Nonsteroidal androgen receptor ligands: Versatile syntheses and biological data

Article abstract of DOI:10.1021/ml3000269

We report herein a stereoselective and straightforward methodology for the synthesis of new androgen receptor ligands with (anti)-agonistic activities. Oxygen-nitrogen replacement in bicalutamide-like structures paves the way to the disclosure of a new class of analogues, including cyclized/nitrogen-substituted derivatives, with promising antiandrogen (or anabolic) activity.

Full text of DOI:10.1021/ml3000269

Letter
pubs.acs.org/acsmedchemlett
Nonsteroidal Androgen Receptor Ligands: Versatile Syntheses and
Biological Data
Greta Varchi,*,† Andrea Guerrini,† Anna Tesei,‡ Giovanni Brigliadori,‡ Carlo Bertucci,§
Marzia Di Donato,∥ and Gabriella Castoria∥
†Istituto CNR per la Sintesi Organica e la Fotoreattivita
‡Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Forlì, Italy
̀
(ISOF), Bologna, Italy
§Dipartimento di Scienze Farmaceutiche, Universita
̀
di Bologna, Bologna, Italy
∥Dipartimento di Patologia Generale II, Universita
̀
di Napoli, Napoli, Italy
S
* Supporting Information
ABSTRACT: We report herein a stereoselective and straightfor-
ward methodology for the synthesis of new androgen receptor
ligands with (anti)-agonistic activities. Oxygen−nitrogen replace-
ment in bicalutamide-like structures paves the way to the disclosure
of a new class of analogues, including cyclized/nitrogen-substituted
derivatives, with promising antiandrogen (or anabolic) activity.
KEYWORDS: androgen receptor, enantiopure antiandrogens, hormone-refractory prostate cancer, sulfinylimino propanamides
onsteroidal androgen receptor (AR) ligands represent a
very important class of molecules acting as either
antagonists or agonists of AR. In particular, nonsteroidal
antiandrogens, such as (R,S)-bicalutamide (1) and nilutamide
(2), have been shown to be effective in the treatment of
prostate cancer (PCa) (Figure 1). These kinds of molecules
that these drugs serve as agonists under resistance circum-
stances.3,4 Sawyers and co-workers showed that a 3−5-fold up-
regulation of the AR is one of the most likely causes of
resistance to antiandrogens.5,6
N
They further demonstrated that hormone-refractory prostate
cancer (HRPC) was still dependent on the AR ligand binding
domain for growth.6 Therapeutic options for HRPC patients
are limited, with lack of evidence for long-term survival. Besides
PCa, it has been widely reported that little structural
modifications of bicalutamide-like molecules provide dramatic
changes on their biological activity, for example, from AR
antagonist to agonist.2−10 Nonsteroidal AR agonists are still in
the early stages of drug discovery, and ostarine 3 (Figure 1) is
one of the most promising for muscle wasting and osteoporosis
treatment.
Indeed, there is an urgent need for novel compounds able to
act as either antiandrogens in HRPC conditions or nonsteroidal
anabolics. In view of these concerns, we developed an effective
and inexpensive methodology, able to easily afford novel
structural hybrids of bicalutamide and/or nilutamide and/or
ostarine.
Figure 1. (R,S)-Bicalutamide (1), nilutamide (2), and ostarine (3).
present several advantages over steroidal antagonists such as
oral bioavailability and lack of cross-reactivity with other steroid
receptors.1 In particular, because bicalutamide showed a
superior pharmacokinetic profile along with minor side effects,2
it has been chosen as the routine clinical treatment.
However, after its long-term use, a subset of patients had to
discontinue bicalutamide treatment. This behavior has been
termed “anti-androgen withdrawal syndrome” and indicates
Received: January 27, 2012
Accepted: April 10, 2012
Published: April 10, 2012
© 2012 American Chemical Society
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tert-Butane sulfinamide (4), introduced by Ellman's
representative for our preliminary studies. LNCaP cells express
a point-mutated AR, which leads to a decrease in steroid-
binding specificity.18,19 Nonetheless, these cells have largely
been used as a model of androgen-responsive growth.20 On the
other hand, human PCa cells LNCaP-AR have been engineered
by Jung et al. with the aim to evaluate the potency of emerging
nonsteroidal antiandrogens (MDV3100).5,20 These cells, in
fact, express 3−5-fold higher levels of the AR, thus mimicking
the clinical scenario of HRPC.5,21,22 Because PCa progression
most frequently correlates with a rise in PSA concentrations,23
we first measured the effect of our compounds on PSA levels in
culture medium of LNCaP and LNCaP-AR cells. Figure 2A
group11−13 (Scheme 1), represents a very attractive precursor
Scheme 1. tert-Butane Sulfinylimino-N-(4-cyano-3-
a
trifluoromethylphenyl)propanamides S(S)- and S(R)-6
a
Reagents and conditions: (a) Ti(OEt)4; THF, 40 °C. (b) p-F-
n
(CH3SO2)C6H4, BuLi, THF, −45 °C. (c) MCPBA, CH2Cl2, rt, 2 h.
