastage, bone density loss, andCNS dysfunction—which are exacerbated in elderly men. There-fore, there is a growing need for selective androgen receptormodulators (SARM) that would demonstrate tissue-selective

1Cardiff China Medical Research Collaborative, Cardiff University School ofMedicine, Cardiff, Wales, United Kingdom. 2Androgen Signaling Laboratory,Department of Surgery and Cancer, Imperial College London, HammersmithHospital Campus, London, United Kingdom. 3School of Pharmacy and Pharma-ceutical Sciences, Cardiff University, Cardiff, Wales, United Kingdom.

D.A. Dart and S. Kandil contributed equally to this article.

Corresponding Author: D. Alwyn Dart, CCMRC, Cardiff University, The HenryWellcome Building, Heath Park Way, Cardiff CF14 4XN, UK. Phone:02920687069; Fax: 02920687069; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-18-0037

�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 17(9) September 20181846

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activity—being inhibitors of the AR in the prostate with tissue-sparing effects elsewhere (11).

Enobosarm (also referred to as Ostarine, GTx-024 or S-22) isa nonsteroidal SARM with a favorable safety and pharmacoki-netic profile (Fig. 1B). It belongs to the class of arylpropiona-mides, which are structurally derived from the antiandrogenbicalutamide. Enobosarm binds to and activates the AR withenantioselective affinity, potency, and efficacy similar to, butsomewhat less than, that of DHT (12). Transient transfectionstudies indicate that enobosarm is remarkably selective for theAR, and it does not cross-react with other nuclear hormonereceptors. This receptor specificity differentiates enobosarm'spharmacology from other steroidal androgens and has likelycontributed to observations that the drug has generally beenwell tolerated in clinical trials. It has selective anabolic effectsand is under clinical development for prevention and treatmentof muscle wasting (cachexia) in cancer patients (13, 14).

ResultsRational design of novel enobosarm analogues

Nonsteroidal antiandrogens such as bicalutamide act asAR antagonists (Fig. 1A), thus preventing androgen-dependentcell growth. In contrast, AR agonists, e.g., enobosarm (Fig. 1B)may act as agonists in all the AR LBD mutants, because theywill fit sterically better with the bulk-reducing AR mutationsthan in ARwt (15). The difference in activity between agonistssuch as enobosarm and antagonists such as bicalutamide inARwt may arise from the linker oxygen's smaller size relativeto the sulfonyl group of bicalutamide (Fig. 1A and B). Thisbulk in AR antagonist structure, though small, is thoughtto push the AR helix 12 away from the binding pocket,disturbing the agonist conformation of the androgen receptor(16). The current theoretical key to antagonism states thatthe differences observed among AR modulators (agonist orantagonist) are not because of their B-ring size or shape butbecause of their linker size and orientation (17). The improvedunderstanding is being applied to rationally designed treat-ment options with novel mechanisms of action. It is proposedthat AR antagonists should have appending molecular exten-sions to the core structure of AR agonists that interfere with theplacement of H12, thereby disrupting coactivator recruitment(16, 18, 19).

In the enobosarm/ARwt cocrystal structure (PDB code; 3RLJ;ref. 17; Fig. 2A and B), the 4-cyanophenyl ether group ofenobosarm is situated between residues of H12 and the indolylside chain of Trp741, suggesting that in the wild-type receptor(and probably the W741L mutant) the larger substitution withthe same orientation of the linker would result in a better ARantagonist by pushing against Helix 12. We tested this hypo-thesis in silico by investigating whether the introduction of3,5-bis-trifluoromethyl (3,5-bis-CF3) into B ring of enobosarmwould provide the geometric bulk needed to keep ring B towardHelix 12 of AR. Preliminary molecular modeling studiesshowed that the bis-trifluoromethyl phenyl moiety (ring B inSK33 and SK51) has significantly changed the orientation ofring B so that the (3,5-bis-CF3) groups are pointing outwardaway from Trp741 and toward helix 12, thus preventing AR fromadapting the AR agonist conformation, while keeping the smallsize of the oxygen linker. The combination of the small oxygenlinker and the steric hindrance of the (3,5-bis-CF3) substitution

on ring B resulted in the optimum size and orientation of ring Bto impose conformational restriction away from Trp741 andtoward helix 12 (Fig. 2C and D). A small library of AR antago-nists having the (3,5-bis-CF3) motif was prepared (20), amongwhich SK33 showed the best antiproliferative and metabolicprofiles, hence it was selected for further studies.

The introduction of fluorinated substituents into drug candi-dates can impart a unique set of properties owing to the uniquecombination of electronegativity, size, and lipophilicity featuresof fluorinated groups (21–25). These factors can have a substan-tial impact on the molecular conformation, which in turn affectthe binding affinity to the target protein (26, 27).

Chemical synthesis of SK33 and SK51Racemic enobosarm derivatives (SK33 and SK51) were pre-

pared by reacting methacryloyl chloride (2) with the corre-sponding substituted anilines 1a and 1b in dimethylacetamide(DMA) solvent to obtain phenylacrylamides 3a and 3b, whichwere converted into the corresponding epoxides 4a and 4b inthe presence of a large excess of hydrogen peroxide andtrifluoroacetic anhydride in dichloromethane (DCM). Open-ing of the epoxide rings of 4a and 4b with substituted 3,5-bis(trifluoromethyl)phenol 5 in tetrahydrofuran (THF) gavethe final compounds SK33 and SK51. The structures of thesynthesized compounds were confirmed using analytical andspectroscopic data (1H NMR, 13C NMR, 19F NMR, and massspectrometry), which were in full accordance with theirdepicted structures. SK33 and SK51 were purified (>95% asconfirmed by high-performance liquid chromatography—HPLC) and tested as racemic mixture (Fig. 1C).

