Novel trifluoromethylated enobosarm analogues with potent antiandrogenic activity in vitro and tissue selectivity in vivo

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

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Novel Trifluoromethylated Enobosarm Analogues with Potent Anti-

androgenic Activity in vitro and Tissue Selectivity in vivo.

a,c☥

b☥

c

D. Alwyn Dart

, Sahar Kandil , Serena Tommasini-Ghelfi , Gilberto Serrano de

c

a

b

Almeida , Charlotte L. Bevan , Wenguo Jiang , and Andrew D. Westwell

a

b

Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, Wales, UK. School of Pharmacy and

c

Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, CF10 3NB Cardiff, Wales, UK. Androgen Signalling

Laboratory, Department of Surgery & Cancer, Imperial College London, Hammersmith Hospital Campus, London, W12 0NN, UK.

☥

These authors contributed equally to this manuscript

Running title: Potent anti-androgenic enobosarm analogues for prostate cancer treatment.

Corresponding author: dartd@cardiff.ac.uk

Keywords: prostate, cancer, enobosarm, anti-androgen, therapeutics

Conflict of Interest

The authors declare that there are no conflicts of interest in relation to the work described.

1

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

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Abstract

Prostate cancer often develops anti‐androgen resistance, possibly via androgen receptor (AR)

mutations which change antagonists to agonists. Novel therapies with increased anticancer activity,

whilst overcoming current drug resistance are urgently needed. Enobosarm has anabolic effects on

muscle and bone whilst having no effect on the prostate. Here we describe the activity of novel

chemically modified enobosarm analogues. The rational addition of bis‐trifluoromethyl groups into

ring B of enobosarm, profoundly modified their activity, pharmacokinetic and tissue distribution

profiles. These chemical structural modifications resulted in an improved AR binding affinity ‐ by

increasing the molecular occupational volume near helix 12 of AR. In vitro, the analogues SK33 and

SK51 showed very potent antiandrogenic activity, monitored using LNCaP/AR‐Luciferase cells where

growth, PSA and luciferase activity were used as AR activity measurements. These compounds were

1

0-fold more potent than bicalutamide and 100-fold more potent than enobosarm within the LNCaP

model. These compounds were also active in LNCaP/BicR cells with acquired bicalutamide

resistance. In vivo, using the AR‐Luc reporter mice, these drugs showed potent AR inhibitory activity

in the prostate and other AR‐expressing tissues e.g. testes, seminal vesicles and brain. These

compounds do not inhibit AR activity in the skeletal muscle, and spleen ‐ thus indicating a selective

tissue inhibitory profile. These compounds were also active in vivo in the Pb-PTen deletion model.

SK33 and SK51 have significantly different and enhanced activity profiles compared to enobosarm,

and are ideal candidates for further development for prostate cancer therapy with potentially fewer

side effects.

2

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

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Introduction

Prostate cancer (PCa) is the most commonly diagnosed male cancer in the Western world (1). Tumor

growth is initially androgen dependent - driven by the androgen receptor (AR). Androgen receptor

(AR) signalling pathway remains a key driver of PCa, implicated throughout the different stages of

the disease, and as such represents an obvious therapeutic target in PCa therapy (2, 3). Androgen

receptor (AR) is a member of the family of steroid hormone receptors, which are ligand-dependent

transcriptional regulators for diverse sets of genes involved in both reproductive and anabolic

actions (4-6). The prototypical model for AR involves ligand binding that induces conformational

changes in the ligand-binding domain (LBD), revealing a co-activator dimerization surface composed

of helices 3, 5, and 12, as the LBD site is adjacent to helix 12 (H12).

Currently, the mainstays of prostate cancer treatment are androgen ablation (to castrate levels)

and/or antiandrogen treatment, which block AR signalling (2, 3). The major anti-androgens in clinical

use are bicalutamide and enzalutamide (and historically flutamide, hydroxyflutamide and

nilutamide) (Figure 1A). However, these compounds bind to AR with low affinity and can induce

escape mechanisms. Furthermore, under AR amplification or mutation conditions some of these

compounds exhibit agonist activity and fail to inhibit AR. This resistance may arise from, among

other mechanisms, mutations to several key residues in the ligand-binding domain (AR-LBD) (7, 8).

Common mutations in AR found in PCa patients include T877A, W741L, and W741C. Bicalutamide

W741L

W741C

(Figure 1A) can act as an agonist in AR

and AR

while hydroxyflutamide is an agonist in

T877A

AR

(7, 8). Gain of function mutations are common in prostate cancers and are correlated with

disease progression, the lack of therapeutic response and poor clinical outcomes. Additionally, AR

mutations may be selected for during anti-androgen therapy – reviewed in (9, 10) .

Additionally, the use of anti-androgens in patients may elicit moderate side effects e.g. muscle

wastage, bone density loss and CNS dysfunction – which are exacerbated in elderly men. Therefore,

there is a growing need for selective androgen receptor modulators (SARM) that would demonstrate

tissue-selective activity – being inhibitors of the AR in the prostate with tissue sparing effects

elsewhere (11).

TM

Enobosarm (also referred to as Ostarine , GTx-024 or S-22) is a non-steroidal selective androgen

receptor modulator (SARM) with a favourable safety and pharmacokinetic profile (Figure 1B). It

belongs to the class of arylpropionamides, which are structurally derived from the antiandrogen

bicalutamide. Enobosarm binds to and activates the AR with enantioselective affinity, potency and

efficacy similar to, but somewhat less than, that of DHT (12). Transient transfection studies indicate

that enobosarm is remarkably selective for the AR and it does not cross-react with other nuclear

hormone receptors. This receptor specificity differentiates enobosarm’s pharmacology from other

3

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steroidal androgens and has likely contributed to observations that the drug has generally been well

tolerated in clinical trials. It has selective anabolic effects and is under clinical development for

prevention and treatment of muscle wasting (cachexia) in cancer patients (13, 14).