to chiral amines. Thus, we exploited this chemistry to provide a
new synthetic and straightforward route to novel and
enantiopure AR modulators. Their synthesis should also allow
us to study the relationship between stereochemistry and
pharmacological activity, the importance of stereochemistry
already having been demonstrated for bicalutamide.14
To the best of our knowledge, no examples of highly
derivatizated N-tert-butane-sulfinyl ketimines bearing an
electron poor aryl propanamide skeleton have been reported
so far. Sulfinylimino propanamides S(S)- and S(R)-6 were
obtained by reaction of oxopropanamide (5)15,16 with (S)/
(R)-4 with acceptable isolated yield (56%), and they represent
the key starting materials for the preparation of the new AR
ligands. In fact, their reactivity allows the reaction with different
carbanions. In particular, an alkylation reaction with p-
fluorophenyl methyl sulfone occurs in good yield (78%) and
with 76% diastereomeric excess, which was evaluated by NMR
analysis of the crude material.17 During the purification step,
only one diastereoisomer could be isolated.
Thus, ketimine S(S)-6 affords compounds (S)-22, (S)-23, (S)-
26, (S)-27, and (S)-28, while their enantiomers are provided by
ketimine S(R)-6 (Schemes 1 and 2). The absolute configuration
of compound (S)-23 was determined by electronic circular
dichroism (ECD) (see the Supporting Information for details).
For in vitro experiments, we selected LNCaP and LNCaP-
AR human PCa cell lines, considering them the most
Figure 2. (A) PSA levels induced by antiandrogens (20 μM). A
significant increment (p < 0.01) of PSA levels in LNCaP (47%) and
LNCaP-AR (63%) was observed upon 48 h of exposure to the
synthetic androgen R1881, as expected. (B) Cytotoxic activity of (R)-
bicalutamide and new AR ligands in LNCaP cell lines (average of three
independent experiments).
a
Scheme 2. Synthesis of Amino Bicalutamide Analogues
shows that some of the antiandrogens synthesized by us inhibit
PSA secretion in culture medium of both cell lines, causing a
PSA level reduction significantly higher (p < 0.01) than that
produced by (R)-bicalutamide, the most active enantiomer. In
particular, compound (S)-26 showed the highest PSA reduction
effect (82% on LNCaP and 72% on LNCaP-AR). Chemo-
sensitivity analysis was performed by SRB assay on LNCaP
cells,24 which were incubated for 144 h to scalar drug
concentrations ranging from 0.02 to 20 μM. As shown in
Figure 2B, all compounds showed higher cell growth inhibition,
as compared to (R)-bicalutamide (orange line), in the range of
a
Reagents and conditions: (a) HCl, MeOH, −5 °C to rt, 3 h. (b) CDI,
DPEA, toluene, 100 °C, 3 h or DPTC, DPEA, toluene, 100 °C, 5 h.
(c) p-CH3−C6H4SO2Cl, pyridine.
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2−20 μM, with (S)-26 and (S)-22 being the most potent of the
series. These preliminary data prompted us to further explore
the AR antagonistic capability of derivatives (S)-26 and (S)-22.
A chemosentitivity test on LNCaP-AR cells exhibited a stronger
cytotoxic effect of (S)-22 and (S)-26 with respect to the control
drug, especially between 2 and 20 μM. In fact, even at these
concentrations, (R)-bicalutamide was ineffective in inhibiting
cells proliferation (Figure 3A). Moreover, a high cytocidal effect
Figure 4. Effect of Casodex and (S)-26 on the androgen activated
transcription analyzed by ARE-luc reporter assay established in
LNCaP cells (average of three independent experiments).
due, at least in part, to its antagonistic behavior toward mutated
AR.
Figure 3. (A) Cytotoxic activity of (R)-bicalutamide and (S)-22/26 in
LNCaP-AR cell lines (average of three independent experiments). (B)
Apoptosis analysis by flow cytometry TUNEL assay.
Besides alkylation reaction, we report herein the unprece-
dented, straightforward formation of aziridines 8, which
smoothly occurs in 90% yield (1/1 diastereomeric ratio) by
adding S(S)-6 to a (chloromethyl)lithium solution in the
presence of LiBr.26,27 Aziridine ring opening with thiolates or
phenolates takes place with good yield and complete
regioselectivity (Scheme 3). In the ARE-luc assay established
at 20 mM was observed for (S)-26 in both LNCaP and
LNCaP-AR cell lines (LC50 = 18.3 μM in both cell lines).
