Chiral nonsteroidal affinity ligands for the androgen receptor. 1. Bicalutamide analogues bearing electrophilic groups in the B aromatic ring

Article abstract of DOI:10.1021/jm990027x

A series of chiral analogues of bicalutamide bearing electrophilic groups (isothiocyanate, N-chloroacetyl, and N-bromoacetyl) on aromatic ring B of the parent molecule were synthesized. These compounds were designed as affinity ligands for the androgen receptor (AR). We prepared the (R)- and (S)-optical isomers of these compounds as pure enantiomers. The AR binding affinities of these compounds were measured in a competitive binding assay with the radiolabeled high-affinity AR ligand, [³H]mibolerone. In accordance with our previous results for the enantiomers of bicalutamide, we found that all (R)-isomers demonstrated much higher binding affinity to the AR as compared to their corresponding (S)-isomers. The para-substituted affinity ligands in ring B bound the AR with higher affinities than the corresponding metasubstituted analogues. Oxidation of thioester affinity ligands to their sulfonyl analogues for the para-substituted compounds decreased AR binding affinities and similar modification increased binding affinities for corresponding meta-analogues. The least potent para-substituted sulfonyl compounds had higher AR binding affinities than the most potent meta- substituted sulfonyl compounds. Overall, the para-substituted unoxidized molecules demonstrated the highest AR binding affinity. Subsequent research using AR exchange assays and Scatchard analyses showed that the isothiocyanate affinity ligands (R)-7, (R)-9, and (R)-10 reported herein are the first specific chemoaffinity ligands for the AR.

Full text of DOI:10.1021/jm990027x

J . Med. Chem. 2000, 43, 581-590
581
Ch ir a l Non ster oid a l Affin ity Liga n d s for th e An d r ogen Recep tor . 1.
Bica lu ta m id e An a logu es Bea r in g Electr op h ilic Gr ou p s in th e B Ar om a tic Rin g1
Leonid Kirkovsky, Arnab Mukherjee, Donghua Yin, J ames T. Dalton, and Duane D. Miller*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee-Memphis, Memphis, Tennessee 38163
Received J anuary 19, 1999
A series of chiral analogues of bicalutamide bearing electrophilic groups (isothiocyanate,
N-chloroacetyl, and N-bromoacetyl) on aromatic ring B of the parent molecule were synthesized.
These compounds were designed as affinity ligands for the androgen receptor (AR). We prepared
the (R)- and (S)-optical isomers of these compounds as pure enantiomers. The AR binding
affinities of these compounds were measured in a competitive binding assay with the
radiolabeled high-affinity AR ligand, [3H]mibolerone. In accordance with our previous results
for the enantiomers of bicalutamide, we found that all (R)-isomers demonstrated much higher
binding affinity to the AR as compared to their corresponding (S)-isomers. The para-substituted
affinity ligands in ring B bound the AR with higher affinities than the corresponding meta-
substituted analogues. Oxidation of thioester affinity ligands to their sulfonyl analogues for
the para-substituted compounds decreased AR binding affinities and similar modification
increased binding affinities for corresponding meta-analogues. The least potent para-substituted
sulfonyl compounds had higher AR binding affinities than the most potent meta-substituted
sulfonyl compounds. Overall, the para-substituted unoxidized molecules demonstrated the
highest AR binding affinity. Subsequent research using AR exchange assays and Scatchard
analyses showed that the isothiocyanate affinity ligands (R)-7, (R)-9, and (R)-10 reported herein
are the first specific chemoaffinity ligands for the AR.
In tr od u ction
binding core domain of the GR. [3H]Dex-mes covalently
labeled a single residue (Cys-656) of the rat GR. Using
point mutations and Dex-mes affinity labeling, the
authors were able to generate one of the first known
models of the steroid binding “pocket” of the GR.