4

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Results

Rational design of novel Enobosarm analogues

Nonsteroidal anti-androgens such as bicalutamide act as AR antagonists (Figure 1A), thus preventing

androgen-dependent cell growth. In contrast, AR agonists e.g. enobosarm (Figure 1B) may act as

agonists in all the AR LBD mutants, since they will fit sterically better with the bulk-reducing AR

wt

mutations than in AR (15). The difference in activity between agonists such as enobosarm and

wt

antagonists such as bicalutamide in AR may arise from the linker oxygen’s smaller size relative to

the sulfonyl group of bicalutamide (Figure 1A&B). This bulk in AR antagonist structure, though small,

is thought to 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 that

the differences observed among AR modulators (agonist or antagonist) are not because of their B-

ring size or shape but because of their linker size and orientation (17). The improved understanding

is being applied to rationally designed treatment options with novel mechanisms of action. It is

proposed that AR antagonists should have appending molecular extensions to the core structure of

AR agonists that interfere with the placement of H12, thereby disrupting co-activator recruitment

(16, 18, 19).

wt

In the enobosarm/AR co-crystal structure (PDB code; 3RLJ)(17) (Figure 2A&B), the 4-cyanophenyl

741

ether group of enobosarm is situated between residues of H12 and the indolyl side chain of Trp ,

suggesting that in the wild-type receptor (and probably the W741L mutant) the larger substitution

with the same orientation of the linker would result in a better AR antagonist by pushing against

Helix 12. We tested this hypothesis in silico by investigating whether the introduction of 3,5-Bis-

trifluoromethyl (3,5-Bis-CF ) into B ring of enobosarm would provide the geometric bulk needed to

3

keep ring B towards Helix 12 of AR. Preliminary molecular modelling studies showed that the bis-

trifluoromethyl phenyl moiety (ring B in SK33 and SK51) has significantly changed the orientation of

741

ring B so that the (3,5-Bis-CF ) groups are pointing outwards away from Trp and towards helix 12,

3

thus preventing AR from adapting the AR agonist conformation, whilst keeping the small size of the

oxygen linker. The combination of the small oxygen linker and the steric hindrance of the (3,5-Bis-

CF

conformational restriction away from Trp and towards helix 12 (Figure 2C & D). A small library of

AR antagonists having the (3,5-Bis-CF ) motif was prepared (20) among which SK33 showed the best

3

) substitution on ring B resulted in the optimum size and orientation of ring B to impose

741

3

antiproliferative and metabolic profiles, hence it was selected for further studies.

The introduction of fluorinated substituents into drug candidates can impart a unique set of

properties owing to the unique combination of electronegativity, size and lipophilicity features of

fluorinated groups (21-25). These factors can have a substantial impact on the molecular

5

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conformation, which in turn affect the binding affinity to the target protein (26, 27).

Chemical synthesis of SK33 and SK51

Racemic enobosarm derivatives (SK33 and SK51) were prepared by reacting methacryloyl chloride

(

2) with the corresponding substituted anilines 1a and 1b in dimethylacetamide (DMA) solvent to

obtain phenylacrylamides 3a and 4b which were converted into the corresponding epoxides 4a and

b in the presence of a large excess of hydrogen peroxide and trifluoroacetic anhydride in

dichloromethane (DCM). Opening of the epoxide rings of 4a and 4b with substituted 3,5-bis

4

(trifluoromethyl)phenol (5) in tetrahydrofuran (THF) gave the final compounds SK33 and SK51. The

1

structures of the synthesised compounds were confirmed using analytical and spectroscopic data ( H

13

19

NMR, C NMR, F NMR and mass spectrometry), which were in full accordance with their depicted

structures. SK33 and SK51 were purified (>95% as confirmed by HPLC) and tested as racemic mixture

(Figure 1C).

Cellular activity of enobosarm and the trifluoromethylated analogues SK33 and SK51

The antiproliferative and selective activities of these compounds were tested in vitro in a

panel of prostate cancer cell lines, namely LNCaP, VCaP, PC3 and Du145. LNCaP and VCaP

represent prostate cells which have retained the expression of the androgen receptor (28,

29). However, LNCaP cells overexpress a mutant AR, with a T877A mutation in the ligand

binding site. The VCaP cell line overexpresses wild type AR. Both cell lines are responsive to

androgens and cells will express androgen responsive genes. The PC3 and Du145 cells do

not express the AR nor do they express androgen responsive genes.

Cells were exposed to increasing concentrations of anti-androgens for 96 hours and cell

AR+ve

viability was analysed using the MTT assay. In LNCaP

cells the parental compound

enobosarm, showed insignificant activity with the IC50 = 40 M, however, the

trifluoromethylated enobosarm analogue, SK33, demonstrated a significantly potent activity

with IC50 = 0.2 M (Figure 3A). Moreover, when compared to the antiandrogen

bicalutamide, the novel compounds showed an increased efficacy (Figure 3B). SK51 also

displayed an increased activity compared to bicalutamide (IC50 = 0.7M) (Figure 3B).

AR-ve

In PC3

cells, enobosarm showed activity only at relatively high dose (Figure 3C), whilst

SK33 showed some moderate activity. When compared to bicalutamide, SK33 showed a

significantly increased increased activity. On the other hand, the anti-proliferative activity of

6

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AR+ve

AR-ve

SK33 in LNCaP

cells was over ten times higher than that of its activity against PC3

cell line (Figure 3D).

Furthermore, the panel of prostate cancer cell lines were screened for their expression of

AR+ve

the target androgen receptor (AR) by qPCR. As expected, LNCaP

and VCaP

cells

AR-ve

showed robust expression of AR whilst AR was not detectable in PC3

Figure 3E).

and Du145

(

Figure 3F depicts the summary of the activity profile of the SK33 and SK51 compounds in

the four PCa cell lines where a clear activity difference (selectivity) can be observed

+ve

-ve

between the AR cell lines (LNCaP and VCaP) and the AR cell lines (PC3 and Du145).

SK33 decreases cells entering S-phase in LNCaP cells but not in PC3 cells

To further analyse the activity of these compounds in prostate cancer cell lines we treated

+ve

-ve

LNCaP (AR ) or PC3 (AR ) cells with 10M of antiandrogen (or vehicle DMSO) for 48

hours. These cell lines were collected for FACS analysis and standard cell cycle profiling

using propidium iodide staining for DNA content, was carried out.

