20-Aminosteroids as a novel class of selective and complete androgen receptor antagonists and inhibitors of prostate cancer cell growth

Article abstract of DOI:10.1016/j.bmc.2010.08.029

Here, the synthesis and the evaluation of novel 20-aminosteroids on androgen receptor (AR) activity is reported. Compounds 11 and 18 of the series inhibit both the wild type and the T877A mutant AR-mediated transactivation indicating AR antagonistic function. Interestingly, minor structural changes such as stereoisomers of the amino lactame moiety exhibit preferences for antagonism among wild type and mutant AR. Other tested nuclear receptors are only weakly or not affected. In line with this, the prostate cancer cell growth of androgen-dependent but not of cancer cells lacking expression of the AR is inhibited. Further, the expression of the prostate specific antigen used as a diagnostic marker is also repressed. Finally steroid 18 enhances cellular senescence that might explain in part the growth inhibition mediated by this derivative. Steroids 11 and 18 are the first steroids that act as complete AR antagonists and exhibit AR specificity.

Full text of DOI:10.1016/j.bmc.2010.08.029

Bioorganic & Medicinal Chemistry 18 (2010) 6960–6969
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
20-Aminosteroids as a novel class of selective and complete androgen
receptor antagonists and inhibitors of prostate cancer cell growth
Manolis A. Fousteris a, , Undine Schubert b, Daniela Roell b, Julia Roediger b, Nikolaos Bailis a,
a,
Sotiris S. Nikolaropoulos c, Aria Baniahmad b, , Athanassios Giannis
⇑
⇑
a Institut für Organische Chemie, Fakultät für Chemie und Mineralogie, Universität Leipzig, Johannisallee 29, Leipzig 04103, Germany
b Institute of Human Genetics, Jena University Hospital, Jena D-07743, Germany
c Laboratory of Medicinal Chemistry, Department of Pharmacy, University of Patras, Patras 26500, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Here, the synthesis and the evaluation of novel 20-aminosteroids on androgen receptor (AR) activity is
reported. Compounds 11 and 18 of the series inhibit both the wild type and the T877A mutant AR-med-
iated transactivation indicating AR antagonistic function. Interestingly, minor structural changes such as
stereoisomers of the amino lactame moiety exhibit preferences for antagonism among wild type and
mutant AR. Other tested nuclear receptors are only weakly or not affected. In line with this, the prostate
cancer cell growth of androgen-dependent but not of cancer cells lacking expression of the AR is inhib-
ited. Further, the expression of the prostate specific antigen used as a diagnostic marker is also repressed.
Finally steroid 18 enhances cellular senescence that might explain in part the growth inhibition mediated
by this derivative. Steroids 11 and 18 are the first steroids that act as complete AR antagonists and exhibit
AR specificity.
Received 13 August 2010
Accepted 14 August 2010
Available online 19 August 2010
Keywords:
Aminosteroids
Antihormone
Prostate cancer
Androgen receptor
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
signaling is related with pathological disorders such as BPH and
PCa, thus AR comprises primary therapeutic target for the treat-
Benign prostate hyperplasia (BPH) and prostate cancer (PCa) is a
serious health problem worldwide. PCa has advanced to the second
leading cause of cancer mortality of men in western countries.1
Many factors affect risk of developing PCa, important being age.2
With advancing age the prostate gland increases in size leading
to BPH. In addition older men develop more chances for having a
diagnosis for PCa. Notably, both the proliferation of prostate and
also PCa is enhanced by androgens.1,3
ment of these diseases.6
Antiandrogens (AR-antagonists) are structurally classified as
steroidal and non-steroidal compounds. Structural modifications
of T and DHT afforded many steroidal AR antagonists for PCa treat-
ment like cyproterone acetate, a partial antiandrogen.7 On the
other hand, currently approved antiandrogens for PCa, are flutam-
ide (EulexinÒ), bicalutamide (CasodexÒ), and nilutamide (Nilan-
dronÒ) are of non-steroidal nature.
Androgens mediate effects through the androgen receptor (AR),
a ligand-regulated transcription factor and member of the nuclear
receptor superfamily.4 Activation of AR either by endogenous
Despite the initial tumor regression and the overall clinical
improvement of patients, eventually the tumor develops resistance
to antiandrogens and becomes hormone refractory and castration
resistant. However, the AR is expressed, still remains active and
promotes growth of prostate cells despite androgen ablation and
antagonist treatments.2,8 Particularly, point mutations often lead
to a receptor that has gained the ability to promiscuously bind also
to non-androgenic ligands, be activated by other ligands9 and thus
bypassing the androgen ablation or hormone therapy by androgen
antagonists. For example, the mutation T877A of human AR, iso-
lated from PCa patients, renders the complete non-steroidal antag-
onist hydroxyflutamide (OH-Fl) to an agonist that activates AR.10
Unambiguously, the development of novel, more potent antiandro-
gens with pure antagonistic activities against both the wild-type
(wt) and the mutated forms of AR, would be of a great therapeutic
impact for the treatment of PCa.
androgens such as testosterone (T) and 5a-dehydrotestorenone
(DHT) or by exogenous compounds induces conformational
changes of the receptor resulting to its translocation to the nucleus
and androgen-induced gene expression. The AR plays a fundamen-
tal role in the development and maintenance of primary and sec-
ondary male sexual characteristics including the growth of
normal prostate gland.5 However, excessive stimulation of the AR
⇑
Corresponding authors. Tel.: +49 3641935501; fax: +49 3641935502 (A.B.); tel.:
+49 3419736581; fax: +49 3419736599 (A.G.).
E-mail addresses: aban@mti.uni-jena.de (A. Baniahmad), giannis@uni-leipzig.de
(A. Giannis).
Present address: Laboratory of Medicinal Chemistry, Department of Pharmacy,
University of Patras, Patras 26500, Greece.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.029

M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
6961
Be prompted by this need, we focused our attempts in the field
of steroidal antiandrogens. Especially, we designed and synthe-
sized novel steroidal derivatives bearing a chiral tertiary C-20 ami-
no-moiety and we evaluated their effects on AR activity and PCa
cell growth. We envisaged the introduction of an amino-group at
the C-20 of the steroidal skeleton as an intriguing modification
since close related positions of natural and synthetic ligands are in-
volved in key interactions with amino acid residues of the recep-
tor.11 Particularly, the crystal structures of the ligand-binding
domains (LBDs) of both the wtAR and the T877A mutant AR bound
to DHT revealed that the 17b-hydroxy group is involved in key
interactions with the protein.11a The ligand is located at similar po-
sition within the AR LBD or AR-T877A LBD and interacts similarly
with the amino acid residues except that of threonine. The muta-
tion T877A contributes to the broader ligand specificity of the AR
mutant. For example, cyproterone acetate, a steroidal antiandrogen
bearing a bulky substituent at C-17, induces such conformational
changes to the protein in the presence of T877 that ligand-binding
pocket is expanded and becomes susceptible to accommodate the
bulky group. Consequently, cyproterone is tightly bound to the
AR-T877A LBD and acts as an agonist. On the contrary, other com-
pounds, bearing bulky side chains at the C-16, C-18 or C-20, repo-
sition of the helix-12 and prevent the recruitment of coactivators
leading to the AR inhibition.12 Based on these observations, we in-
tended to exploit the influence of an amino-group insertion at C-20
of pregnenolone in regard to the AR activity. Moreover, although
steroidal skeleton has been exhaustively modified, to the best of
our knowledge, C-20-lactame ring steroids have not been synthe-
sized to date, thus providing a challenging synthetic target with
potential activity against AR.
2. Chemistry
Nucleophilic addition of organometallic reagents to N-tert-
butanesulfinyl imines is a well established method for the stereo-
selective synthesis of a
-branched amines.13 Utilizing this synthetic
methodology we planned to transform initially the C-20 of preg-
nenolone into a new amino-substituted stereogenic center which
was anticipated afterwards to provide us access to novel 20-ami-
no-functionalized steroids.
To that purpose, condensation of 3b-TBS-protected-pregneno-
lone 114 with the chiral auxiliary (S)-(ꢀ)-2-methyl-2-propanesulfi-
namide afforded under optimized conditions the corresponding
(20SS,E)-imine 2 as a single geometrical isomer whose structure
established by X-ray analysis (Scheme 1).15 Nucleophilic addition
of allylmagnesium bromide to 2 completed in a stereoselective
fashion providing the sulfinamide 3 as a single diastereomer in
80% yield. Assignment of stereochemistry to the newly created ste-
reogenic center C-20 was enabled after removal of the chiral aux-
iliary from 3. Thus, treatment with hydrogen chloride resulted in
Scheme 1. Synthesis of (20R)-aminosteroids. Reagent and conditions: (a) (S)-(ꢀ)-2-Methyl-2-propanesulfinamide, Ti(OEt)4, 90 °C, 48 h, 63%; (b) 3: allylMgBr, CH2Cl2 , ꢀ78 °C
to rt, overnight, 80%; 5: 1-methyl-2-propenylmagnesium chloride, CH2Cl2, ꢀ78 °C to rt, overnight, 63%; 7: benzylMgCl, CH2Cl2, ꢀ78 °C to rt, overnight, 73%; (c) HCl, MeOH, rt,
30 min, (4: 91%, 6: 80%, 8: 93%); (d) acryloyl chloride, Et3N, CH2Cl2, H2O, 0 °C to rt, 21 h (9: 23%, 10: 44%); (e) NaOCH3, CH3OH, rt, 6 h, 81%; (f) 2nd generation Grubbs catalyst
(5%), CH2Cl2, reflux, 4.5 h, 92%; (g) Al(i-PrO)3, cyclohexanone, benzene, 80 °C, 16 h, 58%; TBS, tert-butyldimethylsilyl.

