A new avenue toward androgen receptor pan-antagonists: C2 sterically hindered substitution of hydroxy-propanamides

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pubs.acs.org/jmc

A New Avenue toward Androgen Receptor Pan-antagonists: C2

Sterically Hindered Substitution of Hydroxy-propanamides

Andrea Guerrini,^†,#Anna Tesei,^‡,#Claudia Ferroni,^†Giulia Paganelli,^‡Alice Zamagni,^‡Silvia Carloni,^‡

Marzia Di Donato,^§Gabriella Castoria,^§Carlo Leonetti,^∥Manuela Porru,^∥Michelandrea De Cesare,^⊥

Nadia Zaﬀaroni,^⊥Giovanni Luca Beretta,^⊥Alberto Del Rio,^†and Greta Varchi*^,†

^†Institute for the Organic Synthesis and Photoreactivity, Italian National Research Council, Via Gobetti 101, 40129 Bologna, Italy

^‡I.R.S.T., Istituto Scientiﬁco Romagnolo per lo Studio e la cura dei Tumori, Via P. Maroncelli, 40, 47014 Meldola, Forlì, Italy

^§Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Via L. De Crecchio, 7, 80138 Naples, Italy

^∥Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Via delle Messi d’Oro, 156, 00158 Rome, Italy

^⊥Fondazione IRCCS Istituto Nazionale dei Tumori Milano, Via Amadeo, 42, 20133 Milano, Italy

S

* Supporting Information

ABSTRACT: The androgen receptor (AR) represents the

primary target for prostate cancer (PC) treatment even when

the disease progresses toward androgen-independent (AIPC)

or castration-resistant (CRPC) forms. Because small chemical

changes in the structure of nonsteroidal AR ligands determine

the pharmacological responses of AR, we developed a novel

stereoselective synthetic strategy that allows sterically hindered

C2-substituted bicalutamide analogues to be obtained. Bio-

logical and theoretical evaluations demonstrate that C2-

substitution with benzyl and phenyl moieties is a new, valuable

option toward improving pan-antagonist behavior. Among the

synthesized compounds, (R)-16m, when compared to casodex,

(R)-bicalutamide, and enzalutamide, displayed very promising in vitro activity toward ﬁve diﬀerent prostate cancer cell lines, all

representative of CPRC and AIPC typical mutations. Despite being less active than (R)-bicalutamide, (R)-16m also displayed

marked in vivo antitumor activity on VCaP xenografts and thus it may serve as starting point for developing novel AR pan-

antagonists.

1. INTRODUCTION

Prostate cancer (PC) is the second cause of cancer-related

death among the male population of Western society, and

androgen-deprivation therapy (ADT) represents a ﬁrst-line

treatment option. In spite of androgen receptor (AR)

expression throughout the various stages of PC, ADT

frequently fails,^1,2and PC progresses toward the androgen-

independent (AIPC) or castration-resistant (CRPC) forms.^3,4

Because both AIPC and CRPC express AR, therapies

decreasing the level of the receptor reduce tumor growth.⁵⁻⁷

Castration resistance is attributed to the reactivation of AR

transcriptional activity, most likely due to the receptor gene

Figure 1. Nonsteroidal androgen receptor antagonists: bicalutamide

ampliﬁcation^8,9or mutation¹⁰⁻¹³as well as AR activation by

(1), ﬂutamide (2), enzalutamide (MDV3100) (3), and ARN-509 (4).

alternative androgens^14,15or signaling eﬀectors.¹⁶⁻¹⁸Therefore,

AR represents the primary target for the treatment of PC.

Although steroidal and nonsteroidal AR antagonists are used

for the treatment of PC, nonsteroidal antagonists, such as

bicalutamide and ﬂutamide (Figure 1), are considered to be

eﬀorts have been undertaken to develop new AR antagonists

that trigger the death of PC cells either at the early stage of

androgen dependence or at the later stage of androgen

independence.²²

more eﬃcacious because of their selective blockade of androgen

action and because they present fewer side eﬀects.¹⁹⁻²¹Clinical

evidence, however, suggests that classical AR antagonists are

ineﬀective for the treatment of advanced PC. Therefore, many

Received: March 31, 2014

Published: August 14, 2014

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a

Scheme 1. Synthesis of (2S,5R)-7a−n Dioxolanone Bromides

a

Reagents: (a)³⁴LHMDS, THF, HMPA, RX, −78 to −40 °C, 6h; (b) 2-mercapto pyridine-N-oxide, CBrCl₃, DCC, reﬂux, 2 h; (c)³⁵pivalaldehyde,

H₂SO₄, pentane, reﬂux; (d) LHMDS, THF, HMPA, CH₂I₂, −78 to −30 °C, 2 h.

A wide range of literature reports and data evidence that PC

can spontaneously acquire androgen receptor gain-of-function

mutations rather than simply acquiring mutations that preclude

inhibitor binding. This behavior underscores the crucial

challenge of overcoming AR resistance mechanisms.²³This is

also conﬁrmed by the observation that even very small

structural modiﬁcations in nonsteroidal AR ligands greatly

alter the nature of the receptor−ligand interaction and lead to

quite divergent pharmacological responses (i.e., antagonist vs

agonist activity). Agonists and antagonists, for instance,

diﬀerently modulate the interaction of AR with transcriptional

machinery by positioning the AR-H12 helix in such a way that

it seals (agonists) or opens (antagonists) the AR ligand binding

pocket.²⁴⁻²⁷

Structure−activity relationship (SAR) studies on bicaluta-

mide-like molecules have shown that electron-poor A rings,

such as p-CN (or NO₂), as well as m-CF₃substitutions are

required for a strong ligand−AR binding.²⁴Moreover, SAR

studies on novel derivatives with antagonistic activity on both

the wild-type and mutant AR (pan-antagonists) have shown

that ﬁne tuning the B ring size²⁸and properly choosing the

moiety connecting the B ring with the bicalutamide backbone,

e.g., -SO₂- vs -O-,²⁹pushes the AR-H12 helix and induces an

antagonistic conformation even on mutated AR.

With the aim of obtaining more eﬀective AR pan-antagonists,

we synthesized a novel class of C2-substituted bicalutamide-like

molecules by inserting molecular fragments with diﬀerent steric

and electronic properties.³⁰The available procedures for the

synthesis of such derivatives are so far unsuitable. In fact, the

challenging coupling reaction with the very electron-poor

aniline moiety (A_ring-NH₂, Figure 1) signiﬁcantly hampers the

possibility of achieving C2-substituted derivatives. To our

knowledge, only methyl³¹and triﬂuoromethyl³²groups have

been successfully introduced at this position. To this end, we

developed a novel, straightforward, and diastereoselective

methodology able to aﬀord a large variety of C2-substituted

compounds, starting from commercially available and inex-

pensive materials. Among the novel compounds, we have

successfully identiﬁed a new structure, (R)-16m, which is

eﬀective in vitro against various PC-derived cell lines and shows

remarkable in vivo antitumor activity with respect to that of

casodex. Collectively, our results indicate that (R)-16m is a

promising lead derivative for the development of more potent

AR pan-antagonists.

2. RESULTS AND DISCUSSION

2.1. Chemistry. Exploiting the Seebach’s “self-regeneration

of stereocenters” synthetic principle (SRS),³³we synthesized

variously substituted (2S,5S) dioxolanone acids (6a−m)

starting from commercially available and inexpensive (S)-

malic acid, resulting in good yields and diastereoselectivities

(>96%).³⁴Subsequent decarboxylative bromination aﬀorded

the corresponding bromides (2S,5R)-7a−m (Scheme 1). On

the other hand, compound (2R,5R)-7n, bearing a more

sterically demanding phenyl group at the C5 position, was

synthesized as homochiral material by acetalization of (R)-

mandelic acid with pivalaldehyde³⁵followed by addition of

diiodomethane to the corresponding enolate (Scheme 1).

The (2S,5R) dioxolanone derivatives 7a−n are versatile

starting materials for further functionalization. In particular, the

reaction of compounds (2S,5R)-7a,b with hydrochloric acid (6

N) quantitatively aﬀorded the corresponding α-hydroxyl acids

(R)-8a,b that were then coupled^31,36with 4-cyano-3-triﬂuor-

omethylaniline (9) to aﬀord the corresponding amides (R)-

10a,b in good yields. The subsequent nucleophilic substitution

with 4-ﬂuorothiophenol followed by oxidation of the sulfur to

sulfone aﬀorded (R)-12a, namely, (R)-bicalutamide and (R)-

12b (Scheme 2).

Alternatively, a one-pot, two-step reaction of bromides

(2S,5R)-7c−i with NaOH in the presence of 4-ﬂuorothiophe-

nol aﬀorded the corresponding α-hydroxyl acids (R)-13c−i in

very good yields. Afterward, methyl esters (R)-14c−i were

obtained by reaction of (R)-13c−i with a (trimethylsilyl)-

a

Scheme 2. Synthesis of Derivatives (R)-12a and (R)-12b

a

Reagents: (a) 6 N HCl, reﬂux, 4 h; (b) SOCl₂, DMA, from −10 °C to

rt, 16 h; (c) NaH, THF, p-F-C₆H₄SH, rt, 3−5 h; (d) mCPBA, CH₂Cl₂,

rt, 12 h.

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a

Scheme 3. Synthesis of Novel Non-steroidal Antiandrogens (R)-16c−I

a

i

Reagents: (a) p-F-C₆H₄SH, PrOH/NaOH (1 N), rt, 20 h; (b) Me₃SiCHN₂(2 M in Et₂O), MeOH/toluene, rt, 1 h; (c) 4-amino-2-

(triﬂuoromethyl)benzonitrile (9), THF, LHMDS, HMPA, from −10 °C to rt, 13 h; (d) mCPBA, CH₂Cl₂, rt, 12 h.

diazomethane ethereal solution in MeOH/toluene mixed

solvent in good isolated yields (Scheme 3). The reaction of

esters (R)-14c−i with the lithium salt of aniline 9 at low

temperature and in the presence of HMPA provided the

corresponding amides (R)-15c−i in good isolated yields.