LNCaP cells showed a decrease in the number of cells in S phase with all the anti-androgen

compounds. SK33 showed the highest reduction of the number of cells in S phase, followed

by SK51, enzalutamide and then bicalutamide, respectively. (Figure 3G). No apparent cell

toxicity could be visualised as estimated by the absence of a sub-G1 population or

fragmented necrotic cells (Figure 3G). In the PC3 cell line, no significant effect was observed

with any of the tested compound (Figure 3H). However, a small increase in G2/M phase

could be detected with SK33 in PC3 cells.

SK33 reduces AR transcriptional activity

We then analysed the ability of SK33 to inhibit the activity of the AR protein i.e. the ligand

activated transcription factor activity. The LNCaP/Luc cell line, has an integrated luciferase

reporter gene under the specific control of the AR transcription factor, as shown in the

schematic figure 4A (30). Growing these LNCaP/Luc cells in charcoal stripped serum for

7

2hours removes exogenous androgens and reduced the expression of AR-responsive genes

down to a baseline level, as monitored by qPCR. Adding a synthetic androgen (R1881) back

into the media (0-10nM) increased both PSA and Luciferase gene expression in a dose

dependent manner (Figure 4B). Androgen treatment does not affect G418 gene expression

7

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(coexpressed from the same integrated plasmid as the luciferase reporter) nor did it affect

the expression of housekeeping genes L19, -actin and GAPDH. The ability of the anti-

androgens to block this androgen-dependent gene upregulation was then further tested.

Treating hormonally starved LNCaP cells with R1881 showed a 9-fold upregulation of the

endogenous gene PSA. When treated with increasing doses of either bicalutamide or SK33 a

dose dependent inhibition of gene expression was seen. SK33 showed a greater PSA

inhibition than bicalutamide at 1 and 10M (Figure 4C). Conversely, the parental compound,

enobosarm, showed a mild stimulatory activity (Figure 4D).

We then examined the real-time activity of AR in live LNCaP/Luc cells in response to

increasing doses of SK33 compared to the parental compound enobosarm and the anti-

androgen bicalutamide. Live imaging and photon flux measurements of LNCaP/Luc cells

grown on 96 well plates in the presence of 0-100M compound for 24 hours resulted in a

strong inhibition of AR-mediated luciferase activity (Figure 4E). The AR inhibitory

concentration [50%] for both SK33 and bicalutamide were approx. 1M. Bicalutamide

showed no further inhibitory effects past this concentration. However, SK33 maintained a

dose dependent inhibition, completely inhibiting the AR-mediated luciferase activity at 50-

100M concentration (Figure 4F).

SK33 and SK51 are active in LNCaP cells with acquired bicalutamide resistance.

LNCaP cells were grown in increasing concentrations of bicalutamide for 3-6 months, until

proliferation was seen. This bicalutamide resistant cell line (LNCaP/BicR) then grew well in

20M bicalutamide, with cell cycle kinetics similar to that of the parental cell line. An

increased expression level of AR was seen in this cell line upon qPCR analysis (Figure 5A).

Treating this cell line with increasing doses of bicalutamide showed a proliferative response

–

5

at 20-25M, whereas the parental cell line showed sensitivity and an IC50 of 3M (Figure

B). The LNCaP/BicR cell line also showed a partial cross-resistance to enzalutamide (IC50

M approx.), Figure 5C. We then analysed if the mechanism of the acquired resistance to

=

bicalutamide extended to the novel agents SK33 and SK51. Treatment of LNCaP/BicR cells

with increasing concentrations of SK33 and SK51 for 96hours resulted in a dose-dependent

response and an inhibition of cell growth, rather than being proliferative agents as

bicalutamide (Figure 5D), with IC50 of approximately 2M for SK51 and 5M for SK33.

8

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Although not as active as in the parental sensitive cell line, SK51 did show an improved

activity over enzalutamide (& bicalutamide).

In vitro aqueous solubility and microsomal metabolic stability

Subsequently, an early in vitro pharmacokinetic study of the most promising compound,

SK33, was performed. The metabolic stability of SK33 was tested in human liver

microsomes, where compounds were incubated for 45 min with pooled liver microsomes

and the intrinsic clearance (CLint) and half-life (t1/2) values were calculated based on 5 time

points. Compound SK33 is characterised by an increased metabolic stability in comparison

with the standard – bicalutamide (see Table 1). SK33 has microsomal half-life (t1/2) of 32 hr,

which compares to that of standard drugs (bicalutamide and enzalutamide) and represents

a good starting point for optimisation towards a weekly or twice-weekly administrable

product. No observed in vitro cardiotoxicity was observed in our preliminary studies (7.6 %

inhibition of hERG at 25M concentration) (http://cyprotex.com). Because of these

promising in vitro properties and the potent anti-proliferative profile in prostate-cancer cell

lines, SK33 was selected for more detailed pre-clinical in vitro and in vivo studies.

SK33 is a potent inhibitor of AR in the ARE-Luc reporter mouse – and shows tissue

specificity

The transgenic ARE-Luc mouse model shows AR-driven luciferase activity in all tissues in

response to androgen action and activity can be monitored in live animals in real time (31).

Using age-matched male mice (littermates) we analysed the basal AR-driven luciferase

levels in mice prior to treatment. ARE-Luc mice were injected with D-luciferin and imaged

using an IVIS CCD camera in order to obtain baseline visualisation and measurements of

androgen receptor tissue activity. ARE-Luc mice were then injected sub-cutaneously with

5

0mg/kg bicalutamide, SK33 or vehicle (DMSO) control and left for 24 hours (n=3 for each

treatment), after which the mice were imaged again under the exact same conditions. Post

treatment mice were imaged again for luciferase activity (Figure 6A). Luciferase activity was

measured from three regions of interest (ROI), including the head, abdominal and gonadal

region (Figure 6B). Bicalutamide and SK33 treatment resulted in a decreased luciferase

activity in all areas. However, mice treated with SK33 showed a greater reduction of

9

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luciferase activity in these areas when compared to bicalutamide. DMSO vehicle did not

significantly affect luciferase activity in any region (Figure 6C).

Tissues were then removed from sacrificed animals, and RNA was extracted and analysed by

q-PCR for luciferase transcripts in each individual tissue. Bicalutamide and SK33 showed

strong reduction in luciferase transcripts compared to vehicle or untreated mouse tissues.