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M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
the cleavage of the tert-butanesulfinyl moiety and concomitant re-
moval of the 3-TBS-protective group, thereby affording compound
4. The absolute configuration of 4 was determined by X-ray analy-
sis proving the (20R)-stereochemistry.
reactions as described above, 15 was transformed to the steroidal
derivatives 18 and 19 via cyclization of 17.
3. Results and discussion
Extensive trials to substitute the imine 2 with various Grignard
reagents (pentyl-, hexyl-, isohexyl-, phenyl-, 4-methoxybenzyl-,
vinyl- or prenyl-), n-butyl lithium or the Reformatsky reagent de-
rived from ethyl bromoacetate, led either to decomposition or
recovery of the starting material. However, addition of 1-methyl-
2-propenylmagnesium chloride or benzylmagnesium chloride
afforded the corresponding sulfinamides 5 and 7 as single diaste-
reomers which upon acidic methanolysis furnished the hydrochlo-
ric salts 6 and 8, respectively (Scheme 1).
Further attempts to generate functionalized (20R)-aminoster-
oids were pursued using the steroidal amine 4. Acylation with
acryloyl chloride in the presence of triethyl amine under
Schotten–Bauman conditions led to the biacylated 9 and the
monoacylated product 10 (Scheme 1). Basic hydrolysis of the
3-acylgroup of 9 resulted in the recovery of the monoacylated
product 10. Subsequently, ring closing metathesis of 10 using
2nd generation Grubbs catalyst furnished the C-20 lactame ring
steroidal derivative 11 in excellent yield. Eventually, the latter
was easily converted to the corresponding testosterone derivative
12 after Oppenauer oxidation of the 3b-hydroxy-group and subse-
quent migration of the double bond to the more stable enone
system.
The novel 20-aminosteroids were first evaluated for agonistic or
antiandrogenic activity of human AR (Fig. 1) performing transient
transfection assays in CV1 cells expressing human AR with cotrans-
fection of MMTV-luc, a well-known AR responsive promoter.18
None of the tested compounds induced the AR-mediated transacti-
vation (Fig. 1A) suggesting that none of these derivatives possess
androgenic activity for the wtAR. The androgen-induced wtAR
transactivation was potently inhibited by 11, 12, and 19 and weak-
er by 18 suggesting that these compounds act as complete AR
antagonists. Also, we compared 0.1 to 10 micromolar range for
both OH-Fl and Casodex and observed that higher doses of these
AR antagonists activate the wtAR, strongly by OH-Fl and Casodex
at a lower level (Fig. 1S, Supplementary data). Determination of
IC50 values as inhibition of AR-mediated transactivation by 50% re-
vealed for compounds 11 and 12 about 2
and for 19 about 0.5 M. However, comparing the IC50 values of
our new synthesized compounds to those of therapeutically used
AR antagonists like flutamide (IC50 = 110 M) or Casodex
(IC50 = 0.3–0.9
M)19 we achieve similar or better potency. Coinci-
lM, for 18 about 3 lM,
l
l
l
dentally no inhibition of androgen-induced wtAR transactivation
was observed by derivatives 4, 6, 8, and 15 (Fig. 1A).
To investigate the impact of the 20-amino moiety configuration
on the biological activity, we turned our attention to the synthesis
of the diastereomeric (20S)-aminosteroids. We postulated that the
intermediate (20Rs)-imine with opposite chirality at sulfur would
provide us access to these products. Condensation of 1 with the
chiral auxiliary (R)-(+)-2-methyl-2-propanesulfinamide (Scheme
2) afforded exclusively the (20RS,E)-imine 13 (structure verified
by X-ray crystallography).16 All trials to substitute 13 with ethyl-
magnesium bromide under various experimental conditions failed,
confirming the previous findings obtained for the imine 2.
Nevertheless, addition of allylmagnesium bromide proceeded dia-
stereoselectively providing the sulfinylamide 14 as a single diaste-
reomer, whose structure was determined by X-ray analysis of the
hydrochloric salt 15.17 To our delight, assignment of the
stereochemistry revealed the (20S)-configuration confirming our
expectations. Subsequently, following the same sequence of
Using the AR-T877A mutant, which is known to be able to by-
pass the beneficiary effects of androgen ablation and hormone
therapy by androgen antagonists, a different response of the
20-amino steroidal derivatives was provided (Fig. 1B). Interest-
ingly, compounds 4, 6, and 15 exhibited potent agonistic activities.
The compounds 8, 12, and 19 led to weak induction of the AR-
T877A-mediated transactivation whereas 11 and 18 did not reveal
agonistic activity for the mutant AR. Noteworthy, in the presence
of R1881, a well known and highly potent AR agonist, the andro-
gen-induced AR-mediated transactivation was inhibited by 18.
Compound 11 exhibited a partial inhibition of AR-T877A whereas
the other compounds did not show an inhibition. These data,
(Fig. 2S, Supplementary data) reveal that the tested compounds
differently inhibit or activate the wtAR and the AR-T877A mutant
acting as complete or mixed AR antagonists. Clearly, they present
selectivity either for the wtAR (12, 19) or the AR-T877A mutant
Scheme 2. Synthesis of (20S)-aminosteroids. Reagent and conditions: (R)-(+)-2-Methyl-2-propanesulfinamide, Ti(OEt)4, 83 °C, 31.5 h, 85%; (b) allylMgBr, CH2Cl2, ꢀ78 °C to rt,
overnight, 89%; (c) HCl, MeOH, rt, 2 h, 95%; (d) acryloyl chloride, Et3N, CH2Cl2, H2O, 0 °C to rt, 21 h (16: 23%, 17: 46%); (e) NaOCH3, CH3OH, rt, 6 h, 99%; (f) 2nd generation
Grubbs catalyst (5%), CH2Cl2, reflux, 4 h, 84%; (g) Al(i-PrO)3, cyclohexanone, benzene, 80 °C, 15 h, 50%; TBS, tert-butyldimethylsilyl.

M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
6963
Figure 1. AR agonistic and antagonistic activities of 20-aminosteroids comparing wild-type AR (wtAR) and the AR-T877A mutant. (A) CV1 cells were transfected with the
expression vector for human wild-type AR and the reporter MMTV-luciferase either untreated, treated with or without the compounds, or the potent AR-agonist
methyltrienolone (R1881; KD value for nuclear AR lies in between 5 and 12 nM23). Luciferase units obtained were normalized to the cotransfected pCMX-LacZ as internal
control and values are indicated as relative light units (RLU). As control, cells were treated with the AR-antagonist NBBS (N-butylbenzene-sulfonamide; indicated as ‘N’), or
with the solvent DMSO alone. As further controls, cells were incubated without solvent. (B) The compounds were tested individually or combined with R1881 as described
above, except the expression vector for the human AR-T877A mutant was used.
(18) indicating that only one amino acid exchange in the AR ligand-
binding domain has an important impact on the action of these
compounds. Nevertheless, derivative 18 serves as a promising anti-
androgen inhibiting potently both the wtAR and the T877A mutant
AR.
onists displaying a potency comparable to that of the well-known
AR antagonists flutamide and Casodex.
Further we analyzed the growth of LNCaP cells treating them
with 11 and 18 for the indicated days (Fig. 2B). Atraric acid (AA)
was used as positive control known to inhibit LNCaP cell growth.20
Concentration series showed that derivative 18 repressed wtAR
At 10 lM 18 potently inhibited LNCaP cell growth whereas at 1 lM
and AR-T877A mediated transactivation at 1
mentary data).
l
M (Fig. 3S, Supple-
the inhibition was only slight. Also 11 inhibited LNCaP cell growth
albeit a bit weaker compared to 18. Therefore, we conclude that 18
is a potent growth inhibitor of LNCaP cells. To exclude, however, a
general growth inhibition by 18 we used the human PCa cell line
PC3, that lacks functional AR. Treatment of PC3 cells with 18 did
not affect cell growth (Fig. 2C) indicating that 18 is not a general
inhibitor of cell growth.
Taken together, the data suggest that the derivatives 11 and 18
are potent AR antagonists, 18 being a more potent inhibitor of cell
growth.