Affinity labeling is a powerful technique that can
provide valuable information about the binding pocket
of a protein, i.e., the substrate binding site of an enzyme
or the ligand binding site of a receptor. This methodol-
ogy is widely used for mapping the amino acids involved
in ligand interaction. In addition to the basic application
to protein characterization, affinity ligands can be
utilized for practical purposes in cell biology, including
irreversible tagging with a radioactive atom or fluores-
cent group for visualization of a particular protein (e.g.,
in flow cytometry or fluorescence microscopy).2
Steroid hormone receptors (SHR) are an important
class of intracellular regulatory proteins that bind
structurally diverse ligands and modulate estrogen,
glucocorticoid, and androgen action. Chemoaffinity and
photoaffinity ligands have played a critical role in
structural characterization of many of these receptors.
There are two major groups of affinity ligands for
SHR: steroidal and nonsteroidal.
A number of steroidal analogues, including promege-
sterone and triamcinolone acetonide (TA), have been
used to photoaffinity label the glucocorticoid receptor
(GR). Promegesterone and TA labeled identical amino
acids in the ligand binding domain of the GR.3 Dexam-
ethasone mesylate (Dex-mes) has been widely used as
an electrophilic affinity labeling reagent and is known
to bind with cysteine residues in the GR.4,5 Chakraborti
et al.5 recently showed that tryptic digestion of the GR
resulted in a 16-kDa fragment that retained its steroid
binding specificity, justifying its designation as a steroid-
Katzenellenbogen and co-workers6 developed steroidal
compounds bearing electrophilic and photoreactive groups
with the potential to affinity label the estrogen receptor
(ER). However, electrophilic nonsteroidal estrogens
proved superior in terms of efficiency and selectivity of
covalent attachment to the ER.7 Tamoxifen aziridine
(TAZ), which acts as an antagonist for the ER, and
ketononestrol aziridine (KNA), which acts as an estro-
gen agonist, were used to identify Cys-530 as the site
of covalent attachment in the ligand binding domain of
the ER.8 These studies provided the necessary back-
ground for more detailed examination of the effect of
specific amino acids on transcriptional activation and
the discrimination of estrogens and antiestrogens by the
ligand binding domain. Subsequent structural analysis
of proteolytic cleavage products from the affinity-labeled
wild-type and mutated ER allowed this group to propose
a model for interaction of both agonists and antagonists
with the ligand binding domain of the ER.9 These
studies demonstrate the unique advantages that affinity
labels provide for steroid receptor characterization (i.e.,
stringent treatment of the protein without the risk of
ligand dissociation during purification) and indicate that
nonsteroidal affinity labels can serve as efficient, highly
selective tools for steroid receptor characterization.
Affinity labeling studies of the androgen receptor (AR)
have been limited. Chang et al.10,11 used dihydrotest-
osterone 17â-bromoacetate (DHT-BA; Figure 1) for
* To whom correspondence should be addressed. Tel: 901-448-7530.
Fax: 901-448-6828. E-mail: dmiller@utmem.edu.
10.1021/jm990027x CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/01/2000

582 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Kirkovsky et al.
Sch em e 1. Synthesis of Chiral AR Affinity Ligands
7-17 (with (R)-isomers as an example)a
F igu r e 1. Structures of potential nonsteroidal affinity ligands
for the AR.
electrophilic labeling and methyltrienolone (R1881) for
photoaffinity labeling of the AR. However, both of these
steroidal compounds interact with a number of non-
receptor species, limiting their usefulness for AR char-
acterization.12,13 Initial studies suggested that DHT-BA
covalently labeled a 56-kDa proteolytic fragment of the
human AR.14 McCammon et al.15 recently reported that
DHT-BA and R1881 in fact covalently bind to cytosolic
aldehyde dehydrogenase, an enzyme expressed in greater
abundance than the AR in genital skin fibroblasts,
rather than the earlier reported binding to the AR. The
lack of selective and efficient AR affinity labels has
impeded more detailed analysis of the amino acids
involved in ligand discrimination and contributed to the
fact that the human AR remains one of the least
characterized steroid receptors.