Although similar, SK33 reduced luciferase transcripts to a greater extent than bicalutamide

–

with significantly greater effects in the brain. Neither bicalutamide nor SK33 had

significant effects on AR activity in the spleen. However, SK33 did not reduce luciferase

activity in the leg muscle and showed a significant upregulation of luciferase transcripts in

this tissue (Figure 6D). Additionally, both bicalutamide and SK33 have reduced luciferase

enzymatic activity in the prostate, testes and brain. Both anti-androgens showed significant

inhibition compared to vehicle control. Although slightly more inhibition was seen in the

SK33 treated mice, the antiandrogens did not show statistically significant differences from

each other (Figure 6E).

ko

SK33 is a potent inhibitor of AR in the PTen mouse model for prostate intraepithelial

neoplasia (PIN).

PTen loss is one of the most frequent events in prostate cancer both at the initiation stage

and during late stage metastatic development - found in 30%+ of all human primary

prostate cancers, and 60%+ of higher grade metastatic lesions. Wang et al, established

loxp/loxp

Pten

;Pb-Cre4 mice, which have conditional Pten alleles deleted by a Probasin (Pb)

promoter-driven Cre recombinase, limiting Pten deletion to the prostatic epithelial layer

32). The mouse model of prostate-specific probasin-mediated Pten deletion leads to

(

prostate intraepithelial neoplasia (PIN) leading to adenocarcinoma thereafter. Although the

mouse prostate structure is different to human, there are several points of commonality –

specifically with the glandular epithelial AR-driven structure.

loxp/loxp

Pten

;Pb-Cre4 mice with an integrated androgen reporter were injected sub-

cutaneously with 50mg/kg bicalutamide, SK33 or vehicle (DMSO) control and left for

4hours (n=3 for each treatment). Prostate tissues were collected for qPCR and luciferase

2

assay analysis. Upon q-PCR for luciferase transcripts both anti-androgens showed significant

inhibition compared to vehicle control with more inhibition in the SK33 treated mice than

bicalutamide, but the anti-androgens did not show statistically significant difference from

10

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each other (Figure 6F).

+ve

ko

In AR mouse Pten cells growing in culture (PTEN-CaP8 -ATCC CRL-3033), SK33 showed a

stronger activity than enzalutamide or bicalutamide (Figure 6G).

11

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Discussion

Prostate cancer (PCa), like the prostate gland from which it is derived, is androgen

dependent – at least in the initial stages. The inhibition of the androgen receptor activity

either by anti-androgen therapy or by androgen 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 these compounds and the advantages of more

novel agents e.g. the anti-testosterone synthesis agent abiraterone. Resistance has been

historically linked to the development of AR mutations especially in the ligand binding site.

For example, AR T887A, W741L and W741C have been found to arise in patients relapsing

under flutamide 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 such remains a viable target for therapeutic intervention.

The current clinically used AR antagonists bind the receptor ligand binding site, but with far

less efficiency and affinity than natural agonists. The mechanism of the difference between

the structure of AR antagonists and partial AR agonists, such as enobosarm, has been

associated with the sulfonyl group on the antagonist being larger than the oxygen linker

group of the agonist, thus causing a better steric fit. The larger size of the antagonist then

pushes away helix 12, inhibiting the folding of the activated AR.

Here we describe the chemical modification of an AR partial agonist – enobosarm.

Enobosarm has been shown to have anabolic activity in the body e.g. on bone and muscle

mass, but to have minimal effects on the gonadal tissues i.e. an agent with selective AR

modulating effects (SARM). The introduction of the 3,5-Bis-trifluoromethyl groups was

theorized to give bulkier sides to the B ring thus inhibiting helix 12 folding. In essence

changing a high affinity agonist to a high affinity antagonist. Two bis-trifluoromethyl-

containing enobosarm analogs named SK33 and SK51 were the focus of this study.

Initial screening in prostate cancer cell lines indicated that the parental compound,

enobosarm, was inactive in all of them, whereas the SK33 and SK51, modified enobosarm

+ve

analogues showed higher activity (200 fold) in AR cell lines and modest or low activity in

-

ve

AR cell lines. SK33 showed the highest activity, being more active than the current

clinically used anti-androgen – bicalutamide. Treating LNCaP cells with SK33, resulted in the

inhibition of cell cycle progression, with cells arresting in G1. No apoptotic response or

cellular fragmentation was observed. AR inhibition has been associated with the inability of

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cells to enter the cell cycle, as the AR may act as a replication licensing protein in prostate

cells (33, 34).

The LNCaP cell line is an androgen sensitive cell line with a T877A mutation in the LBD of the

AR. Treating this cell line in culture for an extensive period with bicalutamide gave rise to a

resistant subclone (LNCaP/BicR) which proliferated in the presence of high doses of

bicalutamide, and showed an enhanced expression of AR. This switch to AR agonism is

indicative of clinical prostate cancer as it progresses, and this work has been previously

reviewed (35, 36). We did not detect other LBD mutations in this cell line. Although not as

active as in the parental cell line SK33 and especially SK51 showed moderate activity. At this

stage it is unclear if the concentration of SK33 (&SK51) required to inhibit the acquired

resistance phenotype may be achievable in vivo, but their increased metabolic stability,

resistance to oxidative metabolism and detoxification together with increased solubility may

translate to high levels of drug in circulation. Additionally and of clinical importance, SK51

showed increased activity over enzalutamide in the LNCaP/BicR cells.

It is rare that an inhibitor compound such as an anti-androgen can be tested in a biological

system where there is a highly specific real-time reporter for its target protein activity -

integrated into the host genome. Utilizing an LNCaP cell line with an integrated AR-driven

luciferase reporter (30) we analyzed the effect of these compounds directly on the

transactivation function of the AR. SK33 resulted in a stronger inhibition of AR-mediated

gene activation of both the endogenous gene PSA and the transgene luciferase, than that

mediated by bicalutamide. SK33 could also competitively inhibit the AR in the presence of a

strong synthetic androgen (R1881), whilst enobosarm itself had a mildly stimulating effect.

Further testing in vivo using unique AR-reporter mice, developed by the authors (31),

showed the anti-androgen activity of these compounds in the target tissue i.e. prostate, as

well as in peripheral tissues, in real time within the same animal. Here, side effects, and

bystander effects could be monitored. SK33 showed a stronger or equal anti-androgen

effect in most tissues, when compared to bicalutamide at the same dosage concentration.