Cellular activity of enobosarm and the trifluoromethylatedanalogues SK33 and SK51

The antiproliferative and selective activities of these com-pounds were tested in vitro in a panel of prostate cancer cell lines,namely, LNCaP, VCaP, PC3, and Du145. LNCaP and VCaPrepresent prostate cells that have retained the expression of theAR (28, 29). However, LNCaP cells overexpress amutant AR, witha T877A mutation in the ligand-binding site. The VCaP cell lineoverexpresses wild-type AR. Both cell lines are responsive toandrogens and cells will express androgen responsive genes. ThePC3 and Du145 cells do not express the AR nor do they expressandrogen responsive genes.

Cells were exposed to increasing concentrations of antian-drogens for 96 hours and cell viability was analyzed usingthe MTT assay. In LNCaPARþ cells, the parental compoundenobosarm, showed insignificant activity with the IC50 ¼40 mmol/L; however, the trifluoromethylated enobosarm ana-logue, SK33, demonstrated a significantly potent activity withIC50 ¼ 0.2 mmol/L (Fig. 3A). Moreover, when comparedwith the antiandrogen bicalutamide, the novel compoundsshowed an increased efficacy (Fig. 3B). SK51 also displayedan increased activity compared with bicalutamide (IC50 ¼0.7 mmol/L; Fig. 3B).

In PC3AR-ve cells, enobosarm showed activity only at rela-tively high dose (Fig. 3C), while SK33 showed some moderateactivity. When compared with bicalutamide, SK33 showed asignificantly increased activity. On the other hand, the anti-proliferative activity of SK33 in LNCaPARþ cells was over10 times higher than that of its activity against PC3AR-ve cellline (Fig. 3D).

Potent Antiandrogenic Enobosarm Analogues for Prostate Cancer

www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 1847




A

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Figure 1.

A, Chemical structure of the clinically used nonsteroidal antiandrogens; flutamide, hydroxylflutamide, nilutamide, bicalutamide, and enzalutamide showingrings A and B. B, Chemical structure of enobosarm (left), SK33, and SK51 (right). C, Chemical synthesis of SK33 and SK51 compounds, reagents andconditions: (i) DMA, rt, 24 hours, (ii) H2O2, DCM, rt, 2 hours, (iii) NaH, THF, rt, 24 hours.

Dart et al.

Mol Cancer Ther; 17(9) September 2018 Molecular Cancer Therapeutics1848




Furthermore, the panel of prostate cancer cell lines wasscreened for its expression of the target AR by qPCR. Asexpected, LNCaPARþ and VCaPARþ cells showed robust expres-sion of AR while AR was not detectable in PC3AR-ve andDu145AR-ve cells (Fig. 3E).

Figure 3F depicts the summary of the activity profile of theSK33 and SK51 compounds in the four prostate cancer cell lineswhere a clear activity difference (selectivity) can be observedbetween the ARþ cell lines (LNCaP and VCaP) and the AR� celllines (PC3 and Du145).

SK33 decreases cells entering S-phase in LNCaP cells but notin PC3 cells

To further analyze the activity of these compounds in pros-tate cancer cell lines, we treated LNCaP (ARþ) or PC3 (AR�)cells with 10 mmol/L of antiandrogen (or vehicle DMSO) for 48hours. These cell lines were collected for FACS analysis, andstandard cell-cycle profiling using propidium iodide stainingfor DNA content was carried out.

LNCaP cells showed a decrease in the number of cells in Sphase with all the antiandrogen compounds. SK33 showed thehighest reduction of the number of cells in S phase, followedby SK51, enzalutamide, and then bicalutamide, respectively(Fig. 3G). No apparent cell toxicity could be visualized asestimated by the absence of a sub-G1 population or fragmentednecrotic cells (Fig. 3G). In the PC3 cell line, no significant effectwas observed with any of the tested compounds (Fig. 3H).However, a small increase in G2–M phase could be detectedwith SK33 in PC3 cells.

SK33 reduces AR transcriptional activityWe then analyzed the ability of SK33 to inhibit the activity

of the AR protein, i.e., the ligand activated transcription factoractivity. The LNCaP/Luc cell line has an integrated luciferase

reporter gene under the specific control of the AR transcriptionfactor, as shown in the schematic in Fig. 4A (30). Growingthese LNCaP/Luc cells in charcoal-stripped serum for 72 hoursremoves exogenous androgens and reduced the expressionof AR-responsive genes down to a baseline level, as monitoredby qPCR. Adding a synthetic androgen (R1881) back intothe media (0–10 nmol/L) increased both PSA and luciferasegene expression in a dose-dependent manner (Fig. 4B).Androgen treatment does not affect G418 gene expression(coexpressed from the same integrated plasmid as the lucif-erase reporter) nor did it affect the expression of housekeepinggenes L19, b-actin, and GAPDH. The ability of the antiandro-gens to block this androgen-dependent gene upregulation wasthen further tested. Treating hormonally starved LNCaP cellswith R1881 showed a 9-fold upregulation of the endogenousgene PSA. When treated with increasing doses of either bica-lutamide or SK33 a dose-dependent inhibition of gene expres-sion was seen. SK33 showed a greater PSA inhibition thanbicalutamide at 1 and 10 mmol/L (Fig. 4C). Conversely, theparental compound, enobosarm, showed a mild stimulatoryactivity (Fig. 4D).