To confirm AR antagonism we used the human origin LNCaP
PCa cells expressing endogenously the AR-T877A mutant. The
LNCaP cell line is known to grow androgen-dependently and thus
also useful for androgen-regulated growth studies. To determine
AR antagonism on an endogenous gene we analyzed the expression
of the prostate specific antigen (PSA) known as a diagnostic marker
for PCa. Quantitative real-time reverse transcription PCR (qRT-PCR)
was used to determine the PSA mRNA levels of LNCaP cells
(Fig. 2A). Compounds 11 and 18 showed potent inhibition of
To get insights into the underlying molecular pathway of the
growth inhibition we focused on compound 18 being the most po-
tent inhibitor of LNCaP cell growth. Treating cells with 18 did not
result in enhanced apoptosis. A further possibility for reducing
PSA-expression. Compound
6 increased PSA-expression while
compound 19 exhibited weak AR antagonism, which is in line with
the reporter assays (Fig. 1B). Thus, 11 and 18 emerged as AR antag-

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M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
hormone-induced transactivation of these receptors (data not
shown). Unlike the AR natural ligand DHT which bears a 3-keto-
group both of these derivatives bear a 3b-hydroxy-group. Thus,
the 3b-hydroxy-group emerges as a preferable substituent of these
novel antiandrogens contributing to the maintenance of the AR
specificity.
To our knowledge 11 and 18 are the first steroids that act as
complete AR antagonists and exhibit AR specificity.
Interestingly, 11 and 18 inhibited the growth of the PCa cell line
LNCaP. These cells express the AR-T877A mutant which is unex-
pectedly activated by the non-steroidal antiandrogen OH-F.10 Since
11 and 18 inhibit the growth of LNCaP cells they may serve as
advantageous tumor inhibitors with minimal risk of therapy resis-
tance occurrence in contrast to the currently used antiandrogens.
The obtained results offered evidence that the underlying inhi-
bition mechanism of AR transactivation by derivatives 11 and 18
could be partly based on the induction of cellular senescence, a
phenomenon that is known to induce irreversibly cell cycle
arrest.22
Comparing the efficacy on cell growth inhibition by 18 with
that of Casodex, the compound 18 exhibits a similar inhibition at
one micromolar concentration and a higher inhibition at 10 lM
of LNCaP cell growth (Fig. 3). On the other hand comparing the ef-
fects on inhibition of AR-mediated transactivation, Casodex exhib-
its a more potent antagonism of AR-mediated transactivation at
one micromolar concentration or lower (see Fig. 1S, Supplemen-
tary data) as compared to 18. Therefore, the data suggest that dif-
ferent molecular mechanisms of 18 and Casodex are used to induce
growth inhibition or to inhibit AR-mediated transactivation. Taken
together, compound 18 emerged as an AR antagonist with a higher
potency of growth inhibition compared to the well-known AR
antagonist Casodex.
Figure 2. (A) Compounds 11 and 18 potently inhibit the endogenous androgen-
induced PSA gene expression. Endogenously expressed mRNA levels of the PSA
gene, a direct AR target gene, were detected using qRT-PCR. LNCaP cells were
treated with or without androgens plus the indicated compounds (10 lM). DMSO
treated cells were used as controls. Values were normalized to the expression of
beta-actin. (B) Compounds 18 and 11 inhibit growth of the LNCaP cells. Indicated
concentrations of 18 and 11 were tested for the ability to influence growth of the
model human PCa cell line LNCaP. Equal amounts of cells were seeded out and the
number of cells was counted at the indicated days and normalized to the cells of
day 0. The known AR antagonist atraric acid (AA) served as positive control. (C)
Compound 18 does not affect the growth of the PC3 cells lacking AR expression.
Growth analysis was performed as described in (B).
4. Conclusion
Newly synthesized 20-aminosteroids were evaluated for their
activity against AR. Among them, derivatives 11 and 18, which bear
a C-20 lactame ring moiety, acted as complete AR antagonists and
exhibited AR specificity. Moreover, 11 and 18 inhibited the cell
growth of androgen-dependent prostate cancer cells and the
growth is the phenomenon of cellular senescence leading to an
irreversible cell cycle arrest of metabolic active cells. A very spe-
cific marker for cellular senescence is the activity of the senes-
cence-associated b-galactosidase (SA-b-Gal).21 Cells undergoing
cellular senescence remain metabolically active but exhibit an irre-
versible growth arrest. Interestingly, treating LNCaP cells with 18
induced the number of SA-b-gal expressing cells (Fig. 4S, Supple-
mentary data). This suggests that 18 enhances cellular senescence
that might explain in part the growth inhibition mediated by 18.
Interestingly, whereas the 3-keto derivative 19 activated the
AR-T877A mutant, the corresponding 3b-hydroxy-one 18 inhibited
it, suggesting that the 3b-hydroxy-group is crucial for the AR-med-
iated transactivation. Moreover, while the 3-keto diastereomers 12
and 19 acted almost similarly, only 19 proved significantly able to
inhibit the ligand-activated AR-T877A. On the other hand, the
3b-hydroxy diastereomers 11 and 18, acted very similarly against
both the wtAR and the AR-T877A mutant, indicating that the con-
figuration of the lactamic ring is not critical for the antiandrogenic
activity. Albeit, the C-20 lactame moiety represents a unique struc-
tural feature of the new compounds that contributes substantially
to the biological activity as the simple steroidal amines 4, 6, 8 and
15 exhibited no antiandrogenic effects. Also, these findings confirm
in part our thoughts that a bulky substituent at C-20 might disturb
the right folding of the protein inducing antagonistic effects,
although further studies are necessary to confirm this hypothesis.
Importantly, compounds 11 and 18 showed the highest speci-
ficity for AR without inducing transactivation of glucocorticoid
receptor or progesterone receptor and only weakly inhibiting the
Figure 3. Compound 18 inhibits more potently the growth of human androgen-
dependent PCa cells compared to Casodex. LNCaP cells were treated with Casodex
(Cas) or compound 18 at concentrations of 10ꢀ5 or 10ꢀ6 M for 8 days. The number of
cells treated with the solvent as control was set arbitrarily to 100%. The deviation of
three independent experiments is shown.

M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
6965
expression of the prostate specific antigen. Remarkably, enhance-
ment of the cellular senescence, that might explain in part the
mediated growth inhibition, was induced by compound 18.
In summary, the newly synthesized 20-aminosteroids may
serve not only as a novel class of AR antagonists but also as valu-
able chemical tools for the investigation of novel inhibitory mech-
anisms of the AR transactivation and prostate cell growth.
Specifically, the 20-aminosteroidal derivative 18 figures as a prom-
ising lead compound for the development of more potent AR
antagonists with pure antagonistic activities against AR variants
and as an effective inhibitor of PCa growth.
layers were washed with brine, dried over anhydrous Na2SO4, fil-
tered and evaporated under reduced pressure. The crude residue
was purified by silica gel column chromatography (10–20% EtOAc
in n-hexane) to afford 2 (187 mg, 63%) as a white crystalline solid;
mp 182–184 °C; ½a D23
ꢁ
= +89.9 (c 0.623 in CHCl3); IR (KBr): mmax
2933, 1608, 1094, 1068, 838 cmꢀ1
;
1Y NMR (400 VHz, CDCl3): d
0.05 (s, 6Y), 0.68 (s, 3Y), 0.89 (s, 9Y), 1.00 (s, 3Y), 1.25 (s, 9Y),
2.32 (s, 3Y), 3.44–3.52 (m, 1Y), 5.31 (d, J = 5.2 Hz, 1Y); 13C NMR
(100 MHz, CDCl3): d -4.59, 13.43, 18.24, 19.42, 21.09, 22.38,
23.70, 24.29, 25.06, 25.92, 31.78, 32.02, 32.03, 36.60, 37.37,
38.91, 42.77, 44.64, 50.10, 56.28, 56.99, 63.40, 72.53, 120.81,
141.62, 185.63; HRMS-ESI (m/z): [M+H]+ calcd for C31H56NO2SSi:
534.37955, found: 534.37988.
5. Experimental section
5.1. Chemistry
5.1.2. (20R)-N-[t-Butyl-(S)-sulfinyl]-20-allyl-3b-(t-butyldimeth-
ylsilyloxy)-pregn-5-en-20-amine (3)
All reagents were obtained commercially from Acros, Alfa Aesar,
Sigma-Aldrich or Merck and used without further purification.
Reactions involving moisture-sensitive reactants were run in
flame-dried glassware under an atmosphere of argon. Reagents
and anhydrous solvents were transferred via syringe. Dichloro-
methane was distilled under argon from SICAPENT (phosphorus
pentoxide on solid support with indicator). Benzene and methanol
were purchased in anhydrous form and used without further puri-
fication. Analytical TLC was performed on Merck Silica gel 60 F254
on precoated silica gel plates, with visualization under UV light
(254 and 365 nm) or/and by use of Seebach staining solution. Flash
column chromatography was performed on silica gel (Acros 60A,
0.035–0.070 mm). 1H and 13C NMR spectra were recorded on a Var-
ian Gemini-300BB (300 MHz for 1H; 75 MHz for 13C), Varian Mer-
cury-300BB (300 MHz for 1H; 75 MHz for 13C) and Varian
Mercury-400BB spectrometer (400 MHz for 1H; 100 MHz for 13C).