But, interestingly we observed that the effects of SK33 upon leg muscle AR activity was

stimulatory, and the effects in the spleen were minimal.

The anabolic effects of enobosarm have been well documented, although its mechanism of

action in specifically stimulating skeletal muscle AR and not prostate or gonadal AR remains

elusive. Since SK33 is a modified analog of enobosarm the effects on skeletal muscle was

13

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interesting to observe, especially given its simultaneous high inhibitory activity in the

prostate and other tissues. The rational addition of bis-trifluoromethyl groups into ring B of

enobosarm seems to have successfully exaggerated the SARM activity of enobosarm. The

mechanism of SARM-like compounds remain relatively unknown but locally expressed AR

tissue-specific cofactors have been postulated to govern the activity of AR modulating

compounds such as SARMs. Although our results are limited to the mouse leg skeletal

muscle, the use of such a compound in prostate cancer patients may ease side effects such

as muscle wastage and fatigue, whilst maintaining prostate AR inhibition. The effects on

other muscle tissues e.g. the AR-responsive levator ani muscle, in the mouse was

unavailable.

It should also be noted that SK33 showed very potent inhibitory effects in the brain - this

may reflect the increased lipophilicity of the fluorinated compounds in traversing the blood-

brain barrier or in fact their increased anti-androgen potential. The CNS-specific effects

could not be determined in the mouse model, but no deleterious effects on motor function

or behavior was observed. The effects in humans could well correlate with CNS-related side

effects.

Finally, within a developing hyperplasia / adenocarcinoma in situ in mice with homozygous

PTEN deletion we observed that SK33 was equal to or better than bicalutamide in reducing

AR activity.

Conclusions

We demonstrate that structure-based design can efficiently engineer second-generation AR

antagonists that have clinical potential to circumvent anti-androgen resistance by complementing

receptor mutations at the molecular level. In principle, there is a limited number of mutations that

can cause an AR antagonist to function as an AR agonist, therefore, it is conceivable that such

approaches may ultimately lead to the development of anti-androgens that resist mutations that

cause anti-androgen withdrawal syndrome.

Utilising novel in vitro and in vivo screening techniques and models, ideally suited to analyze and

evaluate novel antiandrogens, we have identified a novel anti-androgen candidate SK33. This bis-

trifluoromethyl-containing enobosarm analog, displays better activity than bicalutamide in vitro with

increased efficacy against acquired anti-androgen resistance. Importantly, SK33 has comparable

activity in vivo, but maintains a degree of tissue selectivity associated with its SARM – enobosarm

parental molecule. The increased stability and potential differential side effect profile of these

14

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

molecules may make them useful drugs for prostate cancer treatment.

15

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Materials and Methods

All chemicals were purchased from Sigma Aldrich (Gillingham, United Kingdom) or Alfa Aesar

(Heysham, United Kingdom) and were used without further purification. All reactions were

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 spectrometer at 25 C. Chemical shifts (δ)

are expressed in parts per million (ppm) and coupling constants (J) are given in hertz. The following

abbreviations are used in the assignment of NMR signals: s (singlet), bs (broad singlet), d (doublet), t

(triplet), q (quartet), m (multiplet), dd (doublet of doublet), dt (doublet of triplet), td (triple doublet),

m (multiplet). Mass spectrometry was run on a Bruker Micromass system in electrospray ionization

mode. Thin Layer Chromatography (TLC): precoated aluminium backed plates (60 F254, 0.2 mm

thickness, Merck) were visualized under both short and long wave UV light (254 and 366 nm). Flash

column chromatography was carried out using silica gel supplied by Fisher (60 A, 35-70 µm). Purity

of prepared compounds was determined by Analytical High-Performance Liquid Chromatography

(HPLC) analysis using either a ThermoScientific or a Varian Prostar system. All compounds tested in

biological assays were > 95% pure. HPLC using the eluents water (eluent A) and acetonitrile (eluent

B). Column; Varian Pursuit, 150 mm × 4.6 mm, 5.0 μm. Flow rate; 1.0 mL/min, gradient 30 min 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) was added over the course of 10 minutes to a stirring solution

of the different aniline 1a, b (3.4 mmol) in N,N-dimethylacetamide (10 mL) at r.t. for 3 h. After the reaction

was complete, the mixture was diluted with ethyl acetate (100 mL), extracted with sat. aq. NaHCO

3

solution

(

3x25 mL) and with cold brine (4x50 mL). The organic layer was dried over Na SO and the solvent was

2

4

removed at reduced pressure. The crude 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) in DCM (7 mL) was added 30% hydrogen peroxide

(3.6 mL, 32.03 mmol). The reaction mixture was put in a water bath at r.t. and trifluoroacetic anhydride (3.7

mL, 26.7 mmol) was added slowly to the mixture, which was then stirred for 24 h. The reaction mixture was

transferred to a separating funnel using DCM (30 mL). The organic layer was washed with distilled water (20

mL), sat. aq. Na

2 2 3 3 2 4

S O (4x20 mL), sat. aq. NaHCO (3x20 mL) and brine (20 mL), dried over Na SO and

concentrated at reduced pressure.

General method for the preparation of compounds SK33 and SK51

To a mixture of NaH (60% in mineral oil, 0.050 g, 1.23 mmol) in anhydrous THF (2 mL) at 0 °C under Ar

atmosphere was added a solution of the 3,5-bis-trifluoromethylphenol 5 (1.11 mmol) in 1 mL of anhydrous

16

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

THF. This mixture was stirred at r.t. for 20 min. A solution of the different intermediates (4a,b) (0.74 mmol) in

anhydrous THF (3 mL) was added slowly. The reaction mixture was stirred at r.t. o.n. The mixture was then

2 4

diluted with ethyl acetate (30 mL), washed with brine (15 mL) and water (30 mL), dried over Na SO and

concentrated under vacuum. The crude residue was purified by flash column chromatography.

3

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

SK33)

Purified by flash column chromatography eluting with n-hexane/EtOAc 100:0 v/v increasing to n-hexane/EtOAc

(

9

0:10 v/v. Obtained in 85 % yield as a white solid.