Overall, (R)-bicalutamide never reached LC50 and IG50 (1.8
μM) only in LNCaP cells.
After 144 h of exposure to 20 μM (R)-bicalutamide, a
moderate fraction of apoptotic cells, 2.1−5.7%, respectively,
was observed in the two cell lines. Conversely, apoptosis
analysis showed that (S)-26 was capable of inducing a strong
LNCaP cell death (80.5%), and this cell-killing capability was
even higher on LNCaP-AR (91.6%) (Figure 3B).
a
Scheme 3. Aziridine Formation and Reaction
The assay of the human androgen receptor (hAR)
transcriptional activity was conducted to evaluate (anti-)
agonistic activities of our compounds. For androgen-stimulated
transcriptional analysis, subconfluent (60−70%) LNCaP or
Cos-7 cells were plated in phenol red-free DMEM containing
5% charcoal-stripped serum. After 48 h, the cells were
transfected with 0.3 μg of 3416-pTK-TATA-Luc construct, as
reported.25 AR-negative Cos-7 cells were also cotransfected
with 1.5 μg of pSG5-empty plasmid (pSG5) or pSG5-hAR
(hAR) expressing plasmid. After 24 h, LNCaP and Cos-7
transfected cells were left unstimulated or stimulated for 24 h
with a 10 nM concentration of the synthetic androgen, R1881,
in the absence or presence of the indicated concentrations of
synthetic compounds. AR was detected by Western blot
analysis, and the luciferase activity in cell lysates was measured
and expressed as fold induction.25 Figure 4 shows that 10 nM
R1881 increases by 4.2-fold the AR-mediated transcriptional
activity in LNCaP cells. Expectedly, the antiandrogen Casodex
(at 10 μM) inhibits such an activation. Notably, inhibition of
the androgen-induced transcriptional activation is observed in
cells challenged with (S)-26, and this effect is similar to that
observed by using Casodex. Similar findings were observed in
ARE-luc reporter assay established in Cos-7 cells ectopically
expressing hAR (see the Supporting Information), indicating
that (S)-26 exerts its antagonistic effect independently of the
cell type. Figure 4 also shows that in the range of concentration
used (from 10 nM to 10 μM), (S)-26 did not exhibit agonistic
activity. Overall, these data confirm that the activity of (S)-26 is
a
Reagents and conditions: (a) (i) LiBr/MeLi, −78 °C; (ii) S(S)-6,
THF. (b) (i)p-F-C6H4SH, DBU; (ii) HCl/MeOH, −5 °C to rt; (iii)
MCPBA/CH2Cl2, rt. (c) (i) p-CN-C6H4OH, toluene, DBU; 80 °C, 36
h; (ii) HCl/MeOH, −5 °C to rt. (d) CDI/DPEA, 100 °C, 3 h.
in Cos-7 cells (Figure 5), compound (S)-7d slightly decreases
the androgen-stimulated ARE-luc activity. These findings
suggest that (S)-7d exerts both agonistic and antagonistic
effects. This behavior might depend on AR levels. Thus, in cells
expressing high levels of hAR, such as transfected Cos-7 cells
(and most likely LNCaP-AR cells), (S)-7d used at 10 μM is
unable to fully antagonize the androgen-induced transcriptional
effect.
On the other hand, at that concentration, (S)-7d does not
behave as a full agonist. Therefore, this compound represents a
promising tool because of its pronounced flexibility, likely due
to its molecular scaffold, which, besides, is susceptible of
targeted synthetic modifications. The possibility that (S)-7d
differently acts on AR-mediated actions (i.e., nongenomic vs
genomic actions) or different types of AR (i.e., membrane or
cytoplasm or nuclear AR) cannot be excluded at moment.
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: +39 051-6398283. Fax: +39 051-6398349. E-mail: g.
varchi@isof.cnr.it.
Funding
We are grateful for the financial support from the University of
Bologna, from the MIUR, Italy (PRIN 2008 National
Program). G.C. and M.D.D. are supported by a grant from
Associazione Italiana per la Ricerca sul Cancro.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Charles L. Sawyers, Investigator at Memorial
Sloan Kettering Cancer Center, Howard Hughes Medical
Institute (HHMI), New York, for kindly providing LNCaP-AR
cell line.
Figure 5. Effect of Casodex and (S)-7d on AR-mediated transcription
analyzed by ARE-luc reporter assay in Cos-7 cells ectopically
expressing hAR (average of three different experiments). Similar
amounts of hAR were detected by Western blot analysis of cell lysates
(not shown).
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* Supporting Information
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