The chemical shifts (d) are reported in parts per million (ppm)
and the residual solvent signal is used as an internal standard.
The following abbreviations are used for the proton spectra multi-
plicities: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), broad signal (br). Coupling constants (J) are given in Hertz
(Hz). High resolution mass spectra were obtained on a Bruker Dal-
tonics APEX II (for ESI). Infrared spectra were obtained on an ATI/
MATTSON Genesis FT-IR in KBr disks. Absorbance frequencies are
reported in reciprocal centimeters (cmꢀ1). Melting points were
measured with a Büchi Melting Point B-540 and are uncorrected.
To a solution of 2 (258.8 mg, 0.4847 mmol) in dry CH2Cl2
(3.1 mL) a solution of allylmagnesium bromide 1.0 M in diethyl
ether (0.97 mL, 0.97 mmol) was added dropwise at ꢀ78 °C under
argon atmosphere. The reaction mixture was allowed to warm
up slowly to rt with stirring overnight. Then it was quenched with
saturated aqueous NH4Cl and diluted with ethyl acetate. The or-
ganic layer was removed and the aqueous layer was extracted
with ethyl acetate (3ꢂ). The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure. The crude residue was purified by silica
gel column chromatography (10–20% EtOAc in n-hexane) to afford
3 (222 mg, 80%) as an oil; ½a D23
ꢁ
= ꢀ25.1 (c 0.303, CHCl3); IR (KBr):
mmax 2931, 1472, 1462, 1436, 1252, 1093, 1071, 835 cmꢀ1
;
1Y
NMR (400 MHz, CDCl3): d 0.05 (s, 6Y), 0.85 (s, 3Y), 0.88 (s, 9Y),
0.99 (s, 3Y), 1.22 (s, 9Y), 1.33 (s, 3Y), 2.39 (dd, J = 13.9, 8.3 Hz,
1Y), 2.53 (dd, J = 14.1, 6.2 Hz, 1Y), 3.34 (s, 1Y), 3.44–3.51 (m,
1Y), 5.12–5.17 (m, 2Y), 5.31 (d, J = 5.2 Hz, 1Y), 5.90–6.00 (m,
1Y); 13C NMR (100 MHz, CDCl3): d -4.59, 15.06, 18.24, 19.39,
20.87, 22.88, 23.01, 23.74, 24.76, 25.92, 31.34, 31.71, 32.04,
36.56, 37.34, 40.60, 42.77, 43.14, 47.56, 49.96, 55.93, 56.55,
57.90, 61.51, 72.52, 118.88, 120.92, 134.03, 141.54. HRMS-ESI
(m/z): [M+H]+ calcd for C34H62NO2SSi: 576.42650, found:
576.42610.
5.1.3. (20R)-N-[t-Butyl-(S)-sulfinyl]-3b-(t-butyldimethylsilyloxy)
-20-(1-methyl-2-propenyl)-pregn-5-en-20-amine (5)
Optical rotations were determined with
a
half-automatic
To a solution of 2 (100 mg, 0.187 mmol) in dry CH2Cl2 (1.2 mL)
a solution of 1-methyl-2-propenylmagnesium chloride 0.5 M in
THF (0.756 mL, 0.378 mmol) was added dropwise at ꢀ78 °C under
argon atmosphere. The reaction mixture was allowed to warm up
slowly to rt with stirring overnight. Then it was quenched with
saturated aqueous NH4Cl and diluted with ethyl acetate. The or-
ganic layer was removed and the aqueous layer was extracted
with ethyl acetate (3ꢂ). The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure. The crude residue was purified by silica
gel column chromatography (10–30% EtOAc in n-hexane) to afford
Schmidt+Haensch Polartronic D MHZ-8 at the sodium-D line
(589 nm) using a 50 mm path-length cell and are reported as fol-
lows ½a 2D3
ꢁ
, concentration (g/100 mL), and solvent. Preparative HPLC
was performed using Varian ProStar 210 solvent delivery module
with a Jasco-Lichrosorb column (Si 60, 10
l
m, 250 ꢂ 20 mm; elu-
ent MeOH 2% in CHCl3; flow rate of 10 ml minꢀ1) and a Varian Pro-
Star 325 UV–vis detector (k 254, 364 nm). Purity of the synthesized
compounds was determined by a combination of 1H, 13C NMR and
HLPC techniques and was found to be >95%.
5 (67.5 mg, 63%) as an oil; ½a D23
ꢁ
+13.6 (c 1.0, CHCl3); IR (KBr):
5.1.1. (20E)-N-[t-Butyl-(S)-sulfinyl]-3b-(t-butyldimethylsilyl-
oxy)-pregn-5-en-20-imine (2)
mmax 2957, 2931, 1471, 1463, 1090, 1067, 836 cmꢀ1
;
1Y NMR
To a mixture of protected-pregnenolone 1 (289 mg, 0.67 mmol)
and (S)-(ꢀ)-2-methyl-2-propanesulfinamide (68 mg, 0.559 mmol)
titanium(IV) ethoxide (1.17 mL, 5.59 mmol) was added at rt under
argon atmosphere. The reaction mixture was stirred at 90 °C for
48 h. After completion, it was allowed to cool to rt, cooled to 0 °C
and quenched with an equal volume of saturated NaCl aqueous
solution while vigorously stirring. The resulting suspension was fil-
tered through a Celite pad and the filtered cake rinsed thoroughly
with ethyl acetate. The two layers were separated and the aqueous
layer was washed with ethyl acetate (3ꢂ). The combined organic
(400 MHz, CDCl3): d 0.06 (s, 6Y), 0.83 (s, 3Y), 0.89 (s, 9Y), 0.99
(s, 3Y), 1.12 (s, 1.5Y), 1.13 (s, 1.5Y), 1.23 (s, 9Y), 1.35 (s, 3Y),
2.53–2.63 (m, 1Y), 3.29 (s, 1Y), 3.44–3.52 (m, 1Y), 5.02–5.09 (m,
2Y), 5.31 (d, J = 5.2 Hz, 1Y), 5.97 (ddd, J = 16.9, 10.3, 8.6 Hz, 1Y);
13C NMR (100 MHz, CDCl3): d -4.61, 14.69, 15.54, 18.26, 19.36,
20.90, 22.97, 24.01, 24.11, 25.92, 29.69, 31.21, 31.74, 32.00,
36.52, 37.30, 40.74, 42.72, 43.43, 48.65, 49.88, 56.12, 56.42,
56.52, 64.00, 72.50, 115.72, 120.98, 141.39, 141.46; HRMS-ESI
(m/z): [M+H]+ calcd for C35H64NO2SSi: 590.44215; found:
590.44161.

6966
M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
5.1.4. (20R)-N-[t-Butyl-(S)-sulfinyl]-20-benzyl-3b-(t-butyldi-
methylsilyloxy)-pregn-5-en-20-amine (7)
mmax 2933, 1472, 1462, 1252, 1093, 1071, 835 cmꢀ1 1Y NMR
;
(400 MHz, CDCl3): d 0.05 (s, 6H), 0.82 (s, 3Y), 0.88 (s, 9Y), 0.98 (s,
3Y), 1.20 (s, 9Y), 1.39 (s, 3Y), 2.41 (dd, J = 13.8, 8.4 Hz, 1Y), 2.53
(dd, J = 13.9, 6.3 Hz, 1Y), 3.31 (s, 1Y), 3.43–3.52 (m, 1Y), 5.06–
5.13 (m, 2Y), 5.30 (d, J = 5.3 Hz, 1Y), 5.81 (dddd, J = 16.6, 10.1,
8.4, 6.3 Hz, 1Y); 13C NMR (100 MHz, CDCl3): d -4.60, 15.02, 18.26,
19.36, 20.83, 22.35, 22.79, 23.63, 24.88, 25.93, 31.31, 31.68,
32.02, 36.53, 37.32, 40.08, 42.75, 42.84, 48.75, 49.94, 55.80,
56.45, 57.75, 60.91, 72.53, 118.30, 120.98, 134.26, 141.51; HRMS-
ESI (m/z): [M+H]+ calcd for C34H62NO2SSi: 576.42650, found:
576.42588.
To a solution of 2 (100 mg, 0.187 mmol) in dry CH2Cl2 (1.2 mL) a
solution of benzylmagnesium chloride 20% wt in THF (1.5 mL,
1.988 mmol) was added dropwise at ꢀ78 °C under argon atmo-
sphere. The reaction mixture was allowed to warm up slowly to
rt with stirring overnight. Then it was quenched with saturated
aqueous NH4Cl and diluted with ethyl acetate. The organic layer
was removed and the aqueous layer was extracted with ethyl ace-
tate (3ꢂ). The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, filtered and evaporated under re-
duced pressure. The crude residue was purified by silica gel col-
umn chromatography (10–30% EtOAc in n-hexane) to afford 7
5.1.7. General procedure for the synthesis of the 20-aminoster-
oids (4), (6), (8) and (15)
(85.8 mg, 73%) as an oil; ½a D23
ꢁ
= +10.8 (c 0.89, CHCl3); IR (KBr): mmax
2931, 2855, 1496, 1472, 1462, 1252, 1093, 1067, 835 cmꢀ1
;
1Y
To a solution of the appropriate sulfinamide (1 equiv) in MeOH
a 5–6 N solution of HCl in iso-propanol (ꢃ2.0 equiv) was added.