This manuscript describes our initial efforts toward
development of novel nonsteroidal chemoaffinity ligands
for the AR.16 We developed several series of potential
chemoaffinity and photoaffinity ligands based on struc-
tural modification of known clinically used AR antago-
nists. We report herein synthesis and AR binding
affinities of bicalutamide analogues bearing electrophilic
functional groups in aromatic ring B, hereafter referred
to as BAL-2.1 We also prepared bicalutamide affinity
ligands with reactive groups in aromatic ring A, here-
after referred to as BAL-1,17 and hydroxyflutamide
affinity ligands, hereafter referred to as FAL-1, FAL-
2,18 and FAL-3,19 as outlined in Figure 1. Detailed
studies demonstrated the irreversible nature of some
of these BAL-2 compounds.20
a
(a) Methacryloyl chloride, NaOH(aq), 5-10 °C, in aqueous
acetone; (b) NBS, in anhydrous DMF; (c) HCl(concd), reflux; (d)
(R)-3/(R)-3a with thionyl chloride, in anhydrous DMA, -5 to -10
°C, then 4-amino-2-trifluoromethylbenzonitrile, in anhydrous
DMA; (e) (R)-4/(R)-4a with NaH, then 4- or 3-aminothiophenol,
in anhydrous THF; (f) CSCl2, CH2Cl2 + NaHCO3; (g) ClCH2COCl
or BrCH2COBr in CH2Cl2, CaCO3, or iPr2NEt; (h) m-chloroper-
benzoic acid or peracetic acid, in CH2Cl2 or acetone.
The reaction sequence began with coupling of a
commercially available chiral auxiliary, (S)- or (R)-
proline, with methacryloyl chloride in aqueous acetone
at 5-10 °C under Schottenn-Baumann conditions
according to the procedure proposed by J ew et al.24 (R)-
or (S)-prolineamide, (R)-1 or (S)-1 (Scheme 1), formed
in the first step, was converted to its corresponding (R)-
or (S)-bromolactone (R)-2 or (S)-2 using Terashima’s
method of asymmetric bromolactonization.24,25 This step
was critical for the reaction sequence due to creation of
the asymmetric center which ultimately determined the
chirality of the final affinity ligands. Unlike the previous
procedure,21 we used 2 equiv of N-bromosuccinimide
(NBS) instead of 1 equiv to perform bromolactonization.
This provided a higher yield of the product (64-80% in
our preparations vs 49% obtained by Tucker).21
Hydrolysis of the bromolactone (R)-2 or (S)-2 in
refluxing concentrated HCl for 8 h gave rise to (R)- or
(S)-bromoacid (R)-3 or (S)-3, critical intermediates for
all further synthetic manipulations in our study. Al-
though we followed the literature procedure and ob-
tained a white crystalline compound (R)-3 with a
melting point 112-113.5 °C (lit.21 mp 109-113 °C), we
observed by NMR that the product contained about 15%
of another compound (see Experimental Section). This
side product appeared to be the corresponding chloro-
acid 3a , formed by displacement of bromine with
chlorine. Indeed, short (1 h) incomplete hydrolysis of
bromolactone (S)-2 showed exclusive formation of the
product assigned as bromoacid (S)-3. Increasing the
Ch em istr y
The (R)-isomers of compounds 5-17 were prepared
according to the general synthetic scheme reported for
the enantiomers of bicalutamide.21-23 This synthetic
route was utilized by Tucker21,22 to prepare the (S)-
isomer of bicalutamide. We successfully extended this
approach to prepare the (R)-isomer of thiobicalutamide
and bicalutamide.23

Chiral Nonsteroidal Affinity Ligands for AR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 583
reaction time caused gradual accumulation of the second
product (S)-3a which reached a 50:50 ratio after 30 h.
After 93 h of hydrolysis, the second product prevailed
over the first one with a ratio of about 85:15.
obtain good quality 13C spectra for isothiocyanate af-
finity ligands, we ran the spectra in DMSO-d6 using
high concentration of a compound and a relatively short
scanning time (1-1.5 h). The NMR spectra for all our
compounds were obtained in DMSO-d6, allowing direct
comparison of spectroscopic data.