We then examined the real-time activity of AR in liveLNCaP/Luc cells in response to increasing doses of SK33compared with the parental compound enobosarm and theantiandrogen bicalutamide. Live imaging and photon fluxmeasurements of LNCaP/Luc cells grown on 96-well plates inthe presence of 0 to 100 mmol/L compound for 24 hoursresulted in a strong inhibition of AR-mediated luciferaseactivity (Fig. 4E). The AR inhibitory concentration (50%) forboth SK33 and bicalutamide were approximately 1 mmol/L.Bicalutamide showed no further inhibitory effects past thisconcentration. However, SK33 maintained a dose-dependentinhibition, completely inhibiting the AR-mediated luciferaseactivity at 50 to 100 mmol/L concentration (Fig. 4F).

Figure 2.

A and B, X-ray structure of enobosarm(red-carbons) cocrystallized with(AR WT) in the agonist conformationshowing the orientation the distalring B extending away from Helix 12(H12) toward Trp741.C andD,Overlaidpredicted binding modes of SK33-R(pink carbons), SK33-S (turquoisecarbons), and enobosarm (graycarbons) inside the AR-LBD showingthe orientation of ring B with the3,5 CF3 group inducing geometricbulk keeping Helix 12 in the antagonistconformation (open) even upon thedevelopment of adaptive (resistant)bulk reducing mutations.






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Figure 3.

Enobosarm analogues SK33 and SK51 show potent and selective activity in AR-positive prostate cancer cells. MTT cytotoxicity assays of LNCaP cells treatedwith increasing doses of enobosarm or SK33 (A) or bicalutamide, SK33 and SK51 (B) at 0–100 mmol/L. MTT cytotoxicity assays of PC3 cells treatedwith increasing doses of enobosarm or SK33 (C) or bicalutamide, SK33 and SK51 (D) at 0–100 mmol/L for 96 hours. E, qPCR analysis of a panel of prostatecancer cells for AR expression. Data represent the mean of three independent experiments. Data were normalized to GAPDH, b-actin, and RPL19housekeeping genes. F, Bar chart representing a summary of the IC50 values for the 4 cell lines listed according to their AR status. G, FACS analysis ofLNCaP cells treated with 10 mmol/L bicalutamide, enzalutamide, SK33, SK51, or vehicle control for 48 hours, and analyzed for S phase (left-hand side).Histograms showing DNA content (PI fluorescence) of treated LNCaP cells (right-hand side). H, FACS analysis of PC3 cells treated with 10 mmol/Lbicalutamide, enzalutamide, SK33, SK51, or vehicle control for 48 hours, and analyzed for S phase (left-hand side). Histograms showing DNA content(PI fluorescence) of treated PC3 cells (right-hand side). Gated cells (10,000) were measured for each experiment.

Dart et al.





SK33 and SK51 are active in LNCaP cells with acquiredbicalutamide resistance

LNCaP cells were grown in increasing concentrations of bica-lutamide for 3 to 6 months, until proliferation was seen. Thisbicalutamide resistant cell line (LNCaP/BicR) then grew well in20 mmol/L bicalutamide, with cell-cycle kinetics similar to that ofthe parental cell line. An increased expression level of ARwas seenin this cell line uponqPCR analysis (Fig. 5A). Treating this cell linewith increasing doses of bicalutamide showed a proliferativeresponse—at 20 to 25 mmol/L—whereas the parental cell lineshowed sensitivity and an IC50 of 3 mmol/L (Fig. 5B). The LNCaP/

BicR cell line also showed a partial cross-resistance to enzaluta-mide (IC50¼5mmol/L approximately; Fig. 5C).We then analyzedif the mechanism of the acquired resistance to bicalutamideextended to the novel agents SK33 and SK51. Treatment ofLNCaP/BicR cells with increasing concentrations of SK33 andSK51 for 96 hours resulted in a dose-dependent response and aninhibition of cell growth, rather than being proliferative agents asbicalutamide (Fig. 5D), with IC50 of approximately 2 mmol/L forSK51 and 5 mmol/L for SK33. Although not as active as in theparental sensitive cell line, SK51 did show an improved activityover enzalutamide (and bicalutamide).

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Figure 4.

SK33 and SK51 inhibit AR-mediated transcriptional activity in prostate cancer cells. A, Schematic diagram of the activation of the synthetic androgen reporterconstruct integrated into the LNCaP/ARE-Luc cell line. Ligand bound AR activates the expression of the luciferase reporter gene which in the presenceof luciferin substrate gives off bioluminescence directly proportional to AR activity. B, qPCR analysis of the endogenous androgen induced gene PSAand the transgenes luciferase and G418 (neomycin resistance gene—aminoglycoside 30-phosphotransferase) in LNCaP/ARE-Luc cells. Cells were grownfor 72 hours in charcoal-stripped FCS and then treated with 0–10 nmol/L R1881. Data represent the mean of three independent experiments. Data werenormalized to GAPDH, b-actin, and RPL19 housekeeping genes. C, qPCR analysis of PSA gene expression in LNCaP cells grown in charcoal-strippedmedium for 72 hours and then treated with R1881 in the presence of increasing concentrations of bicalutamide or SK33 (0–10 mmol/L) or enobosarm (D).Data represent the mean of three independent experiments. Data were normalized to GAPDH, b-actin, and RPL19 housekeeping genes. E, Grayscale imageof bioluminescence from live LNCaP/Luc cells treated with enobosarm, SK33, and bicalutamide (0–100 mmol/L) for 24 hours. F, Line graph indicatingbioluminescence activity measurements from LNCaP/Luc cells treated with increasing concentrations of bicalutamide, enobosarm, and SK33 for 24 hours.�� , P > 0.01.