The mixture was stirred for the indicated time at rt and then con-
centrated to near dryness. Diethyl ether was added and the prod-
uct precipitated as solid. The precipitate was filtered off, washed
with diethyl ether to provide the corresponding amine as hydro-
chloride salt which was used without further purification.
NMR (400 MHz, CDCl3): d 0.05 (s, 6Y), 0.88 (s, 9Y), 0.93 (s, 3Y),
0.98 (s, 3Y), 1.19 (s, 9Y), 1.33 (s, 3Y), 2.94 (dd, J = 78.3, 13.0 Hz,
2H), 3.42–3.50 (m, 1Y), 3.56 (s, 1Y), 5.30 (d, J = 5.1 Hz, 1Y), 7.16–
7.29 (m, 5Y); 13C NMR (100 MHz, CDCl3): d -4.61, 14.75, 18.23,
19.35, 20.75, 22.86, 23.14, 23.85, 25.91, 26.82, 31.27, 31.66,
31.97, 36.49, 37.23, 39.97, 42.73, 43.03, 47.33, 49.73, 56.00,
56.49, 56.64, 61.56, 72.49, 120.86, 126.38, 127.88, 131.06, 137.39,
141.52; HRMS-ESI (m/z): [M+H]+ calcd for C38H64NO2SSi:
626.44215, found: 626.44212, [M+Na]+ calcd for C38H63NNaO2SSi:
648.42410, found: 648.42352.
5.1.7.1. (20R)-20-Allyl-20-amino-pregn-5-ene-3b-ol hydrochlo-
ride (4). Compound 4 was synthesized according to the general
procedure from compound 3 (322 mg, 0.559 mmol), using a 5–
6 N solution of HCl in iso-propanol (ꢃ2.0 equiv, 0.22 mL) in MeOH
(1.78 mL). The reaction mixture was stirred for 30 min at rt. Yield:
5.1.5. (20E)-N-[t-Butyl-(R)-sulfinyl]-3b-(t-butyldimethylsilyl-
oxy)-pregn-5-en-20-imine (13)
201.2 mg (91%), white solid; mp 322–324 °C (dec.); ½a D23
= ꢀ52.8 (c
ꢁ
To a mixture of 1 (517 mg, 1.2 mmol) and (R)-(+)-2-methyl-2-
propanesulfinamide (242.4 mg, 2 mmol) titanium(IV) ethoxide
(2.26 mL, 10.8 mmol) was added at rt under argon atmosphere.
The reaction mixture was stirred at 83 °C for 31.5 h. Then, it was
allowed to cool to rt, cooled to 0 °C and quenched with an equal
volume of saturated NaCl aqueous solution while vigorously stir-
ring. The resulting suspension was filtered through a Celite pad
and the filtered cake rinsed thoroughly with ethyl acetate. The
two layers were separated and the aqueous layer was washed with
a mixture of ethyl acetate–tetrahydrofuran (3ꢂ). The combined or-
ganic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. The crude
residue was purified by silica gel column chromatography (10–20%
EtOAc in n-hexane) to afford 13 (542 mg, 85%) as a white crystal-
0.394, CH3OH); IR (KBr): mmax 3076, 2930, 1461, 1437, 1056,
933 cmꢀ1 1H NMR (400 VHz, CD3OD): d 0.92 (s, 3H), 1.03 (s,
;
3H), 1.41 (s, 3H) 2.43 (dd, J = 14.5, 7.0 Hz, 1H), 2.52 (dd, J = 14.5,
7.9 Hz, 1H), 3.37–3.43 (m, 1H), 5.26–5.31 (m, 2H), 5.35 (d,
J = 5.3 Hz, 1H), 5.84–5.94 (m, 1H); 13C NMR (100 MHz, CD3OD): d
14.62, 19.86, 21.96, 23.87, 24.00, 24.54, 32.30, 32.72, 32.77,
37.70, 38.57, 40.50, 43.02, 44.07, 44.28, 51.44, 58.01, 58.31,
61.08, 72.41, 121.90, 122.21, 132.13, 142.33; HRMS-ESI (m/z):
[M]+ calcd for C24H40NO: 358.31044, found: 358.31032.
5.1.7.2.
(20R)-20-Amino-20-(1-methyl-2-propenyl)-pregn-5-
was synthesized
ene-3b-ol hydrochloride (6). Compound
6
according to the general procedure from compound 5 (59.7 mg,
0.101 mmol), using a 5–6 N solution of HCl in iso-propanol (ꢃ2.0
equiv, 0.04 ml) in MeOH (0.32 mL). The reaction mixture was stir-
red for 30 min at rt. Yield: 33.1 mg (80%), brownish solid; mp 279–
line solid; mp 184.5–186.5 °C; ½a D23
= ꢀ34.0 (c 1.0, CHCl3); IR
ꢁ
(KBr): m
max 2934, 1618, 1081, 839 cmꢀ1; 1Y NMR (400 MHz, CDCl3):
d 0.05 (s, 6Y), 0.65 (s, 3Y), 0.88 (s, 9Y), 0.99 (s, 3Y), 1.24 (s, 9Y), 2.32
(s, 3Y), 3.44–3.52 (m, 1Y), 5.32 (d, J = 5.2 Hz, 1Y); 13C NMR
(100 MHz, CDCl3): d -4.60, 13.50, 18.26, 19.41, 21.11, 22.20,
24.21, 24.23, 24.81, 25.92, 31.73, 32.02, 32.03, 36.58, 37.35,
39.09, 42.75, 44.78, 50.03, 56.09, 57.06, 63.12, 72.51, 120.87,
141.52, 186.23; HRMS-ESI (m/z): [M+Na]+ calcd for C31H55NNaO2S-
Si: 556.36150, found: 556.36200.
280 °C (dec.); ½a D23
ꢁ
= ꢀ9.0 (c 0.621, CH3OH); IR (KBr): mmax 3379,
2932, 1638, 1504, 1071, 937 cmꢀ1
;
1H NMR (400 MHz, CD3OD): d
0.94 (s, 3H), 1.04 (s, 3H), 1.13 (s, 1.5H), 1.15 (s, 1.5H), 1.29 (s,
3H), 2.70 (qd, J = 13.9, 6.9 Hz, 1H), 3.36–3.44 (m, 1H), 5.24–5.31
(m, 2H), 5.36 (d, J = 5.2 Hz, 1H), 5.87 (ddd, J = 17.0, 10.1, 9.1 Hz,
1H); 13C NMR (100 MHz, CD3OD): d 14.17, 15.88, 19.86, 20.57,
22.00, 24.39, 24.48, 32.30, 32.71, 32.79, 37.69, 38.55, 40.45,
43.01, 44.31, 46.70, 51.36, 55.26, 57.83, 63.82, 72.41, 119.52,
122.22, 138.50, 142.31; HRMS-ESI (m/z): [MꢀCl]+ calcd for
5.1.6. (20S)-N-[t-Butyl-(R)-sulfinyl]-20-allyl-3b-(t-
butyldimethylsilyloxy)-pregn-5-en-20-amine (14)
C25H42NO: 372.32609, found: 372.32602.
To a solution of 13 (300 mg, 0.562 mmol) in dry CH2Cl2 (3.6 mL)
a solution of allylmagnesium bromide 1.0 M in diethyl ether
(1.12 mL, 1.12 mmol) was added dropwise at ꢀ78 °C under argon
atmosphere. The reaction mixture was allowed to warm up slowly
to rt with stirring overnight. Then it was quenched with saturated
aqueous NH4Cl and diluted with ethyl acetate. The organic layer
was removed and the aqueous layer was extracted with ethyl ace-
tate (3ꢂ). The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, filtered and evaporated under re-
duced pressure. The crude residue was purified by silica gel col-
umn chromatography (10–20% EtOAc in n-hexane) to afford 14
5.1.7.3. (20R)-20-Amino-20-benzyl-pregn-5-ene-3b-ol hydro-
chloride (8). Compound 8 was synthesized according to the gen-
eral procedure from compound 7 (68.2 mg, 0.109 mmol), using a
5–6 N solution of HCl in iso-propanol (ꢃ2.0 equiv, 0.044 ml) in
MeOH (0.34 mL). The reaction mixture was stirred for 30 min at
rt. Yield: 44.9 mg (93%), yellowish solid; mp 263–265 °C (dec.);
½
a 2D3
ꢁ
= ꢀ47.6 (c 0.807, CH3OH); IR (KBr): mmax 3397, 2930, 1605,
1498, 1060, 750, 705 cmꢀ1 1H NMR (300 VHz, CD3OD): d 0.97 (s,
;
3H), 1.04 (s, 3H), 1.37 (s, 3H), 3.04 (dd, J = 67.9, 13.8 Hz, 2H),
3.34–3.45 (m, 1H), 5.36 (d, J = 5.1 Hz, 1H), 7.28–7.42 (m, 5H); 13C
NMR (75 MHz, CD3OD): d 14.43, 19.86, 22.01, 22.61, 24.08, 24.64,
(287 mg, 89%) as an oil; ½a D23
= ꢀ56.2 (c 0.705, CHCl3); IR (KBr):
ꢁ

M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
6967
32.30, 32.73, 32.82, 37.69, 38.54, 40.90, 43.01, 44.40, 46.13, 51.40,
58.03, 58.62, 61.86, 72.41, 122.19, 128.80, 129.92, 132.06, 135.67,
142.35; HRMS-ESI (m/z): [MꢀCl]+ calcd for C28H42NO: 408.32609,
found: 408.32590.
column chromatography (35–40% EtOAc in n-hexane) to afford
10 (31.6 mg, 81%) as a white solid.