We did not isolate and characterize the product
assigned as chloroacid 3a . Instead, we converted the
mixture of the acids in situ to their acid chlorides and
coupled them with commercially available 4-amino-2-
trifluoromethylbenzonitrile in DMA at -10 to -15 °C
which gave rise to (R)- or (S)-bromoanilide (R)-4 or (S)-4
along with another anilide (likely (R)-4a or (S)-4a ). The
ratio of bromo- and chloroanilides 4 and 4a was the
same as bromo- and chloroacids 3 and 3a (approxi-
mately 85:15). It is interesting to note that bromoanil-
ides 4 in our preparation had a much higher melting
point than that reported by Tucker (134.5-135.5 °C vs
106-107 °C21). We did not separate these two com-
pounds since they were converted to common anilides
5 or 6 when coupled with aminothiophenols.
Resu lts a n d Discu ssion of AR Bin d in g Affin ity
AR binding affinities of the synthesized ligands were
determined by competitive binding28,29 in the presence
of the high-affinity AR ligand, [3H]mibolerone (MIB).
AR binding studies were performed by incubating
increasing concentrations (10-3-10000 nM) of each
ligand with cytosol and a saturating concentration of
[3H]MIB (1 nM) at 4 °C for 18 h. In preliminary
experiments, the equilibrium dissociation constant (Kd)
of MIB was determined under identical conditions by
incubating increasing concentrations of [3H]MIB (0.01-
10 nM) with cytosol. We found that the minimum
concentration of [3H]MIB required to saturate AR sites
in the cytosol preparation was 1 nM. Subsequent
experiments used either 1 or 2 nM [3H]MIB. The
incubates also contained 1000 nM triamcinolone ac-
etonide to block interaction of MIB with progesterone
receptors.30 For the determination of nonspecific bind-
ing, separate experiments were conducted by adding
1000 nM MIB to the incubate. Separation of bound and
free radioactivity at the end of incubation was achieved
by the hydroxyapatite (HAP) method, as described
previously,23 and 0.8 mL of the ethanolic supernatant
was added to 5 mL of scintillation cocktail. Radioactivity
was counted in a Beckman LS 6800 liquid scintillation
counter (Beckman Instruments, Irvine, CA). Reversed-
phase HPLC was used to determine that our ligands
were stable under the experimental conditions used for
AR competitive binding studies. It is important to note
that binding affinity constants (Ki) represent “apparent”
values because they characterized two steps which
might be involved in the interaction of an affinity ligand
with the AR. The first step was reversible binding of
the affinity ligands to the AR, and the second step was
irreversible interaction with formation of a covalent
bond between the ligand and the receptor. Thus, de-
pending on the extent of the AR labeling during the
competitive binding assay, apparent Ki-values may
reflect a combination of these two processes.
The mixture of anilides 4 and 4a was coupled with
commercially available 4-amino- or 3-aminothiophenols.
The reaction was performed in THF at room tempera-
ture using 1.1-1.2 equiv of sodium hydride. The final
products of the reaction, aniline derivatives (R)-5 and
(S)-5 or (R)-6 and (S)-6, were used as the precursors
for preparation of our final affinity ligands. Conversion
of these aniline precursors 5 or 6 to the isothiocyanate
affinity ligands 7 and 8 was achieved by reaction with
thiophosgene in a heterogeneous aqueous-organic mix-
ture according to the procedure of Leclerc.26 The yields
of these reactions were between 80% and 95%. Chloro-
acetyl and bromoacetyl compounds 11-14 were pre-
pared by acylation of the corresponding aniline precur-
sors 5 and 6 by commercially available chloroacetyl
chloride or bromoacetyl bromide in dry ethyl acetate or
methylene chloride solution under argon with solid
CaCO3 powder or N,N-diisopropylethylamine as proton
scavengers. The reaction was completed within 15-30
min in the case of bromoacetyl bromide and required
greater than 3 h in the case of chloroacetyl chloride. All
thioester affinity ligands 7, 8, and 11-14 were oxidized
to their corresponding sulfonyl derivatives 9, 10, and
15-17 by commercially available 3-chloroperbenzoic
acid or peracetic acid in ethyl acetate or methylene
chloride solutions.
We noted some stability problems with the isothio-
cyanate affinity ligands when running 13C NMR spectra
of these compounds in DMSO-d6 for 5-20 h at room
temperature, as evidenced by the appearance of ad-
ditional peaks in proton and carbon NMR spectra. The
p-sulfonyl compound 9 was the least stable in DMSO-
d6, while the p-thiol analogue 7 was the most stable.