It is worth noting that this latter coupling reaction represents

the key synthetic step of the procedure. In fact, unlike all

previously reported procedures for the synthesis of bicaluta-

mide-like compounds,³¹our methodology allows a large variety

of C2-subsituted bicalutamide analogues to be accessed in high

yields and stereoselectivities starting from inexpensive building

blocks (i.e., (S)-malic acid and (R)-mandelic acid). Finally,

oxidation of sulfur derivatives (R)-15c−i with mCPBA aﬀorded

the corresponding sulfones (R)-16c−i in quantitative yields

(Scheme 3).

Scheme 4. Synthesis of Non-steroidal Antiandrogens (R)-

16l−n

a

Whenever milder conditions were required, methyl esters

(R)-14 were prepared by an alternative protocol (Scheme 4).

First, compounds (2S,5R)-7l−n were reacted with 4-

ﬂuorothiophenol in DMF and in the presence of K₂CO₃. The

obtained cyclic sulfur compounds (2S,5R)-17l−n were then

treated with sodium methylate (1 M) at 0 °C to aﬀord the

corresponding methyl esters (R)-14l−n. Derivatives (R)-16l−n

were then obtained as described previously for compounds (R)-

16c−i in very good isolated yields (Scheme 4).

Besides studying the role of C2 substitutions, we also wanted

to evaluate the inﬂuence of the SO₂linker on the antagonistic/

agonistic eﬀect by replacing it with oxygen. In fact, Miller and

co-workers demonstrated that the nature of the linker plays a

pivotal role in controlling the ultimate antagonistic (agonistic)

eﬀect of these kinds of molecules.^29,37To this end, bromides

(2S,5R)-7c and (2S,5R)-7e were reacted with 4-cyanophenol in

DMF and in the presence of K₂CO₃. The obtained cyclic

compounds (2S,5S)-18c and (2S,5S)-18e were then treated

with sodium methylate (1 M) at 0 °C to aﬀord the

corresponding methyl esters (S)-19c and (S)-19e that, upon

reaction with the lithium salt of aniline 9 at low temperature

and in the presence of HMPA, provided the corresponding

amides (S)-20c and (S)-20e in very good isolated yields

(Scheme 5).

a

Reagents: (a) p-F-C₆H₄SH, K₂CO₃, DMF, rt, 4 h; (b) MeONa (1 M

in MeOH), THF, from 0 °C to rt, 2 h; (c) 4-amino-2-

(triﬂuoromethyl)benzonitrile (9), THF, LHMDS, HMPA, from −10

°C to rt, 13 h; (d) mCPBA, CH₂Cl₂, rt, 12 h.

The reported synthetic strategy is a combination of

dioxolanone chemistry with a novel and eﬃcacious coupling

between the methyl ester and the electron-poor aniline 9

lithium salt. This methodology allows the synthesis of

numerous novel and enantiomerically pure bicalutamide-like

derivatives in high yields.

2.2. In Vitro Biological Activity. 2.2.1. AR-Mediated

Transcriptional Activity. We ﬁrst investigated the ability of the

synthesized compounds to interfere with AR-mediated tran-

scriptional activity. Therefore, we established an ARE reporter

assay in two cell types, LNCaP and Cos-7 cell lines. LNCaP

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cells have been widely used as a model of androgen-responsive

growth³⁸and are the most representative for our studies. AR-

negative Cos-7 cells were transfected with pSG5-hAR plasmid,

which allows wild-type human AR (WT hAR) to be expressed.

Upon transient transfection, Cos-7 cells express high levels of

AR, thus mimicking the scenario of CRPC (Figure S1). This

cell model system has been widely used to evaluate the eﬀect of

androgens, growth factors, and newly emerging antiandrogen

compounds.³⁹⁻⁴¹As reported in Figures 2 and 3, all

compounds exhibited an antagonistic eﬀect on ARE-luc activity

in both LNCaP and Cos-7 cells. Importantly, the potency of

(R)-16m was higher than or similar to that exerted by (R)-

bicalutamide and by the second generation antagonist

MDV3100 (Figure S2 and S3).⁴²

Scheme 5. Synthesis of Non-steroidal AR Ligands (S)-20c

and (S)-20e

a

Reagents: (a) p-CN-C₆H₄OH, K₂CO₃, DMF, 100 °C, 12 h; (b)

MeONa (1 M in MeOH), THF, from 0 °C to rt, 2 h; (c) 4-amino-2-

(triﬂuoromethyl)benzonitrile (9), THF, LHMDS, HMPA, from −10

°C to rt, 13 h; (d) mCPBA, CH₂Cl₂, rt, 12 h.

No cytotoxicity was evidenced for (R)-16c−n derivatives

toward either LNCaP and Cos-7 cells, thus indicating that the

results obtained in the gene transcription assay were not a

Figure 2. Eﬀect of hydroxy-propanamide derivatives on AR-mediated gene transcription in LNCaP cells. Cells were co-transfected with ARE-luc

3416 reporter gene and β-galactosidase (β-gal) plasmid and stimulated with 10 nM R1881 in the absence or presence of the indicated compounds

for 24 h. After treatment, samples were analyzed for luciferase activity. The results are expressed as fold induction with respect to untreated cells and

represent the mean SD of three independent experiments.

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Figure 3. Eﬀect of hydroxy-propanamide derivatives on gene transcription in hAR-expressing Cos-7 cells. Cells were co-transfected with pSG5 or

pSG5-hAR encoding plasmids together with ARE-luc 3416 and β-galactosidase plasmids, and then stimulated with 10 nM R1881 in the absence or

presence of the indicated compounds for 24 h. After treatment, samples were analyzed for luciferase activity. The results are expressed as fold

induction with respect to the untreated cells and represent the mean SD of three independent experiments.

result of reduced viability mediated by drug exposure (Figure

S4 and data not shown).

144 h consecutively. (R)-Bicalutamide was more eﬀective in

AR-mutated LNCaP cells (IG₅₀= 1.8 μM) than in WT AR-

overexpressing LNCaP-AR cells. Conversely, MDV3100

showed a similar eﬀect in both cell lines, while reaching an

IG₅₀value only in LNCaP-AR cells (IG₅₀∼ 18.9 μM).

All of the compounds inhibited cell proliferation at a

concentration lower than that of the plasma peak level of

casodex in both cell lines (Table S1). In addition, (R)-16f, (R)-

16g, and (R)-16h exhibited a cytocidal eﬀect at the highest

concentrations tested (LC₅₀values ranging from 16.2 to 19.7

μM) in LNCaP cells only. Conversely, the three compounds

showed cell killing activity toward LNCaP-AR cells without

reaching LC₅₀values (Figure 4A and Table S1).

To gain further insight into the mechanisms of antitumor

activity, an apoptosis assay was performed on LNCaP and

LNCaP-AR cells. TUNEL assay showed strong cell death

induction in both cell lines, ranging from 91.9 to 99.8%, after

144 h exposure to (R)-16f, (R)-16g, and (R)-16h. Conversely,

(R)-16m scantly aﬀected the viability and apoptosis of LNCaP

and LNCaP-AR cells (Figures 5 and S7). This behavior

underpins the selective AR-mediated action of (R)-16m and as

well as its lower extent of oﬀ-target activity.

The cytotoxicity of (R)-bicalutamide and (R)-16c−n

derivatives was evaluated on PC3 and DU145 cells, two AR-

negative prostate cancer cell lines derived from bone and brain

metastasis, respectively. As expected, (R)-bicalutamide did not

produce any cytotoxic eﬀect. In contrast, (R)-16c, (R)-16d,

(R)-16f, and (R)-16e derivatives aﬀected cell proliferation of

both cell lines at concentrations higher than 2 μM (Figure S8).

Furthermore, (R)-16g and (R)-16h displayed remarkable cell

Among the tested compounds, (S)-20c and (S)-20e

exhibited agonistic activity in Cos-7 cells (Figure S5) compared

with the activity of (S)-22, the most advanced and promising

nonsteroidal molecule acting as an androgen receptor agonist.⁴³

In agreement with the ﬁndings of Miller and co-workers,²⁹

these results conﬁrm the essential role of the linker bulk (i.e.,

-SO₂- vs. -O-) in controlling the antagonist/agonist behavior of

nonsteroidal AR ligands.

Because PSA is a speciﬁc marker of proliferation for PC and

is currently used to monitor disease recurrence in patients with

advanced PC, we further investigated the variations in PSA

secretion in culture medium of treated LNCaP and LNCaP-AR

cells (Figure S6). The latter are cells derived from LNCaP that

are engineered to stably express high levels of AR, a condition

considered representative of clinical AR gene ampliﬁcation

reported in 25−30% of patients with CRPC.^7−9,44With the

exception of (R)-12b, a signiﬁcant inhibition of PSA secretion

was observed in the culture medium of both cell lines treated

with all of the compounds at a concentration of 20 μM. (R)-

12b did not aﬀect the PSA level in the culture medium of

LNCaP cells, whereas it stimulated PSA secretion in the

medium of LNCaP-AR cells, thus conﬁrming the agonist

behavior of this derivative.

2.2.2. In Vitro Cytotoxicity. The cytotoxic eﬀect of (R)-16c−

n derivatives was evaluated on LNCaP and LNCaP-AR cells.

Two references, (R)-bicalutamide and MDV3100, were used.

As reported in Figure 4A,B, all of the compounds showed

cytotoxic activity toward PC cells exposed to the derivatives for

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Figure 4. Cytotoxic activity of novel hydroxy-propanamide derivatives in human PC (A) LNCaP and (B) LNCaP-AR cells. Twenty-four hours after

seeding, cells were exposed to the compounds for 144 h. The cytotoxic potency was measured using SRB assay. (R)-Bicalutamide (e.g., (R)-12a) and

MDV3100 were used as references. The mean of three independent experiments is reported.

Quiescent LNCaP cells were incubated with 10 nM [³H]R1881

in the absence or presence of excess of radio-inert compounds.

(R)-Bicalutamide was used as a reference drug. As reported for

(R)-bicalutamide, (R)-16m displaces the [³H]R1881 binding

by about 60 and 65% when used at 1 and 2 μM, respectively

(Figure 6A). Similar ﬁndings were observed using unlabeled

R1881 (see Methods), thus conﬁrming that (R)-16m

speciﬁcally binds AR.