5.1.9. (20S)-20-Acrylamido-20-allyl-pregn-5-en-3b-ol acrylate
(16) and (20S)-20-acrylamido-20-allyl-pregn-5-en-3b-ol (17)
To a solution of 15 (130 mg, 0.33 mmol) in H2O (3.39 ml) and
triethylamine (0.827 mL, 5.939 mmol) dichloromethane (3.39 mL)
was added. The two-phase mixture was cooled at 0 °C and acryloyl
chloride (0.48 mL, 5.939 mmol) was added dropwise. The reaction
mixture was allowed to warm up and stirred at rt for 21 h. Then, it
was diluted with chloroform and the layers were separated. The
aqueous layer was washed with chloroform (4ꢂ) and the combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. The crude
residue was purified by silica gel column chromatography (20–40%
EtOAc in n-hexane) to afford 16 (36 mg, 23%) as a yellow solid and
5.1.7.4. (20S)-20-Allyl-20-amino-pregn-5-ene-3b-ol hydrochlo-
ride (15). Compound 15 was synthesized according to the general
procedure from compound 14 (263 mg, 0.457 mmol), using a
5–6 N solution of HCl in iso-propanol (ꢃ2.0 eq, 0.18 mL) in MeOH
(1.45 mL). The reaction mixture was stirred for 2 h at rt. Yield:
171.5 mg (95%), white solid; mp 282–284 °C (dec.); ½a D23
= ꢀ66.4
ꢁ
(c 0.506, CH3OH); IR (KBr): mmax 3283, 2926, 1613, 1593,
1530,1519, 1450, 1056, 951; 1H NMR (400 MHz, CD3OD): d 0.86
(s, 3H), 1.00 (s, 3H), 1.37 (s, 3H), 2.49 (d, J = 7.2 Hz, 2H), 3.31–
3.41 (m, 1H), 5.25 (d, J = 7.5 Hz, 1H), 5.28 (s, 1H), 5.32 (d,
J = 4.8 Hz, 1H), 5.84–5.94 (m, 1H); 13C NMR (100 MHz, CD3OD): d
14.48, 19.86, 21.99, 22.91, 23.69, 24.60, 32.30, 32.75, 32.80,
37.69, 38.55, 40.77, 43.01, 44.33, 44.96, 51.42, 56.99, 57.97,
61.10, 72.40, 121.98, 122.20, 132.29, 142.33; HRMS-ESI (m/z):
[M]+ calcd for C24H40NO: 358.31044, found: 358.31010.
17 (61.9 mg, 46%) as
= ꢀ71.4 (c 0.639, CHCl3); IR (KBr): mmax 3362, 2936, 1724,
1665, 1530, 1405, 1196 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.83
a white solid; 16: mp 158–160 °C;
½ ꢁ
a 2D3
;
(s, 3Y), 1.02 (s, 3Y), 1.42 (s, 3Y), 3.10 (dd, J = 13.6, 6.5 Hz, 1Y),
4.64–4.72 (m, 1Y), 5.05–5.10 (m, 2Y), 5.37–5.38 (m, 2Y), 5.55
(dd, J = 10.2, 1.5 Hz, 1Y), 5.71–5.81 (m, 2Y), 5.97–6.12 (m, 2Y),
6.18 (dd, J = 16.9, 1.5 Hz, 1Y), 6.37 (dd, J = 17.3, 1.5 Hz, 1Y); 13C
NMR (100 MHz, CDCl3): d 14.44, 19.27, 20.75, 22.85, 23.52, 23.65,
27.69, 31.32, 31.63, 36.54, 36.90, 38.04, 39.59, 42.70, 43.12,
49.76, 56.12, 56.25, 59.62, 74.01, 117.98, 122.48, 125.38, 128.98,
130.24, 132.27, 134.41, 139.60, 164.46, 165.60; HRMS-ESI (m/z):
[M+Na]+ calcd for C30H43NNaO3: 488.31352, found: 488.31390;
5.1.8. (20R)-20-Acrylamido-20-allyl-pregn-5-en-3b-ol acrylate
(9) and (20R)-20-acrylamido-20-allyl-pregn-5-en-3b-ol (10)
To a solution of 4 (201.2 mg, 0.51 mmol) in H2O (5.24 mL) and
triethylamine (1.279 mL, 9.19 mmol) dichloromethane (5.24 mL)
was added. The two-phase mixture was cooled at 0 °C and acryloyl
chloride (0.74 mL, 9.19 mmol) was added dropwise. The reaction
mixture was allowed to warm up and stirred at rt for 21 h. Then,
it was diluted with chloroform and the layers were separated.
The aqueous layer was washed with chloroform (4ꢂ) and the com-
bined organic layers were washed with brine, dried over anhy-
drous Na2SO4, filtered and evaporated under reduced pressure.
The crude residue was purified by silica gel column chromatogra-
phy (20–40% EtOAc in n-hexane) to afford 9 (53.9 mg, 23%) and
10 (91.6 mg, 44%) both as white solids; 9: mp 123–125 °C;
17: mp 194.5–196.5 °C; ½a D23
= ꢀ73.9 (c 0.460, CHCl3); IR (KBr):
ꢁ
mmax 3272, 3075, 2926, 1666, 1625, 1556, 1433, 1251, 1063 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 0.82 (s, 3Y), 0.99 (s, 3Y), 1.40 (s,
3Y), 3.10 (dd, J = 13.6, 6.5 Hz, 1Y), 3.46–3.56 (m, 1Y), 5.04–5.10
(m, 2Y), 5.33–5.35 (m, 2Y), 5.55 (d, J = 10.3 Hz, 1Y), 5.70–5.81 (m,
1Y), 6.00 (dd, J = 16.9, 10.2 Hz, 1Y), 6.18 (d, J = 16.8 Hz, 1Y); 13C
NMR (100 MHz, CDCl3): d 14.49, 19.34, 20.79, 22.87, 23.51, 23.57,
31.36, 31.56, 31.62, 36.44, 37.17, 39.62, 42.15, 42.67, 43.04,
49.85, 55.99, 56.32, 59.67, 71.61, 118.01, 121.42, 125.51, 132.17,
134.40, 140.80, 164.51; HRMS-ESI (m/z): [M+Na]+ calcd for
½
a 2D3
ꢁ
= ꢀ55.4 (c 0.808, CHCl3); IR (KBr): mmax 3351, 2944, 1725,
1696, 1656, 1541, 1413, 1215 cmꢀ1 1H NMR (400 MHz, CDCl3): d
;
0.85 (s, 3Y), 1.02 (s, 3Y), 1.40 (s, 3Y), 2.41 (dd, J = 13.9, 7.4 Hz
1Y), 2.73 (dd, J = 13.8, 7.3 Hz, 1Y), 4.64–4.72 (m, 1Y), 5.04–5.09
(m, 2Y), 5.39 (br s, 2Y), 5.57 (dd, J = 10.2, 1.4 Hz 1Y), 5.66–5.76
(m, 1Y), 5.80 (dd, J = 10.4, 1.5 Hz, 1Y), 5.93–6.19 (m, 2H), 6.21
(dd, J = 16.9, 1.4 Hz, 1H), 6.44 (ddd, J = 44.9, 17.3, 1.4 Hz, 1Y); 13C
NMR (100 MHz, CDCl3): d 14.99, 19.30, 20.80, 22.26, 23.15, 23.53,
27.71, 31.28, 31.60, 36.53, 36.91, 38.04, 39.31, 42.90, 43.05,
49.75, 55.69, 56.12, 59.76, 74.01, 118.35, 122.53, 125.56, 128.98,
130.30, 132.18, 134.01, 139.54, 164.71, 165.64; HRMS-ESI (m/z):
[M+H]+ calcd for C30H44NO3: 466.33157, found: 466.33166; 10:
C
27H41NNaO2: 434.30295, found: 434.30275.