The m-sulfonyl molecule 10 and the m-thiol molecule 8
demonstrated intermediate stability. First-order rate
constants (kobs) of solvolysis of isothiocyanate affinity
labels were roughly determined by integration of peaks
for the starting compounds and their products in proton
NMR spectra. These rate constants demonstrated good
correlation with the Hammett σ-constants calculated
using the σ-values for methylthiol (MeS) and methyl-
sulfonyl (MeSO2) groups in para- and meta-positions of
the phenyl ring.27 Indeed, kobs decreased in the order of
0.025, 0.022, 0.008, and 0.002 h-1 for 9 (σ ) 0.68), 10 (σ
) 0.60), 8 (σ ) 0.15), and 7 (σ ) 0), respectively. To
Synthesized ligands showed a wide range of binding
affinities. In accordance with our previous results for
enantiomers of bicalutamide,23 we found that the (R)-
isomers of our molecules demonstrated much higher
binding affinity to the AR compared to the correspond-
ing (S)-isomers. Thiol analogues of isothiocyanate af-
finity ligands (R)-7 and (S)-7 had the highest eudismic
ratio (ratio of Ki-values for the (R)- and (S)-isomers) in
our AR binding experiments. This ratio was close to 3
orders of magnitude for (R)-7 and (S)-7 (Table 1). The
eudismic ratio was about 2 orders of magnitude less for
thiochloroacetyl analogues (R)-11 and (S)-11. This
value decreased to about 1 order of magnitude for meta-
and para-substituted anilines (R)-6 and (S)-6 and (R)-5
and (S)-5. In fact, we found that the eudismic ratio was
significantly correlated with the AR binding affinity of
(R)-isomers within our series of compounds (r2 ) 0.9809,
P < 0.0001). Compounds with lower Ki-values demon-

584 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Kirkovsky et al.
Ta ble 1. Rat AR Binding Affinities (Ki) of (R)- and (S)-Optical Isomers of Synthesized Analogues 5-17 and Enantiomers of
Bicalutamide (Casodex) 1
R
X
compd
Ki (nM)
compd
Ki (nM)
4-F
SO2
S
S
S
S
SO2
SO2
S
S
S
(R)-bicalutamide23
(R)-5
(R)-6
(R)-7
(R)-8
11.0 ( 1.5
90 ( 15
(S)-bicalutamide23
365 ( 10
4-NH2
3-NH2
4-NCS
3-NCS
4-NCS
3-NCS
4-NHCOCH2Cl
3-NHCOCH2Cl
4-NHCOCH2Br
3-NHCOCH2Br
4-NHCOCH2Cl
3-NHCOCH2Cl
4-NHCOCH2Br
(S)-5
(S)-6
(S)-7
(S)-8
800
65 ( 25
>1000
0.6 ( 0.1
230 ( 50
41 ( 2
140 ( 10
1.65 ( 0.10
>1000
100 ( 10
>1000
10.7 ( 0.6
130 ( 20
360 ( 30
430 ( 30
130 ( 15
(R)-9
(R)-10
(R)-11
(R)-12
(R)-13
(R)-14
(R)-15
(R)-16
(R)-17
(S)-11
200 ( 30
S
SO2
SO2
SO2
F igu r e 2. Correlation between logarithm of the eudismic
ratios of the AR binding (Ki for (S)-isomers/Ki for (R)-isomers)
for compounds 5-7, 11, and bicalutamide (B) with logarithm
of binding affinities (Ki) of (R)-isomers.
F igu r e 3. Correlation between logarithm of the para to meta
AR binding regioselectivity (ratio of Ki-value for meta-
substituted analogue to Ki-value for para-substituted ana-
logue) with logarithm of binding affinity of the para-substi-
tuted counterpart.
strated higher eudismic ratios. This general trend for
pairs of optical isomers to increase eudismic ratio
(enantiomeric ratio) for more potent compounds is called
Pfeiffer’s rule31 and could be quantitatively described
as the correlation between enantiomeric specificity of
the AR binding of compounds 5-7, 11, and bicalutamide
(B) with the binding affinities of (R)-isomers (Figure 2).