Importantly, (R)-16m did not aﬀect the androgen-induced

nuclear import of hAR ectopically expressed in Cos-7 cells, as

was also observed when cells were treated with (R)-

bicalutamide (Figure S10).

We then evaluated the eﬀect of (R)-16m on two important

biological responses challenged by androgens in LNCaP cells:

DNA synthesis (Figure 6B) and cell motility (Figure 6C). As

Figure 5. Apoptosis induction mediated by hydroxy-propanamide

derivatives in LNCaP and LNCaP-AR cells. Twenty-four hours after

demonstrated by Figure 6B, stimulation of quiescent LNCaP

seeding, cells were treated for 144 h with the compounds (20 μM) and

cells with 10 nM R1881 increased BrdU incorporation by 4.5-

analyzed for apoptotic cell death using TUNEL assay. Data represent

fold in comparison with that of unstimulated cells. (R)-16m

the mean SD of three independent experiments.

almost completely inhibited BrdU incorporation of androgen-

treated LNCaP cells. Importantly, this eﬀect was similar/

stronger than that obtained using the same concentration range

(10−20 μM) of (R)-12a and MDV3100 (Figures 7B and S11).

A transwell assay was used to evaluate the inhibitory eﬀects on

cell migration mediated by (R)-16m. Stimulation of quiescent

LNCaP cells with 10 nM R1881 increased the migration by 3.5-

fold in comparison with that of unstimulated cells. A complete

inhibition of cell migration, similar to that produced by (R)-

bicalutamide and MDV3100, was observed when stimulated

LNCaP cells were treated with (R)-16m (Figure 6C and S12).

Lastly, a wound-healing assay was used to evaluate the

capability of (R)-16m to impair cell migration. To this end,

killing activity toward both cells lines. These data indicate a

general, modest selectivity toward AR for those compounds,

whose cytotoxicity appears to be at least partially AR-

independent. Importantly, (R)-16m displayed either no or a

modest cytotoxic eﬀect on DU145 and PC3 cell lines, and a

superimposable dose−eﬀect curve with that of MDV3100 was

observed (Figure S9). On the basis of these ﬁndings, (R)-16m,

the most promising derivative of the series, was selected for

further investigations.

Accordingly, we evaluated the ability of (R)-16m to displace

the ligand binding activity of AR in whole LNCaP cells.

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Figure 6. Biological characterization of antitumor properties of (R)-16m in LNCaP cells. (A) Displacement of the ligand binding assay by (R)-16m

in LNCaP cells. Quiescent LNCaP cells were incubated with 10 nM [³H]R1881 in the absence or presence of excess (from 0.25 to 4 μM) radio-inert

(R)-bicalutamide ((R)-bic) or (R)-16m. After 4 h of treatment, intracellular radioactivity was measured. The data represent the mean SD of the

residual binding expressed as the percent of total AR binding sites of three independent experiments; (B) quiescent LNCaP cells were seeded on

coverslips and treated for 18 h with 10 nM R1881 in the absence or presence of (R)-bic or (R)-16m (10 or 20 μM). Cells were then pulsed with

BrdU, and the amount of BrdU incorporated was evaluated by immunoﬂuorescence and expressed as the percent of total nuclei. (C) Assay on

collagen-coated transwell ﬁlters. Twenty four hours after seeding, quiescent LNCaP cells were treated with 10 nM R1881, in the absence or presence

of 10 μM (R)-bicalutamide or (R)-16m. Cells were stained with Hoechst and counted using a ﬂuorescent microscope. Data were expressed as the

relative increase in the number of migrated cells. (D) After seeding, cells were wounded and then left unstimulated, or stimulated for 24 h with 10

nM R1881, in the absence or presence of 10 μM (R)-16m. Phase contrast images are representative of four diﬀerent experiments.

quiescent LNCaP cells were wounded and allowed to migrate

in the absence or presence of 10 nM R1881. Phase contrast

images show that the wound area is signiﬁcantly reduced in

cells treated with 10 nM R1881 (Figure 6D). The treatment

with (R)-16m consistently inhibits the eﬀect induced by 10 nM

R1881.

As reported in Figure S12, (R)-16m did not aﬀect cell

motility of LNCaP cells stimulated by serum. Additionally, as

observed for (R)-bicalutamide and MDV3100, no change in

BrdU incorporation was evidenced in AR-negative DU145 cells

stimulated with serum and treated with (R)-16m (Figures S13).

These ﬁndings indicated that (R)-16m speciﬁcally targets AR

without inducing oﬀ-target eﬀects.

In order to investigate (R)-16m for possible pan-antagonistic

behavior, we tested its cytotoxic activity on PC cell lines

resistant to bicalutamide, which are thus representative of

CRPC. To this end, LNCaP-Rbic cells, a subline derived from

LNCaP and selected in our laboratories,⁴⁵which are resistant to

(R)-bicalutamide, were used.⁹In addition, the compound was

tested on CWR22Rv1 cells, a model representative of both

androgen responsiveness and androgen insensitivity,⁴⁶and on

VCaP cells, a cell model mimicking PC progression and

metastasis.⁴⁸Indeed, VCaP cells derive from a vertebral bone

metastasis of a patient aﬀected by CRPC and carry an ampliﬁed

AR gene locus while displaying a chromosomal rearrangement

in which the androgen-regulated gene TMPRSS2 is fused to the

oncogenic ERG transcription factor, an alteration found in

about half of the cases of prostate cancer.^47,48As reported in

Figure 7A,B, (R)-16m markedly inhibited cell growth of

LNCaP-Rbic and VCaP cell lines without aﬀecting cell survival,

as also conﬁrmed by the poor cell death induction observed

after 144 h exposure to the drug (Figure 7C). In comparison to

MDV3100 and (R)-bicalutamide, (R)-16m was very eﬀective in

inhibiting the proliferation of the castration-resistant cell lines

tested, thus conﬁrming its pan-antagonistic behavior.

MDV3100 showed superior activity only in VCaP cells, the

cell model overexpressing WT AR, whereas (R)-bicalutamide

was active toward only the LNCaP cell line (Figure 7 and Table

1).

We then sought to investigate whether (R)-16m interferes

with the transcriptional activity of AR by detecting PSA levels

in LNCaP and LNCaP-Rbic cells in the absence or presence of

R1881 (Figure 8). As expected, LNCaP and LNCaP-Rbic cells

exposed for 24 h to 10 nM R1881 showed signiﬁcantly (P <

0.05) increased mRNA levels of PSA (1.5- and 1.7-fold,

respectively). Unstimulated cells treated with (R)-16m showed

a signiﬁcant reduction of PSA mRNA. However, in the

presence of 10 nM R1881, a decreased ability of (R)-16m to

reduce intracellular levels of PSA was evidenced. This behavior

likely depends on competition between the two molecules for a

common binding site on the androgen receptor. Notably, for

high dose exposure, (R)-16m also inhibits R1881-stimulated

PSA expression in the hormone-refractory LNCaP-Rbic cell

line (Figure 8).

In order to compare the transcription inhibitory potency of

(R)-16m with that of (R)-bicalutamide, LNCaP and LNCaP-

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Figure 7. Cytotoxic (A, B) and apoptotic (C) activity of (R)-16m in LNCaP-Rbic cells and VCaP cells. Twenty-four hours after seeding, cells were

exposed to the compound for 144 h. Cell proliferation and apoptosis were evaluated using SRB and TUNEL assays, respectively.

Table 1. Growth Inhibition Mediated by (R)-Bicalutamide, MDV3100, and (R)-16m on a Panel of Hormone-Resistant Prostate

a

Cancer Cells

IG₅₀144 h (μM)

IC₅₀144 h (μM)

b

c

LNCaP

LNCaP-AR

n.r.

LNCaP-Rbic

VCaP

n.r.

CW22Rv1

(R)-Bic

1.8 0.06

n.r.

37.4 4.2

d

MDV3100

(R)-16m

18.9 1.89

4.9 0.08

0.61 0.039

1.42 0.063

9.7 0.81

d

1.6 0.03

6.43 0.26

5.0 1.2

a

b

Twenty-four hours after seeding, cells were exposed to drug for 144 h. The reported values are the IG₅₀expressed in micromolar; n.r., not reached.

c

d

The reported values are the IC₅₀expressed in micromolar. P < 0.05 (ANOVA) (R)-16m vs MDV3100 and (R)-bicalutamide. Data represent mean

values SD of three independent experiments.

Figure 8. Evaluation of mRNA levels of PSA after exposure to (R)-16m in LNCaP (A) or LNCaP-Rbic (B) cell lines. Twenty-four hours after

seeding, cells were treated for 24 h with (R)-16m in the presence or absence of R1881. Cells were then collected, and the extracted mRNA was

analyzed using RT-PCR. The mean SD of two independent experiments is reported (*P < 0.05). NT, not treated.

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Figure 9. Evaluation of PSA mRNA levels after exposure of in LNCaP (A) or LNCaP-Rbic (B) cells to (R)-16m or (R)-bicalutamide. Twenty-four

hours after seeding, LNCaP (A) or LNCaP-Rbic (B) cells stimulated with 10 nM R1881 were treated for 24 h with 10 and 20 μM (R)-16m or (R)-

bicalutamide. Cells were then collected, and the extracted mRNA was analyzed using RT-PCR. The mean SD of two independent experiments is

reported (*P < 0.05).

Figure 10. In vivo antitumor activity of hydroxy-propanamide derivative (R)-16m against VCaP xenografts. Mice were injected subcutaneously with

10⁶VCaP cells/mouse, and treatment started when a tumor mass (50−150 mg) was evident (day 35 after tumor cell injection). Mice were treated

orally by daily gavage at 10 mg/kg for 4 consecutive weeks with (A) vehicle, (B) casodex, (C) (R)-bicalutamide, and (D) (R)-16m. Five mice per

group were evaluated: ﬁrst mouse, white bar; second mouse, light gray bar; third mouse, dark gray bar; fourth mouse, gray striped bar; and ﬁfth

mouse, black striped bar.

Rbic cells were exposed for 24 h at 10 and 20 μM (R)-16m or

(R)-bicalutamide (Figure 9). Although the compounds

signiﬁcantly reduced the mRNA level of PSA, (R)-16m did

so more eﬃciently than (R)-bicalutamide (P < 0.05) in both

LNCaP and LNCaP-Rbic cells.