5.1.9.1. Hydrolysis of compound (16). To a solution of 16
(29.6 mg, 63.6 mol) in dry MeOH (1.39 ml) sodium methylate
(15.1 mg, 279.5 mol) was added and the reaction mixture was
stirred for 6 h at rt under argon atmosphere. The solvent was re-
moved under reduced pressure and the crude residue was purified
by silica gel column chromatography (35–40% EtOAc in n-hexane)
to afford 17 (26 mg, 99%) as white solid.
l
l
mp 188–190 °C; ½a D23
ꢁ
= ꢀ88.8 (c 0.608, CHCl3); IR (KBr): mmax
5.1.10. General procedure for the synthesis of the 20-
aminosteroids (11) and (18)
3314, 2905, 1666, 1624, 1557, 1461, 1438, 1058 cmꢀ1
;
1Y NMR
(400 MHz, CDCl3): d 0.84 (s, 3Y), 0.99 (s, 3Y), 1.38 (s, 3Y), 2.37
(dd, J = 13.8, 7.4 Hz, 1Y), 2.76 (dd, J = 13.8, 7.2 Hz, 1Y), 3.48–3.56
(m, 1Y), 5.03–5.08 (m, 2Y), 5.34 (dd, J = 5.3, 1.7 Hz, 1Y), 5.39 (s,
1Y), 5.56 (dd, J = 10.2, 1.5 Hz, 1Y), 5.72 (tdd, J = 17.8, 10.5, 7.3 Hz,
1Y), 6.01 (dd, J = 16.9, 10.2 Hz, 1Y), 6.20 (dd, J = 16.9, 1.5 Hz, 1Y);
13C NMR (100 MHz, CDCl3): d 15.00, 19.36, 20.82, 22.24, 23.19,
23.54, 31.31, 31.58, 31.59, 36.42, 37.17, 39.29, 42.20, 42.86,
43.04, 49.84, 55.58, 56.16, 59.71, 71.61, 118.33, 121.47, 125.54,
132.16, 134.01, 140.72, 164.68; HRMS-ESI (m/z): [M+H]+ calcd for
To a solution of the appropriate 20-acrylamido derivative
(1 equiv) in dry and previously degassed dichloromethane, 2nd
generation Grubbs catalyst (5 mol %) was added at rt under an ar-
gon atmosphere. After being refluxed for the indicated time, the
reaction mixture was exposed to air with stirring for 1 h and the
solvent was evaporated under reduced pressure. The crude residue
was purified by silica gel column chromatography (2% MeOH in
dichloromethane) to afford the corresponding C-20 lactame ring
steroidal derivative.
C27H42NO2: 412.32101, found: 412.32119.
5.1.10.1.
17-[(6R)-6-Methyl-5,6-dihydropyridin-2(1H)-one-6-
5.1.8.1. Hydrolysis of compound (9). To a solution of 9 (43.9 mg,
0.094 mmol) in dry MeOH (2.06 ml) sodium methylate (22.4 mg,
0.415 mmol) was added and the reaction mixture was stirred for
6 h at rt under argon atmosphere. The solvent was removed under
reduced pressure and the crude residue was purified by silica gel
yl]-androst-5-ene-3b-ol (11). Compound 11 was synthesized
according to the general procedure from compound 10 (90.4 mg,
219.6
11 mol, 5 mol %) in dichloromethane (7 mL). The reaction mixture
was refluxed for 4.5 h. Yield: 77.5 mg (92%), white solid; mp 207–
lmol), using 2nd generation Grubbs catalyst (9.33 mg,
l

6968
M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
209 °C; ½a 2D3
ꢁ
= ꢀ53.5 (c 0.680, CHCl3); IR (KBr): mmax 3388, 2927,
1H NMR (400 MHz, CDCl3): d 0.83
according to the general procedure from compound 18. The reac-
1729, 1667, 1605, 1427 cmꢀ1
;
tion mixture was stirred at 80 °C for 15 h. Yield 10 mg (50%), white
(s, 3H), 1.00 (s, 3H), 1.34 (s, 3H), 2.60 (ddd, J = 18.0, 3.6, 2.3 Hz,
1H), 3.48–3.55 (m, 1H), 5.34 (d, J = 5.2 Hz, 1H), 5.48 (s, 1H), 5.89
(dd, J = 9.9, 1.6 Hz, 1H), 6.51 (td, J = 9.7, 4.2 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 14.36, 19.36, 20.78, 23.62, 23.64, 26.92,
31.26, 31.59, 35.72, 36.43, 37.16, 40.13, 42.20, 42.94, 49.85,
56.59, 57.58, 58.57, 71.65, 121.30, 123.54, 140.01, 140.83,
165.06; HRMS-ESI (m/z): [M+H]+ calcd for C25H38NO2: 384.28971,
found: 384.29008, [M+Na]+ calcd for C25H37NNaO2: 406.27165,
found: 406.27203.
solid; mp 226–228 °C; ½a D23
ꢁ
= +72.90 (c 0.417, CHCl3); IR (KBr): mmax
3431, 2927, 1676, 1612, 1414, 892, 825 cmꢀ1
;
1H NMR (400 MHz,
CDCl3): d 0.91 (s, 3H), 1.18 (s, 3H), 1.37 (s, 3H), 2.60 (td, J = 18.2,
2.8 Hz, 1H), 5.54 (s, 1H), 5.72 (s, 1H), 5.89 (d, J = 10.1 Yz, 1H),
6.49 (ddd, J = 9.9, 5.3, 3.1 Hz, 1H); 13C NMR (100 MHz, CDCl3): d
13.95, 17.34, 20.70, 23.42, 23.54, 27.26, 31.74, 32.73, 33.92,
34.88, 35.66, 36.08, 38.49, 39.79, 43.24, 53.55, 55.73, 57.71,
59.36, 123.17, 123.91, 139.90, 165.46, 170.87, 199.41; HRMS-ESI
(m/z): [M+Na]+ calcd for C25H35NNaO2: 404.25600, found:
404.25584.
5.1.10.2. 17-[(6S)-6-Methyl-5,6-dihydropyridin-2(1H)-one-6-yl]-
androst-5-ene-3b-ol (18). Compound 18 was synthesized
according to the general procedure from compound 17 (55.9 mg,
5.2. Biological assays
135.8
6.79
l
mol), using 2nd generation Grubbs catalyst (5.76 mg,
5.2.1. Chemicals and hormones
lmol, 5 mol %) in dichloromethane (4.9 mL). The reaction
Methyltrienolone (R1881) was obtained from Perkin Elmer and
AA from Merck, Darmstadt. All test compounds were dissolved in
ethanol or/and dimethylsulfoxide (DMSO). These compounds were
added to the culturing medium such that the final concentration of
ethanol and/or DMSO did not exceed 0.1%.
mixture was refluxed for 4 h. Yield: 44 mg (84%), white solid; mp
213–215 °C; ½a 2D3
ꢁ
= ꢀ42.9 (c 0.504, CH3OH); IR (KBr): mmax 3429,
2935, 1673, 1613, 1428 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 0.84
(s, 3H), 0.98 (s, 3H), 1.34 (s, 3H), 2.58 (td, J = 18.2, 2.8 Hz, 1H),
3.44–3.52 (m, 1H), 5.31 (d, J = 5.2 Hz, 1H), 5.61 (s, 1H), 5.86 (d,
J = 10.0 Hz, 1H), 6.47 (ddd, J = 9.8, 5.2, 3.1 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 13.78, 19.31, 20.66, 23.33, 23.55, 27.13,
31.15, 31.46, 31.53, 35.91, 36.36, 37.13, 39.79, 42.12, 43.06,
49.77, 56.47, 57.64, 59.23, 71.44, 121.14, 123.08, 139.90, 140.81,
165.45; HRMS-ESI (m/z): [M+Na]+ calcd for C25H37NNaO2:
406.27165, found: 406.27160.
5.2.2. Plasmids
The plasmid pMMTV-luc, which contains a luciferase reporter
gene driven by the mouse mammary tumor virus long terminal re-
peats responsive to androgens, is described in Gast et al.18 The
expression vector for the human AR or AR T877A, pSG-hAR or
pSG-hAR T877A are described in the literature.24 The plasmid for
expression of human GR is described also in the literature.25 Hu-
man PR-B expression vector was kindly provided by P. Chambon
(Strasbourg, France).
5.1.11. General procedure for the synthesis of the 20-
aminosteroids (12) and (19)
To a solution of the appropriate 3b-hydroxy C-20 lactame ring
steroidal derivative (20.1 mg, 52.4
lmol) in dry benzene (5 mL),
5.2.3. Reporter assays
cyclohexanone (135.7 L, 1.31 mmol) was added and the mixture
l
CV1 cells were seeded onto 6-well tissue culture plates (Nunc,
Roskilde, Denmark) at 1.2 ꢂ 105 cells per well and grown in DMEM
(Invitrogen) supplemented with 5% (v/v) dextran-coated charcoal
stripped serum and 1% (v/v) penicillin and streptomycin.26 18
hours later cells were transfected by using the CaPO4 method.24
was refluxed with a Dean–Stark apparatus until ꢃ2 mL of solvents
were distilled under an argon atmosphere. Then, a suspension of
Al(i-PrO)3 (32.1 mg, 157.2 lmol) in dry benzene (2 mL) was added
and the reaction mixture was refluxed again until ꢃ2 mL of sol-
vents were distilled. Then, the reaction mixture was stirred at
80 °C for the indicated time. After the end of the reaction, water
was added and the mixture was extracted with chloroform. The
aqueous phase was washed (4ꢂ) with chloroform and the com-
bined organic layers were washed with brine, dried over anhy-
drous Na2SO4, filtered and evaporated under reduced pressure.