In most cases, the meta-substituted affinity ligands
bound the AR with affinities lower than that observed
for the para-substituted analogues. This was true for
pairs of compounds (R)-8 and (R)-7, (R)-9 and (R)-10,
(R)-11 and (R)-12, (R)-13 and (R)-14, and (R)-15 and
(R)-16. The advantage in the AR binding for the para-
substitution versus the meta-substitution (regioselec-
tivity of the AR binding) increased with increasing
binding potency of the para-substituted counterpart. For
example, para-substituted molecules (R)-7 and (R)-11
bound the AR with high affinities (Ki-values were about
1 nM). Moving the isothiocyanate or chloroacetyl group
to the meta-position resulted in a significant decrease
(about 400 and 600, respectively). However, moving the
bromoacetyl or chloroacetyl group from the para- to
meta-position in compounds (R)-13/(R)-14 and (R)-15/
(R)-16 resulted in only a 10-fold decrease in the AR
binding. The meta/para-regioselectivity decreased fur-
ther for compounds with less potent AR binding (e.g.,
(R)-9/(R)-10 and (R)-5/(R)-6). The correlation between
para/meta-regioselectivities and binding affinities of
para-substituted analogues is shown in Figure 3 (r2 )
0.8113, P < 0.01).
Oxidation of thioester affinity ligands to their sulfonyl
analogues decreased binding affinities for the para-
substituted compounds and increased it for the meta-
substituted analogues. However, the most potent meta-
substituted sulfonyl compounds had lower AR binding
affinities than the least potent para-substituted sulfonyl
compounds. Thus, the para-substituted unoxidized mol-
ecules provided the best binding AR affinity ligands in
this series of synthesized compounds.
Con clu sion s
We found that nonsteroidal AR ligands bearing elec-
trophilic functional groups in aromatic ring B main-
tained high-affinity binding to the AR. Moreover, these
studies demonstrate for the first time that nonsteroidal
AR ligands demonstrate stereoselective and regioselec-

Chiral Nonsteroidal Affinity Ligands for AR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 585
molecular characterization of the ligand binding domain
of the AR.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Thomas-
Hoover capillary melting point apparatus and are uncorrected.
Infrared spectra were recorded on a Perkin-Elmer system 2000
FT-IR. Proton and carbon-13 magnetic resonance spectra were
obtained on a Bruker AX 300 spectrometer (300 and 75 MHz
for 1H and 13C, respectively). Chemical shift values are
reported as parts per million (δ) relative to DMSO-d6 peak
1
(2.49 and 39.5 for H and 13C, respectively). Spectral data were
consistent with assigned structures. Elemental analyses were
performed by Atlantic Microlab Inc., Norcross, GA, and found
values were within 0.4% of the theoretical values. Specific
rotations were recorded on Autopol III automatic polarimeter
(Rudolph Research, Fairfield, NJ ) in 1-dm sample tube with
use of sodium D-line at ambient temperature. Routine thin-
layer chromatography (TLC) was performed on silica gel
aluminum plates (Whatman Ltd., Maidstone, Kent, England).
Flash chromatography was performed on silica gel (Merck;
grade 60, 230-400 mesh, 60 Å).
F igu r e 4. Exchangeable specific binding of [3H]MIB after
incubation with the indicated ligand. Values are expressed as
mean (( SD) percent of specific [3H]MIB binding to cytosol
preincubated without ligand. Exchangeable specific binding
of [3H]MIB in the absence of any competitor was 2270 ( 267
dpm. Experiments were performed in triplicate for each ligand.
Adapted from ref 20.
(2R)-1-Meth a cr yloylp yr r olid in e-2-ca r boxylic Acid (-
(R)-1). Prolineamide (R)-1 was prepared according to the
general procedure described by J ew et al.24 D-Proline (10.33
g, 89.7 mmol) was dissolved in 2 N NaOH (53 mL) and cooled
in an ice bath, and the resulting alkaline solution was diluted
with acetone (53 mL). An acetone solution (53 mL) of metacryl-
oyl chloride (14.08 g, 134.7 mmol) and the 2 N NaOH solution
(70 mL) were simultaneously added over 40 min to the aqueous
solution of L-proline with stirring in an ice bath. The pH of
the mixture was kept at 10-11 (measured by pH meter with
glass electrode) during the addition of the metacryloyl chloride.