Collectively, these ﬁndings indicate that (R)-16m is very

eﬀective, more than that of (R)-bicalutamide, in reducing the

transcriptional activity of AR in PC cells. Of note, this eﬀect

was evident also in (R)-bicalutamide-resistant cell lines.

2.3. In Vivo Biological Activity. VCaP cells were selected

to perform in vivo studies on (R)-16m because they better

represent the state of androgen signaling most commonly

observed in CRPC.^47,48VCaP cells were subcutaneously

injected in the ﬂank of 5 to 6-week old CD-1 male nude

(nu/nu) mice. When the tumor mass was evident (50−100

mg), mice were randomized in groups of 5 and treated with the

compounds (10 mg/kg for 4 consecutive weeks). As shown in

Figure 10, all the mice treated with vehicle showed progressive

tumor growth (mean tumor weight of about 2500 mg at day

100). A marked increase of tumor mass in two out of ﬁve mice

treated with casodex was evidenced, whereas disease stabiliza-

tion was elicited in the remaining three mice. Three out of ﬁve

mice treated with (R)-bicalutamide evidenced a complete

response, whereas the remaining two mice showed disease

stabilization at day 50 after cell injection. The eﬃcacy of (R)-

bicalutamide further improved, with a complete response

registered in all of the treated animals at day 100 after tumor

cell implantation. No signs of tumor relapse were observed.

Although less eﬀective than (R)-bicalutamide, a marked

antitumor activity, superior to that observed in mice treated

with casodex, was evidenced in animals treated with (R)-16m.

The stabilization of the disease was observed in two out of ﬁve

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mice treated with the compound (day 60). A complete

regression (no palpable tumor) was observed in three mice,

two of which were considered deﬁnitively cured at the end of

the experiment. Finally, the treatment with (R)-16m was well-

tolerated, as no body weight loss was observed during or after

the end of treatment.

2.4. Molecular Modeling. The capacity of AR ligands to

act as agonists or antagonists has been ascribed to their capacity

to position helix α12, one of the 12 helices contained in the

secondary structure of the ligand binding domain (LBD), in

either a closed (agonists) or an open (antagonists) con-

formation in the ligand binding pocket (Figure S14).^29,49Small

chemical changes in AR ligands have been reported to

underline diﬀerent the pharmacological responses of WT AR

and its mutated forms.⁴⁹Thus, in order to better elucidate the

mechanism of (R)-16m antagonism at molecular level, we

performed molecular modeling calculations and compared

them to that of (R)-bicalutamide.

Because structural data of AR in its antagonist conformation

is not yet available, in silico techniques have been used to derive

useful molecular information.^23,49,50In particular, homology

modeling was used to derive AR models (Figure S14B) starting

from the structure of the glucocorticoid receptor⁵¹and

progesterone receptor that were recently exploited to search

for novel AR antagonists with structure-based drug design

techniques.⁵²Nevertheless, as pointed out by other groups,

diﬃculties may arise when accounting for subtle structural

diﬀerences in the antagonist conformation of the LBD when

using structures of other nuclear receptors as the template.²⁹

Several somatic mutations confer speciﬁc conformational

properties to the α12 helix that are believed to ultimately

underlie the pharmacological resistance to current antiandrogen

treatments. Because modeling the conformational transition

from agonist to antagonist forms can be a computationally

expensive task and in order to focus on the above-mentioned

mutation to gain a clearer picture of the binding mechanism of

our C2-substituted ligands, we used structures of the agonist

AR and probed their ﬁtness in the LBD and toward the initial

transition to the antagonist conformation. In particular, we

compared WT AR and two somatic mutations associated with

CRPC, T877A and W741L, to carry out modeling simulations.

To initially highlight whether the C2 substitution creates a

hindrance in the LBD of the agonist AR that would account for

the observed biological properties, we performed a rigid

docking simulation. Interestingly, the rigid docking failed to

provide a binding pose for (R)-16m in all three structures, and

the analysis obtained by overlaying the scaﬀold of this

compound with (R)-bicalutamide revealed that the side chain

of Met787 hindered the putative location of the 4-cyano-benzyl

C ring (Figure 11). The adjustment of the side chain of Met787

allowed binding pose for (R)-16m to be obtained in all

structures, as shown in Figure 12A. Rings A and B are

accommodated in two binding regions, consistent with that

mediated by (R)-bicalutamide, whereas the C ring stretches

toward a hydrophobic pocket, called the α7 pocket, formed by

helix α7 and β1. Interestingly, induced ﬁt docking (IFD)

calculations suggest a binding pose for the T877A and W741L

mutants that diﬀers from that of the WT (Figure S15), where

the rings are interchanged, so the C ring is replaced by the B

ring to bind an adjacent hydrophobic pocket protruding toward

α11 (Figures 11, 12, and S15). Remarkably, this α11 pocket has

been recently described by Sawyers et al. as a putative region

for the binding of new spirocyclic derivatives of MDV3100 that

also improve the biological proﬁles of mutated AR, in particular

the F876L mutation.²³

To gain additional insight into how (R)-16m can overcome

resistance toward AR-mutated cells, mediated by the movement

of helix α12, we modeled this compound by performing

molecular dynamics (MD) simulations.²⁹Probing the initial

displacement of helix α12 by means of MD simulations has

already been described as a useful way to elucidate the

mechanism of AR antagonism^23,49(Figure S14A,B). In our

simulations, we aimed to understand whether the C2

substitution constitutes a valuable scaﬀold optimization to

induce AR antagonism in WT and in somatic mutations that

confer resistance to current antiandrogen treatments. The

analysis of the trajectories shows that the ligand and the protein

backbones maintain a stable conformation during MD

simulations (Figure 13, blue line). On the other hand, the

amino acidic residues corresponding to helix α12 undergo

sensible movements (Figure 13, red line). These occur in all

cases after approximately 20 ns of simulation, indicating that

(R)-16m is able to exert its antagonist activity not only toward

the WT AR but also in the mutated forms, T877A and W741L.

Not surprisingly, the loop connecting helices α11 and α12

(Figure 13, green line) showed a high degree of ﬂexibility as

ﬂuctuations occurs during the entire trajectory.

3. CONCLUSIONS

This study outlines the synthesis and biological in vitro

evaluation of a novel class of bicalutamide-like molecules as

androgen receptor pan-antagonists. On the basis of the

consideration that small chemical changes in the structure of

nonsteroidal AR ligands change the molecular mechanisms

underling the pharmacological responses of AR and its mutated

forms, we developed a novel stereoselective synthetic strategy

that allows a wide range of sterically hindered C2-substituted

bicalutamide analogues to be obtained starting from inex-

pensive and commercially available compounds (i.e., (S)-malic

acid and (R)-mandelic acid). Taking into account the peculiar

behavior of the androgen receptor, which is able to rapidly

circumvent the eﬀect of new therapies, the straightforward,

diastereoselective,and high yielding synthetic protocol outlined

herein might represent an important advance in the ﬁeld of

nonsteroidal pan-antagonists. Collectively, the presented bio-

logical data demonstrate that the newly explored substitution of

the bicalutamide C2-methyl group with more demanding

benzyl and phenyl moieties can be considered a new option

toward the modulation and improvement of the agonist/

antagonist behavior of this class of molecules. Moreover, our

results conﬁrm the pivotal role of the linker group (i.e., SO₂vs

O) in determining AR antagonistic/agonistic activity. Overall,

compound (R)-16m proved to be the most promising

compound of the series, being active toward ﬁve diﬀerent cell

lines, including those bearing CPRC and AIPC typical

mutations, while being scarcely active toward androgen-

independent cell lines, e.g., PC3 and DU145 cells. Although

Figure 11. (R)-16m structure.

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Figure 12. (A) Binding modes of (R)-bicalutamide and (R)-16m in WT AR and its W741L and T877A mutants. Poses of binding of (R)-

bicalutamide were taken from the crystallographic structures, whereas poses of (R)-16m were obtained from molecular docking simulations. The

binding modes of (R)-16m show a remarkable overlay with the scaﬀold of (R)-bicalutamide, indicating that the benzyl substitution in C2 suitably ﬁts

the LBD of AR WT and its mutated forms. (B) Magniﬁed pose of the (R)-16m in the LBD of the WT androgen receptor. The C ring resulting from

the C2 substitution stretches out to a hydrophobic cavity between α7 and β1. This α7 pocket is predicted to be a valuable binding region of AR to

conceive further ligands that overcome CRPC.

Figure 13. Molecular dynamics simulation of AR in complex with compound (R)-16m (WT, left; T877A, middle; W741L, right). Starting structures

were generated from the agonist conformation and the molecular docking poses of (R)-16m. Blue lines correspond to the root-mean-squared

deviation (RMSD) of the backbone, red lines, to the movement of helix α12 (residues 893−907), and green lines, to the movement of the loop

bridging the helices α11 and α12 (residues 885−892). After approximately 20 ns of simulation, the ligand induce a ﬂuctuation of helix α12 in all

cases, indicating the capability of the compound to act as an antagonist of AR.

peak, δ = 77.0 ppm), CD₃OD (δ = 49.0 ppm), etc. as the internal

standard. Optical rotation was detected on an ADP220 automatic

polarimeter from Bellingham & Stanley Limited. Mass spectra were

measured in positive mode electrospray ionization (ESI). ESI-HRMS

were acquired on an Agilent Dual ESI Q TOF 6520, in positive-ion

mode, using methanol. TLC was performed on silica gel 60 F254

plastic sheets. Column chromatography was performed using silica gel

(35−75 mesh). Purity of prepared compounds was determined by

HPLC-UV analysis (Waters 600 HPLC connected with photodiode

array detector 996). All compounds tested in biological assays were

>95% pure. Purity of intermediates was >90%, unless otherwise stated.