In vitro aqueous solubility and microsomal metabolic stabilitySubsequently, an early in vitro pharmacokinetic study of the

most promising compound, SK33, was performed. The meta-bolic stability of SK33 was tested in human liver microsomes,where compounds were incubated for 45 minutes with pooledliver microsomes and the intrinsic clearance (CLint) and half-life (t1/2) values were calculated based on 5 time points.Compound SK33 is characterized by an increased metabolicstability in comparison with the standard—bicalutamide(see Table 1). SK33 has microsomal half-life (t1/2) of 32 hours,which compares with that of standard drugs (bicalutamide andenzalutamide) and represents a good starting point for opti-mization toward a weekly or twice-weekly administrable prod-uct. No observed in vitro cardiotoxicity was observed in ourpreliminary studies (7.6% inhibition of hERG at 25 mmol/Lconcentration; http://cyprotex.com). Because of these promis-ing in vitro properties and the potent antiproliferative profile inprostate cancer cell lines, SK33 was selected for more detailedpreclinical in vitro and in vivo studies.

SK33 is a potent inhibitor of AR in the ARE-Luc reporter mouseand shows tissue specificity

The transgenic ARE-Luc mouse model shows AR-driven lucif-erase activity in all tissues in response to androgen action andactivity can be monitored in live animals in real time (31). Usingage-matched male mice (littermates), we analyzed the basalAR-driven luciferase levels in mice prior to treatment. ARE-Luc

mice were injected with D-luciferin and imaged using an IVISCCD camera in order to obtain baseline visualization and mea-surements of androgen receptor tissue activity. ARE-Luc micewere then injected subcutaneously with 50 mg/kg bicalutamide,SK33, or vehicle (DMSO) control and left for 24 hours (n¼ 3 foreach treatment), after which the mice were imaged again underthe exact same conditions. Posttreatmentmice were imaged againfor luciferase activity (Fig. 6A). Luciferase activity was measuredfrom three regions of interest (ROI), including the head, abdom-inal, and gonadal region (Fig. 6B). Bicalutamide and SK33 treat-ment resulted in a decreased luciferase activity in all areas.However, mice treated with SK33 showed a greater reduction ofluciferase activity in these areas when compared with bicaluta-mide. DMSO vehicle did not significantly affect luciferase activityin any region (Fig. 6C).

Tissues were then removed from sacrificed animals, and RNAwas extracted and analyzed by qPCR for luciferase transcripts ineach individual tissue. Bicalutamide and SK33 showed strongreduction in luciferase transcripts compared with vehicle oruntreated mouse tissues. Although similar, SK33 reduced lucif-erase transcripts to a greater extent than bicalutamide—withsignificantly greater effects in the brain. Neither bicalutamidenor SK33 had significant effects on AR activity in the spleen.However, SK33 did not reduce luciferase activity in the legmuscle and showed a significant upregulation of luciferasetranscripts in this tissue (Fig. 6D). Additionally, both bicalu-tamide and SK33 have reduced luciferase enzymatic activity inthe prostate, testes and brain. Both antiandrogens showedsignificant inhibition compared with vehicle control. Althoughslightly more inhibition was seen in the SK33 treated mice, theantiandrogens did not show statistically significant differencesfrom each other (Fig. 6E).

SK33 is a potent inhibitor of AR in the PTenko mouse model forprostate intraepithelial neoplasia (PIN)

PTen loss is one of the most frequent events in prostate cancerboth at the initiation stage and during late-stage metastatic

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Figure 5.

SK33 is active in LNCaP cells withacquired bicalutamide resistance. A,Bar graph indicating the relativeexpression levels of AR in LNCaP andLNCaP/BicR cells. Data represent themean of three independentexperiments. Data were normalized toGAPDH, b-actin, and RPL19housekeeping genes. B and C, MTTcytotoxicity assays of LNCaP orLNCaP/BicR cells treated withincreasing concentrations ofbicalutamide (B) or enzalutamide (C)for 96 hours. D, MTT cytotoxicityassays of LNCaP/Bic20 cells treatedwith increasing concentrations ofbicalutamide, SK33, and SK51for 96 hours.

Table 1. t1/2 calculated in human liver microsomes; SK33 is metabolically stablewith no loss of parent compound detected for the duration of the assay

Metabolic stability(t1/2 minutes) PPB %

%hERG inhibitionat 25 mmol/L

Bicalutamide 214 n.d. 96a

SK33 1,930 94.7 7.64

NOTE: Plasma protein binding (PPB) assay; results are expressed in terms ofmean%bound fromduplicate experiments. Cardiotoxicity expressed in terms of% hERG inhibition at 25 mmol/L compound concentration.

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Figure 6.

SK33 inhibits AR transcriptional activity in vivo and in PIN. A, Bioluminescent imaging of live ARE-Luc mice pre and posttreatment with bicalutamide orSK33 (or DMSO vehicle) for 24 hours. Image represents a grayscale image overlaid with pseudocolor bioluminescent activity data (scale representsflux rate of photons/sec/cm2). B, Grayscale image of an ARE-Luc mouse, white circles represent the three ROI measurements for each mouse. C, Bargraph representing relative bioluminescent data emanating from the three ROI—head, abdomen, and gonadal regions. Data are normalized to mousebioluminescence pretreatment with bicalutamide, SK33 (50 mg/kg), or DMSO vehicle. D, qPCR analysis of luciferase expression from various body tissues asindicated. Data are presented as luciferase/housekeeping genes (GAPDH, RPL19, and b-actin). E, Bar graph indicating relative luciferase enzymaticactivity from various tissues from ARE-Luc mice treated with either bicalutamide, SK33, or DMSO. F, qPCR analysis of luciferase expression from themouse PTenko prostate tissues. Data are presented as luciferase/housekeeping genes (GAPDH, RPL19, and b-actin). G, MTT assay of PTen–null mouseprostate cells treated with increasing doses of bicalutamide, enzalutamide, and SK33 for 96 hours. � , P ¼ 0.05; �� , P ¼ 0.01.






development—found in 30%þ of all human primary prostatecancers and 60%þ of higher grade metastatic lesions. Wangand colleagues established Ptenloxp/loxp;Pb-Cre4 mice, which haveconditional Pten alleles deleted by a Probasin (Pb) promoter-driven Cre recombinase, limiting Pten deletion to the prostaticepithelial layer (32). The mouse model of prostate-specificprobasin-mediated Pten deletion leads to PIN leading to adeno-carcinoma thereafter. Although the mouse prostate structure isdifferent to human, there are several points of commonality—specifically with the glandular epithelial AR-driven structure.