The crude residue was purified by silica gel column chromatogra-
phy (1–3% MeOH in dichloromethane) to afford the corresponding
3-keto-4,5-en-steroidal derivative. Analytical samples of the final
products were provided after HPLC purification (Preparative HPLC,
CHCl3–MeOH 2%, 10 mL/min, 30 min, 254–364 nm).
The DNA mixture for transfections consisted of 1
priated luciferase reporter constructs, 0.2 g of the appropriated
mammalian steroid receptor expression vector and 0.2 g of the
lg of the appro-
l
l
cytomegalovirus (CMV)-driven b-galactosidase expressions vector,
as internal control for transfection efficiency. After 24 h media
were replaced either with or without the addition of the appropri-
ate hormones together with the indicated compounds. After addi-
tional 96 h cells were harvested and assayed for luciferase and
b-galactosidase activity. All transfection assays shown were per-
formed in duplicate and were repeated at least twice.
5.2.4. Cell growth assays
5.1.11.1.
17-[(6R)-6-Methyl-5,6-dihydropyridin-2(1H)-one-6-
Human prostate carcinoma LNCaP cells27 were cultured in
RPMI-1640 medium (Invitrogen), supplemented with 10% (v/v)
fetal calf serum (FCS) (Invitrogen), 1% (v/v) penicillin and strepto-
yl]-androst-4-ene-3-one (12). Compound 12 was synthesized
according to the general procedure from compound 11. The reaction
mixture was stirred at 80 °C for 16 h. Yield 11.6 mg (58%), white so-
mycin (Invitrogen), 1% (v/v) L-glutamin (Invitrogen), 1% (v/v) so-
lid; mp 248–250 °C; ½a D23
ꢁ
= +101.2 (c 0.253, CHCl3); IR (KBr): mmax
dium pyruvate (Sigma). PC3 were kindly provided by Dr. A. Cato,
Karlsruhe, Germany28 and were cultured in DMEM supplemented
with 10% (v/v) FCS, 1% (v/v) penicillin and streptomycin and 1%
3431, 2927, 1682, 1619, 1427, 877, 824 cmꢀ1
;
1H NMR (400 MHz,
CDCl3): d 0.86 (s, 3H), 1.18 (s, 3H), 1.34 (s, 3H), 2.60 (ddd, J = 18.0,
3.7, 2.2 Hz, 1H), 5.39 (s, 1H), 5.72 (s, 1H), 5.90 (ddd, J = 9.9, 3.7,
1.8 Hz, 1H), 6.51 (td, J = 9.9, 4.2 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d 14.40, 17.36, 20.75, 23.53, 27.07, 31.73, 32.75, 33.92, 34.93, 35.64,
35.77, 38.49, 39.99, 43.02, 53.56, 55.77, 57.47, 58.42, 123.54,
123.93, 139.92, 165.01, 170.90, 199.43; HRMS-ESI (m/z): [M+H]+
calcd for C25H36NO2: 382.27406, found: 382.27431.
(v/v) L-glutamin. For cell growth assays, cells were seeded onto
six-well tissue culture plates (Nunc, Roskilde, Denmark) at
5 ꢂ 103 (PC3) or 25 ꢂ 103 (LNCaP) cells per well in appropriated
medium in triplicates containing 5% FCS. After 2 days, cells were
fed with fresh medium and treated with DMSO or with the indi-
cated compounds. Every second/third day the media was replaced
with fresh media together with freshly added compounds. The
5.1.11.2. 17-[(6S)-6-Methyl-5,6-dihydropyridin-2(1H)-one-6-yl]-
androst-4-ene-3-one (19). Compound 19 was synthesized
cells of the same two 8
indicated times.
l
m2 areas were counted per well at the

M. A. Fousteris et al. / Bioorg. Med. Chem. 18 (2010) 6960–6969
6969
11. (a) Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An, Y.; Wu, G. Y.;
Scheffler, J. E.; Salvati, M. E.; Krystek, S. R., Jr.; Weinmann, R.; Einspahr, H. M.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4904; (b) Bohl, C. E.; Wu, Z.; Miller, D. D.;
Bell, C. E.; Dalton, J. T. J. Biol. Chem. 2007, 282, 13648.
12. (a) Cantin, L.; Faucher, F.; Couture, J. F.; de Jésus-Tran, K. P.; Legrand, P.;
Ciobanu, L. C.; Fréchette, Y.; Labrecque, R.; Singh, S. M.; Labrie, F.; Breton, R. J.
Biol. Chem. 2007, 282, 30910; (b) Labrie, F.; Singh, S.; Gauthier, S.; Frechette, Y.;
Chenard, S.; Breton, R. US Patent 0250749 A1, 2005.; (c) Roy, J.; Breton, R.;
Martel, C.; Labrie, F.; Poirier, D. Bioorg. Med. Chem. 2007, 15, 3003; (d)
Narayanan, R.; Mohler, M. L.; Bohl, C. E.; Miller, D. D.; Dalton, J. T. Nucl. Recept.
Signal. 2008, 6, e010.
5.2.5. Real time RT-PCR
The real time RT-PCR (qRT-PCR) was performed essentially as
described previously20 with specific primers for detection of PSA
mRNA and beta-actin mRNA for normalization. As modifications
1.28 ꢂ 106 cells were seeded out directly in media containing
10% charcoal stripped FCS. After two days cells were treated with
the indicated compounds or solvent (DMSO) or androgen (R1881)
was added to a final concentration of 3 ꢂ 10ꢀ11 M.
13. (a) Guo-Qiang, L.; Ming-Hua, X.; Yu-Wu, Z.; Xing-Wen, S. Acc. Chem. Res. 2008,
41, 831; (b) Ellman, J. A. Pure Appl. Chem. 2003, 75, 39; (c) Ellman, J. A.; Owens,
T. D.; Tang, T. P. Acc. Chem. Res. 2002, 35, 984.
14. Drew, J.; Letellier, M.; Morand, P.; Szabo, A. G. J. Org. Chem. 1987, 52, 4047.
15. During optimization trials, we noticed unexpectedly the formation of the
5.2.6. Detection of SA-b galactosidase
Senescence-associated b-Gal (SA-b-Gal) staining was carried
out as described previously21,29 using 40,000 cells per well in a
6-well plate. The treatment was performed for 3 days.
(20E)-sulfonylimine
derivative
2a
when
(S)-(ꢀ)-2-methyl-2-
propanesulfinamide was used in excess to the steroidal ketone. The structure
of 2a was verified by X-ray analysis. For spectroscopic data of 2a see
Supplementary data.
Acknowledgments
16. In the case of imine 13 excess of the chiral auxiliary was necessary for the
complete consumption of the steroidal ketone 2.
17. CCDC-753934, 753933, 753936, 753935 and 753937 (2, 2a, 4, 13 and 15,
respectively) contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Professor Dr. J. Sieler is gratefully acknowledged for X-ray crys-
tallographic assistance. We thank Dr. Lothar Hennig for recording
nuclear magnetic resonance-spectra.
18. Gast, A.; Schneikert, J.; Cato, A. C. J. Steroid Biochem. Mol. Biol. 1998, 65, 117.
19. Handratta, V. D.; Vasaitis, T. S.; Njar, V. C.; Gediya, L. K.; Kataria, R.; Chopra, P.;
Newman, D., Jr.; Farquhar, R.; Guo, Z.; Qiu, Y.; Brodie, A. M. J. Med. Chem. 2005,
48, 2972.
20. Papaioannou, M.; Schleich, S.; Prade, I.; Degen, S.; Roell, D.; Schubert, U.;
Tanner, T.; Claessens, F.; Matusch, R.; Baniahmad, A. J. Cell Mol. Med. 2009, 13,
2210.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.029.
21. Dimri, G. P.; Lee, X.; Basile, G.; Acosta, M.; Scott, G.; Roskelley, C.; Medrano, E.
E.; Linskens, M.; Rubelj, I.; Pereira-Smith, O.; Peacocke, M.; Campisi, J. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 9363.
22. (a) Campisi, J. Aging Cell 2007, 6, 261; (b) Campisi, J. Cell 2005, 120, 513.
23. Komada, S.; Itami, S.; Kurata, S.; Takayasu, S. Arch. Dermatol. Res. 1989, 280,
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24. Dotzlaw, H.; Moehren, U.; Mink, S.; Cato, A. C.; Iñiguez Lluhi, J. A.; Baniahmad,
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