4.2. Synthetic Procedures. 4.2.1. General Procedure for the

Synthesis of (2S,5R)-5-(Bromomethyl)-2-(tert-butyl)-5-substituted-

1,3-dioxolan-4-one 7a−m. A solution of dicyclohexylcarbodiimide

(DCC) in CBrCl₃(2.4 equiv) was added to a suspension of (2S,5R)-6

(1 equiv) and 2-mercapto pyridine-N-oxide (1.4 equiv) in CBrCl₃kept

under reﬂux. The addition was performed over 30 min; afterward, the

reaction mixture was stirred under reﬂux for 2 h. After cooling, the

solvent was removed under reduce pressure, and the crude material

was puriﬁed by silica gel column chromatography. Characterization

exempliﬁed by (2S,5R)-7m: eluent: cHex/Et₂O, 7:2; yield = 88%;

less eﬀective than (R)-bicalutamide, (R)-16m exhibited a

marked antitumor activity, superior to that observed when

mice were treated with casodex.

Collectively, the results of induced ﬁt docking suggest that

the large hydrophobic region comprising the α7 and α11

pockets is highly relevant for the binding of ligands to WT AR

and its mutated forms and that C2-substituted molecules may

represent a viable strategy toward the optimization of novel

antiandrogens able to circumvent resistance to CRPC.

4. EXPERIMENTAL SECTION

4.1. General Chemistry Methods. All solvents and reagents were

used as obtained from commercial sources unless otherwise indicated.

All reactions were performed under a nitrogen atmosphere unless

1

otherwise stated. The H and ¹³C NMR spectra were recorded on a

1

Varian spectrometer operating at 400 MHz for H and 100 MHz for

¹³C. Deuterated chloroform was used as the solvent for NMR

experiments, unless otherwise stated. ¹H chemical shifts values (δ) are

referenced to the residual nondeuterated components of the NMR

solvents (δ = 7.26 ppm for CHCl₃and δ = 3.31 ppm for CHD₂OD,

etc.). The ¹³C chemical shifts (δ) are referenced to CDCl₃(central

[α]²_D⁰+39 (c 0.5, CHCl₃); H NMR (400 MHz, CDCl₃) δ 7.64−7.62

1

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(m, 2H), 7.40−7.38 (m, 2H), 4.50 (s, 1H), 3.60−3.50 (m, 2H), 3.29−

3.19 (m, 2H), 0.92 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 171.5,

139.3, 132.7, 131.4, 118.6, 112.1, 109.5, 82.0, 40.2, 34.9, 34.4, 23.7.

HRMS (m/z) calcd for C₁₆H₁₈BrNO₃[M]⁺, 351.0470; found,

351.0467.

134.0, 128.7, 121.8, 117.3 (q, J = 5 Hz), 116.4 (d, J = 22.1 Hz), 115.7,

104.7, 78.2, 45.1, 32.7, 8.0 HRMS (m/z) calcd for C₁₉H₁₆F₄N₂O₂S

[M]⁺, 412.0869; found, 412.0867.

4.2.6. General Procedure for the Synthesis of (R)-12a,³¹b. Three

equivalents of m-chloro perbenzoic acid (mCPBA) were added to a

solution of (R)-11a in CH₂Cl₂(0.1 mM). The reaction mixture was

stirred at room temperature for 12 h. The solution was then diluted

with EtOAc, and the organic layer was washed with aqueous Na₂SO₃

followed by saturated NaHCO₃. The crude compound was puriﬁed by

silica gel column chromatography or by crystallization. (R)-12b: yield,

90%; [α]²_D⁰−57 (c 0.4, EtOH; mp 98−103 °C); ¹H NMR (400 MHz,

CDCl₃) δ 9.06 (br s, 1H), 7.97 (s, 1H), 7.85−7.91 (m, 2H), 7.90−7.87

(m, 2H), 7.79−7.77 (m, 2H), 7.16 (t, J = 8.0 Hz, 2H), 4.94 (s, 1H),

3.95 (d, J = 14.4 Hz, 1H), 3.45 (d, J = 14.4 Hz, 1H), 1.93−1.83 (m,

2H), 0.95 (t, 3H, J = 7.2 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 171.2,

166.5 (d, J = 257 Hz), 141.1, 136.1, 135.2, 134.8 (q, J = 35 Hz)

131.1(d, J = 37.2 Hz), 122.0, 121.9, 117.3 (q, J = 5 Hz), 117.0 (d, J =

22.1 Hz), 115.5, 104.8, 77.1, 60.8, 33.9, 7.5. HRMS (m/z) calcd for

C₁₉H₁₆F₄N₂O₄S [M]⁺, 444.0767; found, 444.0764.

4.2.2. Synthesis of (2S,5R)-5-(Iodomethyl)-2-(tert-butyl)-5-phenyl-

1,3-dioxolan-4-one 7n. To a solution of (2R,5R)-2-tert-butyl-5-

phenyl-1,3-dioxolan-4-one 5⁵³(1 equiv) in THF (0.6M), cooled at

−78 °C, was added LHMDS (1.5 equiv) dropwise. The reaction

mixture was stirred at this temperature for 30 min, and then a solution

of HMPA/THF (0.4 mL, 1.8:1) was added via syringe followed by a

solution of CH₂I₂(3.3 equiv) in THF (3 mL/mmol). The temperature

was allowed to rise to −30 °C over 2 h, and the mixture was quenched

with a saturated solution of NH₄Cl and extracted with Et₂O. Silica gel

column chromatography of the crude material (eluent: cHex/Et₂O,

7:2) aﬀorded the pure compound as sticky oil in 70% yield. [α]²_D⁰+33

1

(c 0.2, CH₂Cl₂); H NMR (400 MHz, CDCl₃) δ 7.70−7.68 (m, 2H),

7.44−7.38 (m, 3H), 4.92 (s, 1H), 3.61 (d, J = 11.6 Hz, 1H), 3.48 (d, J

= 11.6 Hz, 1H), 0.97 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 171.2,

161.2, 133.6, 128.5, 128.4, 81.5, 34.9, 33.2, 32.2, 23.8. HRMS (m/z)

calcd for C₁₄H₁₇IO₃[M]⁺, 360.0222; found, 360.0226.

4.2.7. General Procedure for the Synthesis of (R)-13c−i. A

i

solution of (2S,5R)-7 (1 equiv) in a 1:1 PrOH (1 N)/NaOH mixed

4.2.3. General Procedure for the Synthesis of (R)-8a,⁵⁴b. Bromide

(2S,5R)-7a was dissolved in a large excess of 6 N HCl (6 equiv) and

reﬂuxed for 4 h. The reaction mixture was then cooled at room

temperature, treated with brine, and extracted with ethyl acetate

(EtOAc). The organic layer was then washed with a saturated solution

of NaHCO₃, and the aqueous solution was acidiﬁed with HCl (pH 2)

and extracted with EtOAc. The compound was processed to the next

step without any further puriﬁcation. The procedure used for the

synthesis of derivatives (R)-8a and (R)-8b can be, in principle, applied

to all other derivatives. (R)-8b: yield, 81%; [α]²_D⁰−18.2 (c 0.45, EtOH;

solvent was stirred at room temperature for 3 h; then, 4-

ﬂuorobenzenethiol (1.6 equiv) was added dropwise. The reaction

mixture was additionally stirred at room temperature for 16 h. The

solution was then treated with 1 M HCl (until pH = 8) and extracted

twice with CH₂Cl₂. The pure compound was obtained by

crystallization (CHCl₃/petrol ether). Characterization exempliﬁed by

1

(R)-13e: yield, 98%; H NMR (400 MHz, CDCl₃) δ 7.45−7.42 (m,

2H), 7.20 (d, J = 5.2 Hz, 1H), 7.0−6.93 (m, 3H), 6.86 (d, J = 3.6 Hz,

1H), 3.40 (d, J = 14.0 Hz, 1H), 3.36 (d, J = 14.8 Hz, 1H), 3.27 (d, J =

14.0 Hz, 1H), 3.23 (d, J = 14.4 Hz, 1H). ¹³C NMR (100 MHz,

CDCl₃) δ 177.7, (163.7, 161.3; d), 135.8, (134.0, 133.9; d), (130.5,

130.4; d), 127.9, 127.0, 125.7, 116.4 (d, J = 20 Hz), 78.3, 44.5, 39.2.

HRMS (m/z) calcd for C₁₄H₁₃FO₃S₂[M]⁺, 312.0290; found,

312.0294.

1

mp 107−111 °C); H NMR (400 MHz, CDCl₃) δ 3.74 (d, J = 10.4

Hz, 1H), 3.52 (d, J = 10.4 Hz, 1H), 1.94−1.90 (m, 1H), 1.84−1.77

(m, 1H), 1,03 (t, J = 7.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ

174.4, 89.2, 40.1, 32.4, 7.1. HRMS (m/z) calcd for C₅H₉BrO₃[M]⁺,

195.9735; found, 195.9738.

4.2.8. General Procedure for the Synthesis of (R)-14c−i. To a

solution of (R)-13 (1 equiv) in MeOH/toluene mixed solvent (1:1, 1

mL/mmol) was added Me₃SiCHN₂(2 M in Et₂O; 1.5 equiv)

dropwise at room temperature. The reaction mixture was then stirred

for 1 h and then carefully quenched with acetic acid and extracted with

EtOAc. The crude material was puriﬁed by silica gel column

chromatography. Characterization exempliﬁed by (R)-14f: yield,

87%; ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J = 7.6 Hz, 2H),

7.43−7.40 (m, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.98 (t, J = 8.8 Hz, 2H),

3.51 (s, 3H), 3.40 (d, J = 13.6 Hz, 1H), 3.21 (d, J = 13.6 Hz, 1H), 3.10

(d, J = 13.6 Hz, 1H), 3.04 (d, J = 13.6 Hz, 1H). ¹³C NMR (100

MHz,CDCl₃) δ 173.9, 162.4 (d, J = 20 Hz), 139.4, 134.0 (d, J = 20

Hz), 130.6, 129.6 (d, J = 20 Hz), 125.4 (d, J = 20 Hz), 116.2 (d, J = 20

Hz), 78.3, 52.9, 45.4, 44.5. HRMS (m/z) calcd for C₁₈H₁₆F₄O₃S [M]⁺,

388.0756; found, 388.0759.

4.2.4. General Procedure for the Synthesis of (R)-10a,⁵⁵b. To a

solution of (R)-8a in DMA (dimethylacetamide) (0.55 M) cooled at

−10 °C was added 1.3 equiv of SOCl₂dropwise under a nitrogen

atmosphere. The solution was stirred at this temperature for 3 h, and

then a solution of 4-amino-2-(triﬂuoromethyl)benzonitrile (9) in

DMA (1.2 equiv of amine in 1.5 mL of DMA) was added dropwise.