Ptenloxp/loxp;Pb-Cre4 mice with an integrated androgenreporter were injected subcutaneously with 50 mg/kg bicalu-tamide, SK33, or vehicle (DMSO) control and left for 24 hours(n ¼ 3 for each treatment). Prostate tissues were collected forqPCR and luciferase assay analysis. Upon qPCR for luciferasetranscripts both antiandrogens showed significant inhibitioncompared with vehicle control with more inhibition in theSK33 treated mice than bicalutamide, but the antiandrogensdid not show statistically significant difference from each other(Fig. 6F).

In ARþ mouse Ptenko cells growing in culture (PTEN-CaP8-ATCC CRL-3033), SK33 showed a stronger activity than enzalu-tamide or bicalutamide (Fig. 6G).

DiscussionProstate cancer, like the prostate gland fromwhich it is derived,

is androgen dependent—at least in the initial stages. The inhibi-tion of the AR activity either by antiandrogen therapy or byandrogen ablation remains the mainstay of treatment choices.Recently, there has been a movement away from classical anti-androgens due in part to the development of resistance to thesecompounds and the advantages of more novel agents, e.g., theantitestosterone synthesis agent abiraterone. Resistance has beenhistorically linked to the development of ARmutations, especiallyin the ligand-binding site. For example, AR T887A, W741L, andW741C have been found to arise in patients relapsing underflutamide or bicalutamide therapy (7, 8).

However, even in apparent castrate-resistant prostate cancer,the AR may still be a key molecule to cell growth and as suchremains a viable target for therapeutic intervention. The currentclinically used AR antagonists bind the receptor ligand-bindingsite, but with far less efficiency and affinity than natural agonists.The mechanism of the difference between the structure of ARantagonists and partial AR agonists, such as enobosarm, has beenassociated with the sulfonyl group on the antagonist being largerthan the oxygen linker group of the agonist, thus causing a bettersteric fit. The larger size of the antagonist then pushes away helix12, inhibiting the folding of the activated AR.

Here, we describe the chemical modification of an AR partialagonist—enobosarm. Enobosarm has been shown to have ana-bolic activity in the body, e.g., on bone and muscle mass, but tohave minimal effects on the gonadal tissues, i.e., an agent withselective AR modulating effects (SARM). The introduction of the3,5-bis-trifluoromethyl groups was theorized to give bulkier sidesto the B ring, thus inhibiting helix 12 folding. In essence, changinga high-affinity agonist to a high-affinity antagonist. Two bis-trifluoromethyl-containing enobosarm analogues named SK33and SK51 were the focus of this study.

Initial screening in prostate cancer cell lines indicated that theparental compound, enobosarm, was inactive in all of them,

whereas the SK33 and SK51, modified enobosarm, analoguesshowed higher activity (200-fold) in ARþ cell lines andmodest orlow activity in AR� cell lines. SK33 showed the highest activity,beingmore active than the current clinically used antiandrogen—bicalutamide. Treating LNCaP cells with SK33 resulted in theinhibition of cell-cycle progression, with cells arresting in G1.Noapoptotic response or cellular fragmentationwas observed. ARinhibition has been associated with the inability of cells to enterthe cell cycle, as the ARmay act as a replication licensing protein inprostate cells (33, 34).

The LNCaP cell line is an androgen-sensitive cell line with aT877A mutation in the LBD of the AR. Treating this cell line inculture for an extensive period with bicalutamide gave rise to aresistant subclone (LNCaP/BicR) which proliferated in thepresence of high doses of bicalutamide and showed anenhanced expression of AR. This switch to AR agonism isindicative of clinical prostate cancer as it progresses, and thiswork has been previously reviewed (35, 36). We did not detectother LBD mutations in this cell line. Although not as active asin the parental cell line, SK33 and especially SK51 showedmoderate activity. At this stage, it is unclear if the concentrationof SK33 (&SK51) required to inhibit the acquired resistancephenotype may be achievable in vivo, but their increased met-abolic stability, resistance to oxidative metabolism and detox-ification together with increased solubility may translate tohigh levels of drug in circulation. Additionally and of clinicalimportance, SK51 showed increased activity over enzalutamidein the LNCaP/BicR cells.

It is rare that an inhibitor compound such as an antiandro-gen can be tested in a biological system where there is a highlyspecific real-time reporter for its target protein activity—inte-grated into the host genome. Utilizing an LNCaP cell line withan integrated AR-driven luciferase reporter (30), we analyzedthe effect of these compounds directly on the transactivationfunction of the AR. SK33 resulted in a stronger inhibition ofAR-mediated gene activation of both the endogenous gene PSAand the transgene luciferase, than that mediated by bicaluta-mide. SK33 could also competitively inhibit the AR in thepresence of a strong synthetic androgen (R1881), while eno-bosarm itself had a mildly stimulating effect.

Further testing in vivo using unique AR-reporter mice, devel-oped by the authors (31), showed the antiandrogen activity ofthese compounds in the target tissue, i.e., prostate, as well as inperipheral tissues, in real time within the same animal. Here, sideeffects and bystander effects could be monitored. SK33 showed astronger or equal antiandrogen effect in most tissues, whencompared with bicalutamide at the same dosage concentration.But, interestingly, we observed that the effects of SK33 upon legmuscle AR activity was stimulatory, and the effects in the spleenwere minimal.