1

H NMR (CDCl

3

-d

1

) δ 9.20 (bs, 1H, NH), 8.16 (d, J = 2 Hz, 1H, ArH), 8.00 (dd, J = 2, 8.5 Hz, 1H, ArH), 7.82 (d, J =

), 4.13 (d, J = 9 Hz, 1H, CH2), 3.49

8

.5 Hz, 1H, ArH), 7.53 (s, 1H, ArH), 7.36 (s, 2H, ArH), 4.59 (d, J= 9 Hz, 1H, CH

2

1

9

13

(

bs, 1H, OH), 1.67 (s, 3H, CH3); F NMR (CDCl

3

-d

1

) δ -62.24, -63.09; C NMR (CDCl

3

-d

1

) δ 172.03 (C=O), 158.28

2

1

(

ArC), 141.29 (ArC) , 135.92 (ArCH), 134.18 (q, JC-F = 32.6 Hz, ArC), 133.16 (q, JC-F = 33.3 Hz, ArC), 122.94 (q, JC-

1

3

F

= 264.5 Hz, CF

3

), 122.05 (q, JC-F = 272.6 Hz, CF

3

), 121.84 (ArCH), 117.33 (q, JC-F = 5.1 Hz, ArCH), 115.56 (q, JC-F

), 23.04

3

=

3.6 Hz, ArCH), 115.38 (ArC), 115.14 (q, JC-F = 3.8 Hz, ArCH), 104.92 (ArC), 75.83 (COH), 73.10 (CH

2

3

(CH ). MS [ESI, m/z]: 501.1 [M+H], 523.1 [M+Na]. HPLC: retention time = 25.18 min.

3

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

(

SK51)

Purified by flash column chromatography eluting with n-hexane/EtOAc 100:0 v/v increasing to n-hexane/EtOAc

9

0:10 v/v. Obtained in 78 % yield as a white solid.

1

H NMR (CDCl

3

-d

1

) δ 9.68 (bs, 1H, NH), 8.76 (d, J = 9 Hz, 1H, ArH), 8.59 (d, J = 3 Hz, 1H, ArH), 8.47 (dd, J = 2.5,

), 3.25

) δ 172.02 (C=O), 158.15

9

.5 Hz, 1H, ArH), 7.56 (s, 1H, ArH), 7.36 (s, 2H, ArH), 4.59 (d, J= 9 Hz, 1H, CH2), 4.15 (d, J = 9 Hz, 1H, CH

2

1

3

13

(bs, 1H, OH), 1.69 (s, 3H, CH

3

); F NMR (CDCl

3

-d

1

) δ -61.59, -63.06; C NMR (CDCl

3

-d

1

2

(

ArC), 143.17 (ArC), 140.38 (ArC), 133.16 (q, JC-F = 33.8 Hz, ArC), 133.03 (q, JC-F = 32.8 Hz, ArC), 128.38 (ArCH),

1

3

1

23.22 (q, JC-F = 268.3 Hz, CF

3

), 122.75 (q, JC-F = 271.3 Hz, CF

3

),122.62 (ArCH), 122.46 (q, JC-F = 6.3 Hz, ArCH),

3

15.67 (q, JC-F = 3.8 Hz, ArCH), 115.11 (ArCH), 75.86 (COH), 73.93 (CH

2

), 22.94 (CH

3

). MS [ESI, m/z]: 521.1

[

M+H], 543.1 [M+Na]. HPLC: retention time = 26.42 min.

Cell culture

All cell lines were purchased LGC Standards (Teddington, UK) and used within 6 passages, and

subjected to regular mycoplasma screening (Geneflow PCR test, Lichfield, UK). LNCaP cells were

o

maintained at 37 C, 5% CO

2

in RPMI medium with 10% foetal bovine serum (First Link Ltd, Brierley

Hill, U.K.). PC3, VCaP and Du145 and mouse Pten null cells were maintained in DMEM medium

Sigma) with 10% foetal bovine serum (First Link UK,). All media was supplemented with 2mM L-

(

glutamine, 100units/ml penicillin, 100mg/ml streptomycin (Sigma). For hormone depletion

experiments, 72 hours before androgen exposure, medium was replaced with ‘starvation medium’

17

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

consisting of phenol red-free RPMI (or DMEM) medium, supplemented with 5% charcoal-stripped

foetal bovine serum (First Link UK). All cell lines were obtained from the ATCC cell bank.

MTT Assay

The MTT assay was used as a cell viability assay for the all the cell lines listed using the anti-

4

androgen compounds. Briefly, cells (5 × 10 cells/ml) were seeded into 96-well plates (200ul/well),

allowed to attach and grow for 24hr and subsequently treated with varying concentrations of

antiandrogens (0–100 μM) for 96 hours. Optimal seeding densities were premeasured for linear

growth over the 96hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,

o

5

mg/ml in PBS) was added to a final concentration of 0.5mg/ml for 4 h at 37 C. After four hours,

purple formazan crystals, formed by mitochondrial reduction of MTT, were solubilized in acidified

isopropanol (200μl/well) and the absorbance was read at 570 nm after 10 min incubation. Percent

inhibition of viability was calculated as a % of untreated control and the cytotoxicity / cell growth

inhibition was expressed as IC50

.

Luciferase Assays

Cells were washed and lysed in reporter lysis buffer (Promega). Lysate was mixed with D-luciferin

substrate (Promega) and light emission measured using the Promega Glomax multi luminometer.

For live cell imaging D-luciferin substrate was added directly into the cell media and plates were

imaged on a Syngene G:Box imager.

Tissue was pulverized by grinding in liquid nitrogen and then completely homogenised, using a

microfuge pestle, in reporter lysis buffer with protease inhibitors (Promega). Lysate (20µl) was mixed

with luciferin substrate (20µl) and light emission measured using the Promega Glomax multi

luminometer. Light emission was then normalised to protein content as measured by a Bradford

Assay.

RNA extraction and RT-PCR

Total RNA samples were prepared using Trizol reagent (Sigma) and converted to cDNA using the

GoScript™ Reverse Transcription System (Promega).