The reaction mixture was then allowed to react at room temperature

for 16 h. The solvent was then removed under reduced pressure, and

the crude material was treated with a saturated solution of NaHCO₃

and extracted with EtOAc. The reaction crude was puriﬁed by silica gel

column chromatography. Yield, 84%. (R)-10b: yield: 65%; [α]²_D⁰−31.7

(c 1.1, EtOH; mp 106−109 °C); ¹H NMR (400 MHz, CDCl₃) δ 9.03

(br s, 1H), 8.10 (s, 1H), 7.96 (dd, J = 8.4, 1.6 Hz, 1H), 7.80 (d, J = 8.8

Hz, 1H), 3.99 (d, J = 10.8 Hz, 1H), 3.58 (d, J = 10.8 Hz, 1H), 3.07 (br

s, 1H), 2.06−1.99 (m, 1H), 1.86−1.78 (m, 1H), 1.00 (t, J = 7.6 Hz,

3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.6, 141.4, 136.1, 134.6,

134.1(q, J = 32.5 Hz), 122.2, 117.5 (q, J = 20 Hz), 115.7, 105.0, 78.7,

40.6, 31.3, 8.3. HRMS (m/z) calcd for C₁₃H₁₂BrF₃N₂O₂[M]⁺,

364.0034; found, 364.0031.

4.2.9. General Procedure for the Synthesis of (2S,5R)-17l−n. To a

solution of (2S,5R)-7 (1 equiv) in dry dimethylformamide (DMF)

(6.5 mL/mmol), was added 2.2 equiv of dry K₂CO₃followed by 2.0

equiv of 4-ﬂuorobenzenethiol. The reaction mixture was then stirred at

room temperature for 3 to 4 h. Reaction completion was followed by

TLC using a cerium−molybdenum solution as stain and treated with

distilled water when the starting material disappeared. The organic

phase was extracted with EtOAc, and the crude material was puriﬁed

by silica gel column chromatography. Characterization exempliﬁed by

4.2.5. General Procedure for the Synthesis of (R)-11a,³¹b. To a

suspension of NaH (60% mineral oil; 1.3 equiv) in dry THF cooled at

0−5 °C was added a solution of 4-ﬂuorobenzenethiol (1.0 equiv) in

THF dropwise. The reaction mixture was then stirred at room

temperature for 30 min; after that, a solution of (R)-10a (1 equiv) in

THF was added dropwise at 0−5 °C. The solution was then stirred at

room temperature for 3−5 h and then quenched with distilled H₂O

and saturated NH₄Cl. The organic phase was then extracted with

EtOAc, and the crude material puriﬁed by silica gel column

chromatography. (R)-11b: yield, 85%; [α]²_D⁰−45.2 (c 3.0, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 8.97 (br s, 1H), 7.90 (s, 1H), 7.74 (s,

2H), 7.39−7.36 (m, 2H), 6.85 (t, J = 8.4 Hz, 2H), 3.74 (d, J = 14.0 Hz,

1H), 3.45 (br s, 1H), 3.08 (d, J = 14.0 Hz) 1.86−2.00 (m, 1H), 1.63−

1.76 (m, 1H), 0.93 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

δ 172.8, 161.5 (d, J = 256 Hz), 141.3, 135.9, 134.8 (q, J = 35 Hz),

1

(2S,5R)-17m: yield, 79%; H NMR (400 MHz, CDCl₃) δ 7.61−7.58

(m, 2H), 7.45−7.41 (m, 2H), 7.37−7.34 (m, 2H), 6.99 (t, J = 8.4 Hz,

2H), 3.35 (d, J = 13.6 Hz, 1H), 3.28 (d, J = 14.0 Hz, 1H), 3.19 (d, J =

14.0 Hz, 1H), 3.16 (d, J = 14.0 Hz, 1H), 0.79 (s, 9H). ¹³C NMR (100

MHz, CDCl₃) δ 173.2, 163.8, 161.3, 139.9, 134.1 (d, J = 20 Hz), 132.7,

131.4, 130.9, 118.7, 116.5, 111.9, 109.6, 83.6, 43.0, 40.4, 34.7, 23.5.

HRMS (m/z) calcd for C₂₂H₂₂FNO₃S [M]⁺, 399.1304; found,

399.1301.

4.2.10. General Procedure for the Synthesis of Methyl Esters (R)-

14l−n. To a solution of (2S,5R)-17 (1 equiv) in dry THF (7 mL/

mmol) was added 1.8 equiv of sodium methylate (1 M) dropwise at 0

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°C. The reaction mixture was then stirred at room temperature for 2 h

and then quenched with a 0.1 N solution of HCl. The organic layer

was extracted with EtOAc and dried over anhydrous Na₂SO₄. After

ﬁltration, the crude material was puriﬁed by silica gel column

chromatography. Characterization exempliﬁed by (R)-14m: yield,

were grown in DME supplemented with phenol red, 10% fetal calf

serum (FCS), insulin (6 ng/mL), ^L-glutamine (2 mM), penicillin (100

U/mL), streptomycin (100 U/mL), and hydrocortisone (3.75 ng/

mL). The cells were made quiescent using phenol red-free DME and

dextran charcoal-treated calf serum, as described.⁴⁰LNCaP-AR cells,

derived from LNCaP cells, were kindly provided by Dr. Charles L.

Sawyers (Howard Hughes Medical Institute Investigator at Memorial

Sloan Kettering Cancer Center, NY, USA), grown in RPMI medium

supplemented with 10% fetal bovine serum and glutamine (2 mM),

and checked periodically for mycoplasma contamination by MycoAlert

mycoplasma detection kit (Lonza). LNCaP-Rbic, the bicalutamide-

resistant cell line derived from LNCaP and isolated in our laboratory,⁴⁶

was maintained in the same way in continuous exposure to 20 μM

(R)-bicalutamide.

4.4. Transfection, Nuclear Translocation, and Transactiva-

tion Assay. For AR nuclear translocation analysis, Cos-7 cells were

plated on coverslips at 70% conﬂuence. Cells were then made

quiescent and then transfected with 1 μg of puriﬁed pSG5-hAR-

expressing plasmid using the Superfect reagent (Qiagen, Hilden,

Germany). Cells were left unstimulated or stimulated with 10 nM

R1881 for 60 min in the absence or presence of the study compounds.

For the transactivation assay, LNCaP cells and Cos-7 cells were plated

at 70% conﬂuence in phenol red-free RPMI (LNCaP) or DME (Cos-

7) containing 10% charcoal-stripped serum. After 48 h, cells were

transfected by Superfect with 0.8 μg of 3416-pTK-TATA-Luc alone or

with 0.2 μg of pSG5-hAR-expressing plasmid. After 24 h, transfected

cells were stimulated with 10 nM R1881 (dissolved in 0.001% ethanol,

ﬁnal concentration) for 24 h in the absence or presence of the

indicated compounds. Control cells were treated with the vehicle

alone. Cell lysates were prepared, and luciferase activity was measured

using a luciferase assay system (Promega). The results were corrected

using CH110-expressed-galactosidase activity.

4.5. AR Ligand Binding Displacement Studies in LNCaP

Cells. LNCaP cells were made quiescent using phenol red-free

medium and dextran charcoal-stripped serum.⁵⁸Cells at 70%

conﬂuence were incubated with 10 nM [³H]R1881 (98 Ci/mmole;

PerkinElmer) added to the medium in the absence or presence of the

indicated excess of radio-inert compounds. After a 4 h incubation at 37

°C, cells were washed three times with ice-cold PBS and collected by

gently scraping in a cold room using 600 μL of ice-cold PBS

containing 0.05% EDTA (w/v). The number of cells in an aliquot of

100 μL was counted. An aliquot (200 μL) of the cell suspension was

submitted in duplicate to the extraction of intracellular radioactivity

using 500 μL ice-cold ethanol (100%) for 1 h, as reported.⁴¹After 24 h

at 37 °C, radioactivity was counted in a liquid scintillation counter.

Nonspeciﬁc binding of [³H]R1881 was determined in separate wells

by adding the indicated excess of unlabeled R1881 to the incubation

medium. The statistical signiﬁcance of results was also evaluated by

paired Student’s t test. P values < 0.005 were considered to be

signiﬁcant. No signiﬁcance was attributed to the diﬀerence in the

residual binding between the cells incubated with 10 nM [³H]R1881

in the presence of (R)-16m and that incubated with 10 nM

[³H]R1881 in the presence of (R)-bicalutamide. LNCaP cells were

also incubated with 10 nM [³H]R1881 in the absence or presence of a

100-fold excess (1 μM) of unlabeled R1881 or casodex (Sigma-

Aldrich). Residual binding was 30 and 35% for unlabeled R1881 or

casodex, respectively.

4.6. BrdU Incorporation Analysis, Wound Scratch Analysis,

and Transwell Assay. After in vivo labeling with 100 μM BrdU

(Sigma), quiescent cells on coverslips were ﬁxed and permeabilized.

BrdU incorporation was analyzed by immunoﬂuorescence using

diluted (1:50 in PBS) mouse monoclonal anti-BrdU antibody (clone

BU-1, from GE Healthcare), as reported.⁵⁹Mouse antibody was

detected using diluted (1:200 in PBS) Texas red-conjugated goat anti-

mouse antibody (Jackson Laboratories). No signiﬁcance was attributed

to the diﬀerence in BrdU incorporation between the control cells and

cells stimulated with 10 nM R1881 in the presence of (R)-bic or (R)-

16m. For wound-healing assay, quiescent LNCaP cell monolayers at

conﬂuence were wounded using sterile pipet tips and then washed

with PBS. To avoid proliferation, the cells were treated with cytosine

1

98%; H NMR (400 MHz, CDCl₃) δ 7.54−7.51 (m, 2H), 7.39−7.43

(m, 2H), 7.29−7.25 (m, 2H), 6.98−6.93 (m, 2H), 3.49 (s, 3H), 3.39

(d, J = 14.0 Hz, 1H), 3.19 (d, J = 13.6 Hz, 1H), 3.09 (d, J = 13.6 Hz,

1H), 3.05 (d, J = 13.6 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 173.8,

163.7, 161.2, 140.9, 134.1, 134.0, 132.2, 132.1, 131.3, 131.1, 118.9,

116.4, 116.2, 111.3, 78.2, 52.9, 45.5, 44.7. HRMS (m/z) calcd for

C₁₈H₁₆FNO₃S [M]⁺, 345.0835; found, 345.0838.