The anabolic effects of enobosarm have been well documen-ted, although its mechanism of action in specifically stimulat-ing skeletal muscle AR and not prostate or gonadal AR remainselusive. Because SK33 is a modified analogue of enobosarm theeffects on skeletal muscle was interesting to observe, especiallygiven its simultaneous high inhibitory activity in the prostateand other tissues. The rational addition of bis-trifluoromethylgroups into ring B of enobosarm seems to have successfullyexaggerated the SARM activity of enobosarm. The mechanismof SARM-like compounds remains relatively unknown, butlocally expressed AR tissue-specific cofactors have been


Dart et al.




postulated to govern the activity of AR modulating compoundssuch as SARMs. Although our results are limited to the mouseleg skeletal muscle, the use of such a compound in prostatecancer patients may ease side effects such as muscle wastage andfatigue, while maintaining prostate AR inhibition. The effectson other muscle tissues, e.g., the AR-responsive levator animuscle, in the mouse was unavailable.

It should also be noted that SK33 showed very potentinhibitory effects in the brain—this may reflect the increasedlipophilicity of the fluorinated compounds in traversing theblood–brain barrier or in fact their increased antiandrogenpotential. The CNS-specific effects could not be determined inthe mouse model, but no deleterious effects on motor functionor behavior was observed. The effects in humans could wellcorrelate with CNS-related side effects.

Finally, within a developing hyperplasia/adenocarcinomain situ in mice with homozygous Pten deletion we observedthat SK33 was equal to or better than bicalutamide in reducingAR activity.

ConclusionsWe demonstrate that structure-based design can efficiently

engineer second-generation AR antagonists that have clinicalpotential to circumvent antiandrogen resistance by complement-ing receptor mutations at the molecular level. In principle, thereare a limitednumber ofmutations that can cause anARantagonistto function as an AR agonist; therefore, it is conceivable that suchapproaches may ultimately lead to the development of antian-drogens that resist mutations that cause antiandrogen withdrawalsyndrome.

Utilizing novel in vitro and in vivo screening techniques andmodels, ideally suited to analyze and evaluate novel antiandro-gens, we have identified a novel antiandrogen candidate SK33.This bis-trifluoromethyl-containing enobosarm analogue, dis-plays better activity than bicalutamide in vitro with increasedefficacy against acquired antiandrogen resistance. Importantly,SK33 has comparable activity in vivo, but maintains a degree oftissue selectivity associated with its SARM—enobosarm parentalmolecule. The increased stability and potential differential sideeffect profile of these molecules may make them useful drugs forprostate cancer treatment.

Materials and MethodsAll chemicals were purchased from Sigma Aldrich or Alfa

Aesar and were used without further purification. All reactionswere performed under a nitrogen atmosphere. H-NMR(500 MHz), C-NMR (125 MHz), and F-NMR (470 MHz)spectra were recorded on a Bruker Avance 500 MHz spectrom-eter at 25�C. Chemical shifts (d) are expressed in parts permillion (ppm) and coupling constants (J) are given in hertz.The following abbreviations are used in the assignment of NMRsignals: s (singlet), bs (broad singlet), d (doublet), t (triplet), q(quartet), m (multiplet), dd (doublet of doublet), dt (doubletof triplet), td (triple doublet), and m (multiplet). Mass spec-trometry was run on a Bruker Micromass system in electrosprayionization mode. Thin-layer chromatography: precoated alu-minum backed plates (60 F254, 0.2 mm thickness, Merck) werevisualized under both short and long wave UV light (254 and366 nm). Flash column chromatography was carried out using

silica gel supplied by Fisher (60 A�, 35–70 mm). Purity ofprepared compounds was determined by Analytical HPLCanalysis using either a Thermo Scientific or a Varian Prostarsystem. All compounds tested in biological assays were >95%pure. HPLC using the eluents water (eluent A) and acetonitrile(eluent B). Column; Varian Pursuit, 150mm� 4.6 mm, 5.0 mm.Flow rate; 1.0 mL/min, gradient 30 minutes 10% ! 100%eluent B in eluent A.

General method for the preparation of intermediates (3a,b)Methacryloyl chloride 2 (2.63 mL, 27.16 mmol/L) was added

over the course of 10 minutes to a stirring solution of thedifferent aniline 1a, b (3.4 mmol/L) inN,N-dimethylacetamide(10 mL) at room temperature for 3 hours. After the reaction wascomplete, the mixture was diluted with ethyl acetate (100 mL),extracted with sat. aq. NaHCO3 solution (3 � 25 mL) and withcold brine (4 � 50 mL). The organic layer was dried overNa2SO4 and the solvent was removed at reduced pressure. Thecrude residue was purified by flash column chromatography.

General method for the preparation of intermediates (4a,b)To a stirred solution of the intermediates (3a,b; 3 mmol/L) in

DCM (7 mL) was added 30% hydrogen peroxide (3.6 mL,32.03 mmol/L). The reaction mixture was put in a water bathat room temperature and trifluoroacetic anhydride (3.7 mL,26.7 mmol/L) was added slowly to the mixture, which was thenstirred for 24 hours. The reaction mixture was transferred to aseparating funnel using DCM (30 mL). The organic layer waswashed with distilled water (20 mL), sat. aq. Na2S2O3 (4 �20 mL), sat. aq. NaHCO3 (3 � 20 mL) and brine (20 mL), driedover Na2SO4 and concentrated at reduced pressure.