Q-PCR

Reactions were performed in triplicate on cDNA samples in 96-well optical plates on an ABI Prism

StepOne System (ThermoFisher, U.K.). Reactions consisted of 2 μl cDNA, 7 μl PCR-grade water, 10 μl

2

× TaqMan Universal PCR Master Mix (Applied Biosystems), 1 μl Taqman specific assay probes

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Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

(Applied Biosystems) for PSA, TMPRSS2, KLK2, and L19. Parameters were: 50°C for 2 min, 95°C for 10

min, 40 cycles of 95°C for 15 sec and 60°C for 1 min. Data was recorded using Sequence Detector

Software (SDS version 2.3; PE Applied Biosystems). Levels were normalised to GAPDH, -actin and

RPL19.

Animal experiments

All mouse procedures were performed in accordance with the UK Animals (Scientific Procedures) Act

1

986 under Home Office license. ARE-Luc strains were made and available ‘in house’ (31), and

loxp/loxp

Pten

;Pb-Cre4 were obtained from The Jackson Laboratory.

Luciferase imaging

Anaesthetized mice (3% isofluorane with O carrier, Abbott Animal Health UK) were injected i.p. or

2

s.c. with D-luciferin (Caliper Life Sciences Ltd, Runcorn, UK) at 150 mg/kg, 10 min before imaging.

Light emission from luciferase was detected by the IVIS Imaging System 100 series (Xenogen

Corporation), and overlaid as a pseudocolour image with reference scale, upon a greyscale optical

image.

For ex vivo imaging, mice were sacrificed 10 min after luciferin injection, and immediately dissected.

Target organs were rinsed briefly in PBS and placed under the bioluminescent camera. Tissues were

then collected for luciferase assays, RNA extraction or fixed in 10% formaldehyde before wax

embedding and sectioned using standard procedures.

Cell cycle analysis (FACS)

Cells were grown on 10cm dishes for 24-96ꢀh ± treatments. Cells were then trypsinized, washed

twice in PBS, fixed in 70% ethanol at 4ꢀ°C, were stained with 5ꢀg/ml propidium iodide and RNA

removed using 50ꢀmg/ml RNase A. FACS analysis was carried out using a FACS Calibur (Beckton-

Dickinson, Oxford, UK), using linear scale representation of forward and side scatter during flow

analysis, as well as DNA content. Single cells were gated and the cell cycle profiles measured using

FCS Express4 software, using the built in kinetics & cell cycle analysis best-fit model for cell cycle

phase identification. A total of 10ꢀ000 events were measured per sample.

Acknowledgments

The authors would like to pay a tribute to Professor Christopher McGuigan (the Drug Hunter) for all

his contributions for the success of this project. Professor McGuigan was an exceptional and

19

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

dedicated scientist who has remarkable achievements in the anticancer and antiviral drug discovery

field.

The authors would like to thank Cyprotex (U.K.) for performing the metabolic stability, PPB %

recovery, hERG inhibition assays as an outsourced service. We would also like to thank our funders -

The Welsh Government (A4B-Academic Expertise for Business grant), The Cardiff University – Peking

University Cancer Institute, Prostate Cancer UK, Cancer Research UK and the Harvey Spack memorial

Fellowship (GSA).

20

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tables:

Metabolic stability

t1/2 mins)

Plasma protein

binding assay (PPB)

%

%hERG inhibition at

25μM

(

Bicalutamide

SK33

214

n.d.

96 *

7.64

1930

94.7

Table 1. t1/2 calculated in human liver microsomes; SK33 is metabolically stable with no loss

of parent compound detected for the duration of the assay. Plasma protein binding (PPB)

assay; results are expressed in terms of mean % bound from duplicate experiments.

Cardiotoxicity expressed in terms of % hERG inhibition at 25 μM compound concentration.

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Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure legends

Figure 1.

A, Chemical structure of the clinically used non-steroidal antiandrogens; flutamide,

hydroxylflutamide, nilutamide, bicalutamide and enzalutamide showing rings A and B. B,

Chemical structure of enobosarm (left), SK33 and SK51 (right). C, Chemical synthesis of SK33

and SK51 compounds, reagents and conditions: i) DMA, rt, 24h, ii) H2O2, DCM, rt, 2h, iii)

NaH, THF, rt, 24 h.

Figure 2.

(

A, B) X-ray structure of enobosarm (red-carbons) co-crystallised with (AR wt) in the agonist

conformation showing the orientation the distal ring B extending away from Helix 12 (H12)

towards Trp741. (C, D) Overlaid predicted binding modes of SK33-R (pink carbons), SK33-S

(turquoise carbons) and enobosarm (grey carbons) inside the AR-LBD showing the

orientation of ring B with the bis-CF3 groups inducing geometric bulk keeping Helix 12 in the

antagonist conformation (open) even upon the development of adaptive (resistant) bulk

reducing mutations.

Figure 3. Enobosarm analogues SK33 and SK51 show potent and selective activity in AR

positive prostate cancer cells.

MTT cytotoxicity assays of LNCaP cells treated with increasing doses of enobosarm or SK33

(

A) or Bicalutamide, SK33 and SK51 (B) at 0-100M. MTT cytotoxicity assays of PC3 cells

treated with increasing doses of enobosarm or SK33 (C) or Bicalutamide, SK33 and SK51 (D)

at 0-100M for 96hr. E, Q-PCR analysis of a panel of prostate cancer cells for AR expression.

Data represents the mean of three independent experiments. Data was normalised to

GAPDH, -actin and RPL19 housekeeping 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 of LNCaP

cells treated with 10M bicalutamide, enzalutamide, SK33, SK51 or vehicle control for

4

8hours, and analysed 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M bicalutamide, enzalutamide, SK33, SK51 or vehicle control for 48hours, and

24

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

analysed for S phase (left hand side). Histograms showing DNA content (PI fluorescence) of

treated PC3 cells (right hand side). 10,000 gated cells were measured for each experiment.

Figure 4. SK33 and SK51 inhibit AR-mediated transcriptional activity in prostate cancer

cells.