4.2.11. General Procedure for the Synthesis of (R)-15c−n. To a

solution of 4-amino-2-(triﬂuoromethyl)benzonitrile (9) (1.6 equiv) in

THF (8.5 mL/mmol), cooled at −10 °C, was added LHMDS (4.5

equiv) dropwise. The reaction mixture was then stirred at this

temperature for 40 min, and HMPA (10% of the ﬁnal amount of

THF) was added to the solution. After 5 min stirring, a solution of the

ester (R)-14 (1 equiv) in THF (7 mL/mmol) was added to the

reaction mixture. After 30 min at −10 °C, the solution was stirred 12 h

at room temperature. The solution was then quenched with 0.1 N HCl

and extracted with EtOAc. The crude material was puriﬁed by silica gel

column chromatography. Characterization exempliﬁed by (R)-15m:

1

yield, 77%; H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 7.71−7.68

(m, 2H), 7.53.7.48 (m, 3H), 7.39−7.35 (m, 3H), 7.20−7.18 (m, 1H),

6.84−6.82 (m, 2H), 3.90 (d, J = 14.4 Hz, 1H), 3.78 (br s, 1H), 3.26 (d,

J = 13.6 Hz, 1H), 3.07 (d, J = 14.4 Hz, 1H), 2.96 (d, J = 13.2 Hz, 1H).

¹³C NMR (100 MHz, CDCl₃) δ 171.4, 162.5 (d, J = 248.7 Hz), 140.4,

140.1, 135.6, 134.1, 134.0, 131.9, 131.1, 127.41 (d, J = 3.4 Hz), 121.5,

118.5, 117.1, 117.0, 116.5, 116.3, 115.2, 111.3, 77.4, 44.9, 44.7. HRMS

(m/z) calcd for C₂₅H₁₇F₄N₃O₂S [M]⁺, 499.0978; found, 499.0974.

4.2.12. Synthesis of Compounds (R)-16c−n Was Achieved by the

Same Procedure Reported for the Preparation of (R)-12a,b.

Characterization exempliﬁed by (R)-16m: yield, 87%; [α]²_D⁰+142.5

1

(c 0.40, CHCl₃); H NMR (400 MHz, CDCl₃) δ 8.67 (s, 1H), 7.90−

7.88 (m, 2H), 7.79−7.75 (m, 2H), 7.56−7.54 (m, 3H), 7.33−7.30 (m,

2H), 7.17 (d, J = 8.0 Hz, 2H), 5.33 (s, 1H), 4.02 (d, J = 14.4 Hz, 1H),

3.41 (d, J = 14.4 Hz, 1H), 3.22 (d, J = 13.6 Hz, 1H), 3.13 (d, J = 13.6

Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 170.1, 166.6 (d, J = 258.1

Hz), 140.4, 139.0, 136.0, 132.3, 131.6, 131.3, 131.2, 122.1, 118.6, 117.3

(q, J = 4.7 Hz), 115.3, 112.1, 105.8, 77.4, 60.4, 46.1. HRMS (m/z)

calcd for C₂₅H₁₇F₄N₃O₄S [M]⁺, 531.0876; found, 531.0873.

4.2.13. General Procedure for the Synthesis of Derivatives

(2S,5S)-18c and (2S,5S)-18e. To a solution of (2S,5R)-7 (1 equiv)

in dry dimethylformamide (DMF) (6.5 mL/mmol) was added 2.2

equiv of dry K₂CO₃followed by 2.0 equiv of 4-cyanophenol. The

reaction mixture was then stirred at 100 °C for 12 h. Reaction

completion was followed by TLC using a cerium−molybdenum

solution as stain and treated with distilled water when the starting

material disappeared. The organic phase was extracted with EtOAc,

and the crude material was puriﬁed by silica gel column

chromatography. Characterization exempliﬁed by (2S,5S)-18c: yield,

1

30%; H NMR (400 MHz, CDCl₃) δ 7.58−7.56 (m, 2H), 7.35−7.26

(m, 5H), 6.91−6.89 (m, 2H), 4.41 (s, 1H), 4.25 (d, J = 10.4 Hz, 1H),

4.13 (d, J = 10.4 Hz, 1H), 3.20 (d, J = 14.0 Hz, 1H), 3.05 (d, J = 14.0

Hz, 1H), 0.89 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 1661.5,

134.3, 133.6, 130.4, 129.0, 128.1, 115.5, 109.6, 105.1, 86.2, 70.0, 38.7,

34.8, 23.6. HRMS (m/z) calcd for C₂₂H₂₃NO₄[M]⁺, 365.1627; found,

365.1624.

4.3. Constructs and Cell Lines. cDNA encoding the WT hAR

was in pSG5.⁵⁷The 3416 construct, containing four copies of the WT

slp-HRE2 (59-TGGTCAgccAGTTCT-39), was cloned into the NheI

site of pTKTATA-Luc.⁵⁶The human LNCaP prostate cancer-derived

cell lines (American Tissue Culture Collection; Rockville, MD, USA)

were maintained in RPMI medium supplemented with 10% fetal calf

serum (FCS), 1% glutamine, insulin (6 ng/mL), ^L-glutamine (2 mM),

penicillin (100 U/mL), streptomycin (100 U/mL), and hydro-

cortisone (3.75 ng/mL). The cells were made quiescent by using

phenol red-free RPMI and dextran charcoal-treated calf serum.⁵⁸Cos-

7 (American Tissue Culture Collection; Rockville, MD, USA) cells

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arabinoside (at 100 μM; Sigma) and then left unstimulated or

stimulated for 6 h with 10 nM R1881 in the absence or presence of the

indicated compounds. Fields were analyzed with a DMIRB inverted

microscope (Leica) using N-Plan 10× objective (Leica). Transwell

assay was done as reported.⁶⁰

4.7. Immunoﬂuorescence Analysis. Cells on coverslips were

ﬁxed and permeabilized.⁴⁰WT hAR ectopically expressed in Cos-7

cells was visualized⁶⁰using the rabbit polyclonal anti-C19 antibody

(Santa Cruz). The primary antibody was detected using diluted (1:100

in PBS) Texas red-conjugated goat anti-rabbit antibody (Jackson

Laboratories). Coverslips were ﬁnally stained with Hoechst 33258,

inverted, and mounted in Mowiol (Calbiochem). Fields were analyzed

as detailed described⁵⁹using a DMBL Leica (Leica) ﬂuorescent

microscope equipped with an HCXPL Apo 63× oil objective. Images

were captured using a DC480 camera (Leica) and acquired using

FW4000 (Leica) software.

TACCTTGA-3′); primers were designed using Beacon Designer

Software (version 7.2, BioRad). Real-time PCR was carried out in

triplicate reactions at a ﬁnal volume of 25 mL containing 50 ng of

cDNA template, SYBR Green Mix, and 200 nM of forward and reverse

primers. Samples were maintained at 95 °C for 10 min and 30 s,

followed by 40 ampliﬁcation cycles at 95 °C for 15 s, and then at 60

°C for 30 s for GAPDH, HPRT, and PSA. Product speciﬁcity was

controlled by melting point analysis. Ampliﬁcation eﬃciency, which

never varied by 5% in the diﬀerent experiments, was used to determine

the relative expression of mRNA obtained using Gene Expression

Macro Software (version 1.1) (BioRad). The amount of mRNA was

normalized to the endogenous reference genes GAPDH and HPRT

and expressed as n-fold levels relative to untreated samples. RT-PCR

reactions were performed in triplicate, and the coeﬃcient of variation

(CV), calculated from the three Ct values, was always 1.5%.

Reproducibility of the relative mRNA expression was calculated

from the results of two experiments in which the procedure was

carried out on diﬀerent retrotranscription products derived from the

same mRNA sample. CV was always <10%. For the analysis of

quantitative real-time PCR experiments, one-way ANOVA with

Dunnett’s post-test was carried out using GraphPad Prism version

4.00 for Windows (GraphPad Software, San Diego, CA). A P value <

0.05 was considered to be signiﬁcant.

4.8. Lysates and Western Blot Analysis. Cell lysates (2 mg/mL

protein concentration) were prepared,⁵⁹and AR was detected as

reported⁶⁰using rabbit polyclonal anti-AR antibodies (C-19 or N-20;

Santa Cruz). Immune-reactive proteins were revealed using the ECL

detection system (from GE Healthcare).

4.9. In Vitro Chemosensitivity Assay. Sulforhodamine B (SRB)

assay was used according to the method by Skehan et al.⁶¹Brieﬂy, cells

were collected by trypsinization, counted, and plated at a density of

5000 cells/well in 96-well ﬂat-bottomed microtiter plates (100 μL of

cell suspension/well). Experiments were run in octuplicate, and each

experiment was repeated three times. The optical density of cells was

determined at a wavelength of 490 nm by a colorimetric plate reader.

Growth inhibition and cytocidal eﬀect of drugs were calculated

4.12. Determination of PSA Levels. A PSA ELISA kit supplied

by Abnova (Taiwan Corporation, Taiwan) was used according to the

manufacturer’s instructions. The concentration of PSA was measured

spectrophotometrically at 450 nm in culture medium.

4.13. In Vivo Experiments. Five- to six-week old CD-1 male nude

(nu/nu) mice were purchased from Charles River Laboratories (Calco,

Italy). All animal experiments were performed at the Animal Facility of

Regina Elena National Cancer Institute in Rome, Italy. The procedures

involving animals were in compliance with our institutional animal

care guidelines and with international directives (directive 2010/63/

EU of the European Parliament and of the Council; Guide for the

Care and Use of Laboratory Animals, United States National Research

Council, 2011). The animals were euthanized for ethical reasons by

cervical dislocation when tumors reached a mean of 3.0 g in weight or

when they became moribund during the observation period. For

antitumor activity experiments, mice were injected subcutaneously in

the ﬂank with VCaP prostate cancer cells at 10⁶cells/mouse in 100 μL

of solution composed of 50% Matrigel and 50% serum-free medium.