General method for the preparation of compounds SK33and SK51

To a mixture of NaH (60% in mineral oil, 0.050 g,1.23 mmol/L) in anhydrous THF (2 mL) at 0�C under Aratmosphere was added a solution of the 3,5-bis-trifluoro-methylphenol 5 (1.11 mmol/L) in 1 mL of anhydrous THF.This mixture was stirred at room temperature for 20 minutes. Asolution of the different intermediates (4a,b; 0.74 mmol/L) inanhydrous THF (3 mL) was added slowly. The reaction mixturewas stirred at room temperature overnight. The mixture wasthen diluted with ethyl acetate (30 mL), washed with brine(15 mL) and water (30 mL), dried over Na2SO4, and concen-trated under vacuum. The crude residue was purified by flashcolumn chromatography.

3-(3,5-bis(trifluoromethyl)phenoxy)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide(SK33)

Purified by flash column chromatography eluting withn-hexane/EtOAc 100:0 v/v increasing to n-hexane/EtOAc90:10 v/v. Obtained in 85 % yield as a white solid.

1HNMR (CDCl3-d1) d 9.20 (bs, 1H, NH), 8.16 (d, J¼ 2Hz, 1H,ArH), 8.00 (dd, J ¼ 2, 8.5 Hz, 1H, ArH), 7.82 (d, J ¼ 8.5 Hz, 1H,ArH), 7.53 (s, 1H, ArH), 7.36 (s, 2H, ArH), 4.59 (d, J¼ 9 Hz, 1H,CH2), 4.13 (d, J¼ 9Hz, 1H, CH2), 3.49 (bs, 1H,OH), 1.67 (s, 3H,CH3); 19F NMR (CDCl3-d1) d�62.24,�63.09; 13CNMR (CDCl3-d1) d172.03 (C¼O), 158.28 (ArC), 141.29 (ArC), 135.92 (ArCH),134.18 (q, 2JC-F¼ 32.6Hz, ArC), 133.16 (q, 2JC-F¼ 33.3Hz, ArC),122.94 (q, 1JC-F ¼ 264.5 Hz, CF3), 122.05 (q, 1JC-F ¼ 272.6 Hz,






CF3), 121.84 (ArCH), 117.33 (q, 3JC-F¼5.1Hz, ArCH), 115.56 (q,3JC-F ¼ 3.6 Hz, ArCH), 115.38 (ArC), 115.14 (q, 3JC-F ¼ 3.8 Hz,ArCH), 104.92 (ArC), 75.83 (COH), 73.10 (CH2), 23.04 (CH3).MS [ESI, m/z]: 501.1 [M þ H], 523.1 [M þ Na]. HPLC: retentiontime ¼ 25.18 minutes.

3-(3,5-bis(trifluoromethyl)phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-2-(trifluoromethyl)phenyl) propanamide (SK51)

Purified by flash column chromatography eluting withn-hexane/EtOAc 100:0 v/v increasing to n-hexane/EtOAc90:10 v/v. Obtained in 78 % yield as a white solid.

1HNMR (CDCl3-d1) d 9.68 (bs, 1H, NH), 8.76 (d, J¼ 9Hz, 1H,ArH), 8.59 (d, J ¼ 3 Hz, 1H, ArH), 8.47 (dd, J ¼ 2.5, 9.5 Hz, 1H,ArH), 7.56 (s, 1H, ArH), 7.36 (s, 2H, ArH), 4.59 (d, J¼ 9 Hz, 1H,CH2), 4.15 (d, J ¼ 9 Hz, 1H, CH2), 3.25 (bs, 1H, OH), 1.69 (s,3H, CH3);

19F NMR (CDCl3-d1) d �61.59, �63.06; 13C NMR(CDCl3-d1) d 172.02 (C¼O), 158.15 (ArC), 143.17 (ArC), 140.38(ArC), 133.16 (q, 2JC-F¼33.8Hz, ArC), 133.03 (q, 2JC-F¼32.8Hz,ArC), 128.38 (ArCH), 123.22 (q, 1JC-F ¼ 268.3 Hz, CF3), 122.75(q, 1JC-F ¼ 271.3 Hz, CF3),122.62 (ArCH), 122.46 (q, 3JC-F ¼ 6.3Hz, ArCH), 115.67 (q, 3JC-F ¼ 3.8 Hz, ArCH), 115.11 (ArCH),75.86 (COH), 73.93 (CH2), 22.94 (CH3).MS [ESI,m/z]: 521.1 [Mþ H], 543.1 [M þ Na]. HPLC: retention time ¼ 26.42 minutes.

Cell cultureAll cell lines were purchased LGC Standards and used within

6 passages, and subjected to regular mycoplasma screening(Geneflow PCR test). LNCaP cells were maintained at 37�C,5% CO2 in RPMI medium with 10% fetal bovine serum (FirstLink Ltd). PC3, VCaP, and Du145 and mouse Pten-null cellswere maintained in DMEM medium (Sigma) with 10% fetalbovine serum (First Link UK). All media were supplement-ed with 2 mmol/L L-glutamine, 100 units/mL penicillin,100 mg/mL streptomycin (Sigma). For hormone depletionexperiments, 72 hours before androgen exposure, medium wasreplaced with "starvation medium" consisting of phenol red-free RPMI (or DMEM) medium, supplemented with 5% char-coal-stripped fetal bovine serum (First Link UK). All cell lineswere obtained from the ATCC cell bank.