A, Schematic diagram of the activation of the synthetic androgen reporter construct

integrated into the LNCaP/ARE-Luc cell line. Ligand bound AR activates the expression of the

luciferase reporter gene which in the presence of luciferin substrate gives off

bioluminescence directly proportional to AR activity. B, Q-PCR analysis of the endogenous

androgen induced gene PSA and the transgenes luciferase and G418 (Neomycin resistance

gene - aminoglycoside 3‘-phosphotransferase) in LNCaP/ARE-Luc cells. Cells were grown for

7

2hours in charcoal stripped FCS and then treated with 0-10 nM R1881. Data represents the

mean of three independent experiments. Data was normalised to GAPDH, -actin and

RPL19 housekeeping genes. C, Q-PCR analysis of PSA gene expression in LNCaP cells grown

in charcoal stripped medium for 72hours and then treated with R1881 in the presence of

increasing concentrations of bicalutamide or SK33 (0-10 M) or enobosarm (D). Data

represents the mean of three independent experiments. Data was normalised to GAPDH, -

actin and RPL19 housekeeping genes. E, Grey scale image of bioluminescence from live

LNCaP/Luc cells treated with enobosarm, SK33 and bicalutamide (0-100 M) for 24 hours. F,

Line graph indicating bioluminescence activity measurements from LNCaP/Luc cells treated

with increasing concentrations of bicalutamide, enobosarm and SK33 for 24 hours. ** =

p>0.01.

Figure 5. SK33 is active in LNCaP cells with acquired bicalutamide resistance.

A, Bar graph indicating the relative expression levels of AR in LNCaP and LNCaP/BicR cells.

Data represents the mean of three independent experiments. Data was normalised to

GAPDH, -actin and RPL19 housekeeping genes. B&C, MTT cytotoxicity assays of LNCaP or

LNCaP/BicR cells treated with increasing concentrations of bicalutamide (B) or enzalutamide

25

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

(

C) for 96 hours. D, MTT cytotoxicity assays of LNCaP/Bic20 cells treated with increasing

concentrations of bicalutamide, SK33 and SK51 for 96 hours.

Figure 6. SK33 inhibits AR transcriptional activity in vivo and in prostate intraepithelial

neoplasia (PIN).

A, Bioluminescent imaging of live ARE-Luc mice pre and post treatment with bicalutamide or

SK33 (or DMSO vehicle) for 24 hours. Image represents a grey scale image overlaid with

pseudo-colour bioluminescent activity data (scale represents flux rate of photons/sec/cm2).

B, grey scale image of an ARE-Luc mouse, white circles represent the three regions of

interest measurements for each mouse. C, Bar graph representing relative bioluminescent

data emanating from the three regions of interest - head, abdomen and gonadal regions.

Data is normalised to mouse bioluminescence pre-treatment with bicalutamide, SK33 (50

mg/kg) or DMSO vehicle. D, Q-PCR analysis of luciferase expression from various body

tissues as indicated. Data is presented as luciferase / housekeeping genes (GAPDH, RPL19

and -actin). E, Bar graph indicating relative luciferase enzymatic activity from various

tissues from ARE-Luc mice treated with either bicalutamide, SK33 or DMSO. F, Q-PCR

ko

analysis of luciferase expression from the mouse PTen prostate tissues. Data is presented

as luciferase / housekeeping genes (GAPDH, RPL19 and -actin). G, MTT assay of PTen null

mouse prostate cells treated with increasing doses of bicalutamide, enzalutamide and SK33

for 96hours. *= p value 0.05, ** = p value 0.01.

26

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 1.

A.

A

B

B.

CF ₃

CN

O

B

A

F ₃C

O

N

H

CF ₃

NC

CN

HO

O

B

A

R ational Des ign

SK33

O

N

H

^C^F3

CF ₃

HO

NO ₂

O

B

A

Enobosarm

F ₃C

N

H

HO

SK51

CF₃

C.

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2.

A.

B.

Helix 12

C.

D.

Helix 12

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3.

A.

LNCaP

B.

Enobosarm

SK33

Bicalutamide

SK33

SK51

1

00

100

50

0

5

0

IC50

Eno = 40uM

SK33 = 0.2uM

IC50

Bic = 3.0uM

SK33 = 0.2uM

SK51 = 0.7uM

-

1

0

1

2

-1

0

1

2

Log10 concentration (µM )

Log₁₀concentration (µM )

PC3

D.

C.

PC3

Bicalutamide

SK51

SK33

Enobosarm

SK33

1

00

1

00

5

0

5

0

IC50

Eno = 87uM

SK33 = 3.2uM

IC50

Bic = 30uM

SK33 = 3.2uM

SK51 = 5.0uM

-

1

0

1

2

-

1

0

1

2

Log10 concentration (µM )

E.

F.

IC50

1

0

.5

.0

.5

.0

LNCaP

VCaP

Du145

PC3

0

.1

1

10

100

1000 µM

Eno

Bic

SK51

SK33

C ell L in e

G.

Untreated

SK33

2

1

5

0

5

0

5

0

t

O

c

z

3

1

n

i

3

5

S

n

U

B

E

K

M

S

D

Fluorescence (PI)

C o m p o u n d

H.

2

5

Untreated

SK33

2

1

0

5

0

5

0

t

O

c

z

3

1

n

i

3

5

S

n

U

B

E

K

M

S

D

Fluorescence (PI)

C o m p o u n d

Author Manuscript Published OnlineFirst on June 12, 2018; DOI: 10.1158/1535-7163.MCT-18-0037

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4.

A.

B.

C.

D.

*

0

- 10nM R1881

1

5

0

5

0

1

0

5

2

1

0

.0

.5

.0

.5

0

0 .0

D M SO

100uM

A

e

8

1

d

h

1

₀.5

1

0

1

₀.5

1

0

s

e

t

8

1

S

a

4

t

E

8

Enobosarm

P

r

a

1

+

e

G

r

e

R

f

i

t

c

n

+

Bic

SK33

u

U

L

T r e a tm e n t

Gene of interest

E.

F.

0

- 100µM

Enobosarm

SK33

Bicalutamide

Enobosarm

SK33

1

00

5

0

Bicalutamide

-

1

0

1

2

L o g₁₀co n cen tratio n (µM )

Article Doi

Novel trifluoromethylated enobosarm analogues with potent antiandrogenic activity in vitro and tissue selectivity in vivo

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

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Article abstract of DOI:10.1158/1535-7163.MCT-18-0037

Full text of DOI:10.1158/1535-7163.MCT-18-0037

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