After about 1 month (when a tumor mass of 50−100 mg was evident),

mice were randomized, divided in groups, and treatment was started.

As previously reported,⁴¹mice were treated orally by daily gavage for 4

consecutive weeks with casodex or (R)-bicalutamide at 10 mg/kg. In

addition, the antitumor eﬃcacy of (R)-16m, given with the same

treatment schedule, was evaluated. All compounds were dissolved in

80% PEG-400 and 20% Tween-80. Control mice were treated with

vehicle for the same treatment period. Five mice for each group were

evaluated. Tumor size was measured three times a week in two

dimensions by caliper, and tumor weight was calculated using the

following formula: a × b²/2, where a and b are the long and short

diameters of the tumor, respectively.

4.14. Theoretical Calculations Method. Structures of WT AR

(PDB ID: 2AXA), T877A mutant (PDB ID: 2AX7), and W741L

mutant (PDB ID: 2AX8) were used as starting points for molecular

modeling calculations. Missing loops of residues 844−851 and 918−

919 were built with homology models by using the sequence of the AR

ligand binding domain (UniProt code: P10275, residues 664−919).

Coordinates of progesterone receptor (PDB ID: 2OVM) were used to

generate the homology model of AR antagonist conformation. Prime

software, version 3.1.023, was used in its standard settings. All protein

models were processed with the standard protein preparation wizard

routine as present in Maestro software, version 9.3.023. The docking

software Glide (version 5.7) and the induced ﬁt docking (IFD) were

used in their standard settings to generate binding poses of AR ligands.

Molecular dynamics simulations of complexes between AR forms and

compound (R)-16m were performed starting from the binding poses

of docking calculation with a standard multistep relaxation protocol, as

implemented in Desmond software (version 3.1.023) of the

according to the formula reported by Monks et al.:⁶²[(OD_treated

−

OD_zero)/(OD_control− OD_zero)] × 100%, when OD_treatedis > OD_zero. If

OD_treatedis below OD_zero, then cell killing has occurred. OD_zerodepicts

the cell number at the moment of drug addition, OD_controlreﬂects the

cell number in untreated wells, and OD_treatedreﬂects the cell number in

treated wells on the day of the assay. Cytotoxic potency on CW22Rv1

was assessed by growth inhibition assay. Cells were cultured in RPMI

1640 containing 10% FCS. Twenty-four hours after seeding, cells were

exposed to the drugs (concentration range 0.5−100 μM) and counted

144 h later by coulter counter. IC₅₀is deﬁned as the drug

concentration causing 50% cell growth inhibition, determined by

dose−response curves. Experiments were performed in triplicate.

4.10. Tunel Assay. At the end of drug exposure, the percentage of

apoptotic cells was evaluated by ﬂow cytometric analysis. Brieﬂy, after

treatment, cells were trypsinized, ﬁxed in PBS + 1% paraformaldehyde

on ice for 15 min, suspended in ice-cold ethanol (70%), and stored

overnight at −20 °C. Cells were then washed twice in PBS and

incubated with 50 μL of TUNEL reaction mixture (Roche Diagnostic

GmbH, Mannheim, Germany) containing TdT and FITC-conjugated

dUTP deoxynucleotides in a humidiﬁed atmosphere for 60 min at 37

°C in the dark. Samples were then washed in PBS containing Triton X-

100 (0.1%), counterstained with 2.5 μg/mL propidium iodide (Sigma-

Aldrich) and RNase (10 Kunits/ml, Sigma-Aldrich) for 30 min at 4 °C

in the dark, and ﬁnally analyzed (FACS Vantage Becton Dickinson).

Data acquisition and analysis were performed using CELLQuest

software (Becton Dickinson). For each sample, 10 000 events were

recorded.

4.11. Quantitative Real-Time PCR. Total cellular RNA was

isolated using RNeasy minikit (Qiagen). One microgram of RNA was

reverse-transcribed into cDNA using iScript (BioRad, Hercules, CA)

according to the manufacturer’s instructions. Real-time PCR was

performed using the MyiQ single color real-time PCR detection

system (BioRad) and SYBR green I dye chemistry. The stably

expressed endogenous β2-microglobulin gene was ampliﬁed as a

control for quality and quantity of input RNA. Primers for mRNA

ampliﬁcation are as follows: GAPDH forward 5′-

CGCTACTCTCTCTTTCTGGC-3′, reverse 5′-AGACACATAG-

C A A T T C A G A A T - 3 ′ ; H P R T f o r w a r d 5 ′ -

AGACTTTGCTTTCCTTGGTCAGG-3′, reverse 5′-

GTCTGGCTTATATCCAACATTCG-3′; PSA forward 5′-GCAG-

CATTGAACCAGAGGAG-3′, reverse 5′- CCATGACGTGA-

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Schrodinger suite 9.3 update 1, followed by 50 ns of production

trajectory. MD simulations were carried out on a Linux x86_64

platform machine equipped with an Intel Core2 Quad CPU (Q9400,

2.66 GHz).

(8) Visakorpi, T.; Hyytinen, E.; Koivisto, P.; Tanner, M.; Keinanen,

̈

R.; Palmberg, C.; Palotie, A.; Tammela, T.; Isola, J.; Kallioniemi, O. P.

In Vivo Amplification of the Androgen Receptor Gene and

Progression of Human Prostate Cancer. Nat. Genet. 1995, 9, 401−406.

(9) Edwards, J.; Krishna, N. S.; Grigor, K. M.; Bartlett, J. M. S.

Androgen Receptor Gene Amplification and Protein Expression in

Hormone Refractory Prostate Cancer. Br. J. Cancer 2003, 89, 552−

556.

ASSOCIATED CONTENT

■

S

* Supporting Information

Additional experimental procedures and characterization data

are provided for compounds (2S,5S)-6a−m, (2S,5R)-7a-l, (R)-

13c−d, (R)-13f−i, (R)-14c−e, (R)-14g−i, (2S,5R)-17l,

(2S,5R)-17n, (R)-14l, (R)-14n, (R)-15c−l, (R)-15n, (R)-

16c−l, (R)-16n, (2S,5S)-18e, (S)-19c, (S)-19e, (S)-20c, and

(S)-20e; ¹H and ¹³C NMR spectra of all ﬁnal adducts; cytotoxic

eﬀects of derivatives (R)-16c−n; transactivation assay of

derivatives (S)-20c and (S)-20e on Cos-7 cells; eﬀects of

compounds on PSA secretion; and eﬀect of (R)-bicalutamide

and (R)-16m on nuclear translocation of AR. This material is

available free of charge via the Internet at http://pubs.acs.org.

(10) Taplin, M. E.; Bubley, G. J.; Ko, Y. J.; Small, E. J.; Upton, M.;

Rajeshkumar, B.; Balk, S. P. Selection for Androgen Receptor

Mutations in Prostate Cancers Treated with Androgen Antagonist.

Cancer Res. 1999, 59, 2511−2515.

(11) Veldscholte, J.; Ris-Stalpers, C.; Kuiper, G. G.; Jenster, G.;

Berrevoets, C.; Claassen, E.; van Rooij, H. C.; Trapman, J.; Brinkmann,

A. O.; Mulder, E. A Mutation in the Ligand Binding Domain of the

Androgen Receptor of Human LNCaP Cells Affects Steroid Binding

Characteristics and Response to Anti-Androgens. Biochem. Biophys.

Res. Commun. 1990, 173, 534−540.

(12) Wilding, G.; Chen, M.; Gelmann, E. P. Aberrant Response in

Vitro of Hormone-Responsive Prostate Cancer Cells to Antiandro-

gens. Prostate 1989, 14, 103−115.

AUTHOR INFORMATION

(13) Hara, T.; Miyazaki, J.; Araki, H.; Yamaoka, M.; Kanzaki, N.;

Kusaka, M.; Miyamoto, M. Novel Mutations of Androgen Receptor: A

Possible Mechanism of Bicalutamide Withdrawal Syndrome. Cancer

Res. 2003, 63, 149−153.

■

Corresponding Author

*Phone: +390516398283; Fax: +390516398349; E-mail: greta.

varchi@isof.cnr.it.

(14) Tan, J.-a.; Sharief, Y.; Hamil, K. G.; Gregory, C. W.; Zang, D. Y.;

Sar, M.; Gumerlock, P. H.; deVere White, R. W.; Pretlow, T. G.;

Harris, S. E.; Wilson, E. M.; Mohler, J. L.; French, F. S.

Dehydroepiandrosterone Activates Mutant Androgen Receptors

Expressed in the Androgen-Dependent Human Prostate Cancer

Xenograft CWR22 and LNCaP Cells. Mol. Endocrinol. 1997, 11,

450−459.

Author Contributions

^#Anna Tesei and Andrea Guerrini contributed equally to this

work.

Notes

The authors declare no competing ﬁnancial interest.

(15) Culig, Z.; Hobisch, A.; Cronauer, M. V.; Cato, A. C.; Hittmair,

A.; Radmayr, C.; Eberle, J.; Bartsch, G.; Klocker, H. Mutant Androgen

Receptor Detected in an Advanced-Stage Prostatic Carcinoma Is

Activated by Adrenal Androgens and Progesterone. Mol. Endocrinol.

1993, 7, 1541−1550.

ACKNOWLEDGMENTS

■

Italian Ministry of University and Scientiﬁc Research

(2010NFEB9L_002 to Gabriella Castoria) supported this

work. We thank F. Claessens for the ARE-luc 3416 construct.

(16) Weber, M. J.; Gioeli, D. Ras Signaling in Prostate Cancer

Progression. J. Cell. Biochem. 2004, 91, 13−25.

ABBREVIATIONS USED

■

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Therapy. Cancer Res. 2001, 61, 4315−4319.

CCR2, CC chemokine receptor 2; CCL2, CC chemokine

ligand 2; CCR5, CC chemokine receptor 5; TLC, thin-layer

chromatography; AR, androgen receptor; PC, prostate cancer

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Vessella, R. L.; Said, J. W.; Isaacs, W. B.; Sawyers, C. L. Inactivation of

the Tumor Suppressor PTEN/MMAC1 in Advanced Human Prostate

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