MTT assayThe MTT assay was used as a cell viability assay for all the cell

lines listed using the antiandrogen compounds. Briefly, cells(5� 104 cells/mL) were seeded into 96-well plates (200 mL/well),allowed to attach and grow for 24 hours, and subsequently treat-ed with varying concentrations of antiandrogens (0–100 mmol/L)for 96 hours. Optimal seeding densities were premeasuredfor linear growth over the 96 hours. MTT (3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL in PBS)was added to a final concentration of 0.5 mg/mL for 4 hoursat 37�C. After 4 hours, purple formazan crystals, formedby mitochondrial reduction of MTT, were solubilized in acid-ified isopropanol (200 mL/well) and the absorbance was readat 570 nm after 10 minutes incubation. Percent inhibition ofviability was calculated as a percentage of untreated control andthe cytotoxicity/cell growth inhibition was expressed as IC50.

Luciferase assaysCells were washed and lysed in reporter lysis buffer (Pro-

mega). Lysate was mixed with D-luciferin substrate (Promega)

and light emission was measured using the Promega Glomaxmultiluminometer.

For live cell imaging D-luciferin substrate was added directlyinto the cell media, and plates were imaged on a Syngene G:Boximager.

Tissue was pulverized by grinding in liquid nitrogen and thencompletely homogenized, using a microfuge pestle, in reporterlysis buffer with protease inhibitors (Promega). Lysate (20 mL)was mixed with luciferin substrate (20 mL) and light emissionmeasured using the Promega Glomax multiluminometer. Lightemission was then normalized to protein content as measured bya Bradford Assay.

RNA extraction and RT-PCRTotal RNA sampleswere prepared using TRIzol reagent (Sigma)

and converted to cDNA using the GoScript Reverse TranscriptionSystem (Promega).

qPCRReactions were performed in triplicate on cDNA samples in

96-well optical plates on an ABI Prism StepOne System(Thermo Fisher). Reactions consisted of 2 mL cDNA, 7 mLPCR-grade water, 10 mL 2 � TaqMan Universal PCR MasterMix (Applied Biosystems), 1 mL Taqman specific assay probes(Applied Biosystems) for PSA, TMPRSS2, KLK2, and L19. Para-meters were: 50�C for 2 minutes, 95�C for 10 minutes, 40 cyclesof 95�C for 15 seconds and 60�C for 1 minute. Data wererecorded using Sequence Detector Software (SDS version 2.3;PE Applied Biosystems). Levels were normalized to GAPDH,b-actin, and RPL19.

Animal experimentsAll mouse procedures were performed in accordance with the

UK Animals (Scientific Procedures) Act 1986 under HomeOffice license. ARE-Luc strains were made and available"in house" (31), and Ptenloxp/loxp;Pb-Cre4 were obtained fromThe Jackson Laboratory.

Luciferase imagingAnesthetized mice (3% isofluorane with O2 carrier, Abbott

Animal Health UK) were injected i.p. or s.c. with D-luciferin(Caliper Life Sciences Ltd) at 150 mg/kg, 10 minutes beforeimaging. Light emission from luciferase was detected by the IVISImaging System 100 series (Xenogen Corporation), and overlaidas a pseudocolor image with reference scale, upon a grayscaleoptical image.

For ex vivo imaging, mice were sacrificed 10 minutes afterluciferin injection and immediately dissected. Target organs wererinsed briefly in PBS and placed under the bioluminescent cam-era. Tissues were then collected for luciferase assays, RNA extrac-tion or fixed in 10% formaldehyde before wax embedding andsectioned using standard procedures.

Cell-cycle analysis (FACS)Cells were grown on 10-cm dishes for 24 to 96 hour �

treatments. Cells were then trypsinized, washed twice in PBS,fixed in 70% ethanol at 4�C, were stained with 5 mg/mL propi-dium iodide, and RNA was removed using 50 mg/mL RNase A.FACS analysis was carried out using a FACS Calibur (Beckton-Dickinson) using linear scale representation of forward andside scatter during flow analysis, as well as DNA content. Single


Dart et al.




cells were gated and the cell-cycle profiles measured using FCSExpress4 software, using the built in kinetics and cell-cycleanalysis best-fit model for cell-cycle phase identification. A totalof 10,000 events were measured per sample.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D.A. Dart, S. Kandil, C.L. Bevan, A.D. WestwellDevelopment of methodology: D.A. Dart, S. KandilAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):D.A. Dart, S. Kandil, S. Tommasini-Ghelfi, G. Serranode Almeida, C.L. BevanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, com-putational analysis): D.A. Dart, S. Kandil, G. Serrano de Almeida, C.L. BevanWriting, review, and/or revision of the manuscript: D.A. Dart, S. Kandil,G. Serrano de Almeida, C.L. Bevan, W. Jiang, A.D. WestwellAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.A. Dart, S. KandilStudy supervision: D.A. Dart, C.L. Bevan, W. Jiang, A.D. Westwell

AcknowledgmentsThe authors would like to pay a tribute to Professor Christopher

McGuigan (the Drug Hunter) for all his contributions for the success ofthis project. Professor McGuigan was an exceptional and dedicated scientistwho has remarkable achievements in the anticancer and antiviral drug-discovery field.

The authors would like to thank Cyprotex (UK) for performing themetabolic stability, PPB % recovery, hERG inhibition assays as an out-sourced service. We would also like to thank our funders—The WelshGovernment (A4B-Academic Expertise for Business grant), The CardiffUniversity–Peking University Cancer Institute, Prostate Cancer UK, CancerResearch UK, and the Harvey Spack Memorial Fellowship (G. Serrano deAlmeida).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 18, 2018; revised April 10, 2018; accepted June 7, 2018;published first June 12, 2018.

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Dart et al.




2018;17:1846-1858. Published OnlineFirst June 12, 2018.Mol Cancer Ther D. Alwyn Dart, Sahar Kandil, Serena Tommasini-Ghelfi, et al.

In Vivo and Tissue Selectivity In VitroAntiandrogenic Activity Novel Trifluoromethylated Enobosarm Analogues with Potent

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