After the stirring was continued for 3 h at room temperature,
the mixture was evaporated in vacuo on a rotary evaporator
at a slightly elevated temperature (35-45 °C) to remove
acetone. The residual solution was washed with ether and
acidified to pH 2 with concentrated HCl. The acidic mixture
was saturated with NaCl and extracted with EtOAc (3 × 100
mL). The combined EtOAc extracts were dried over MgSO4,
reduced in volume by evaporation in vacuo, mixed with
hexanes (with about 1:1 ratio), and further concentrated until
the beginning of crystallization to give 12.50 g (76%) of the
title compound as white crystals: mp 102.5-103.5 °C (lit.32
mp 103-104.5 °C for the (S)-prolineamide). NMR spectrum
of this compound demonstrated existence of two rotamers of
the title compound: 1H NMR (DMSO-d6) δ 5.28 (s) and 5.15
(s) (first rotamer), 5.15 (s) and 5.03 (s) (second rotamer) (totally
2H for both rotamers, vinyl CH2), 4.24-4.20 (m) (first rotamer),
4.48-4.44 (m) (second rotamer) (totally 1H for both rotamers,
CH at the chiral center), 3.57-3.38 (m, 2H, aliphatic CH2),
2.27-2.12 (m, 1H, aliphatic CH), 1.97-1.72 (m, 6H, aliphatic
CH2, CH and Me); 13C NMR (DMSO-d6) δ for the first (major)
rotamer 173.26, 169.12, 140.87, 116.38, 116.38, 58.26, 48.73,
28.89, 24.70, 19.51; for the second (minor) rotamer 174.04,
170.03, 141.61, 115.24, 60.26, 45.94, 30.95, 22.34, 19.66. Mass
spectra data were obtained on Bruker Daltonics Esquire LC
multipole ion trap mass spectrometer with ESI: [R]D25 +80.8°
(c ) 1, MeOH). Anal. (C9H13NO3) C, H, N.
tive binding to the AR. (R)-Isomers demonstrated much
higher binding affinity to the AR as compared to their
corresponding (S)-isomers. The para-substituted affinity
ligands in ring B bound the AR with higher affinities
than the corresponding meta-substituted analogues.
Oxidation of thioester affinity ligands to their sulfonyl
analogues for the para-substituted compounds de-
creased AR binding affinities and similar modification
increased binding affinities for corresponding meta-
analogues. The least potent para-substituted sulfonyl
compounds had higher AR binding affinities than the
most potent meta-substituted sulfonyl compounds. Over-
all, the para-substituted unoxidized molecules demon-
strated the highest AR binding affinity. In the synthe-
sized series of bicalutamide affinity ligands BAL-2,
compounds combining two features (an electrophilic
group located at the para-position of the aromatic ring
B and an unoxidized sulfur bridge) provided the best
binding AR affinity.
Further, we recently reported the results of detailed
studies to examine the potential of these ligands to
covalently label the AR.20 In exchange assays, the
isothiocyanate affinity ligands (R)-7, (R)-9, and (R)-10
reduced exchangeable specific binding to an average of
16%, 15%, and 50%, respectively (Figure 4). (R)-Bi-
calutamide (B) and the aniline derivatives (R)-5 and
(R)-6 were used as reversible controls and did not
demonstrate significant decreases in exchangeable bind-
ing. The thiochloroacetyl affinity ligand (R)-11 also
decreased exchangeable binding to 26% of control.
However, Scatchard analyses with this ligand showed
that (R)-11 did not irreversibly bind the AR (data not
shown).20 The results of AR exchange assay and Scat-
chard analysis indicated that isothiocyanate affinity
ligands (R)-7, (R)-9, and (R)-10 are the first specific
chemoaffinity ligands for the AR.20
(2S)-1-Meth a cr yloylp yr r olid in e-2-ca r boxylic Acid ((S)-
1). Prolineamide (S)-1 was prepared using L-proline ([R]20
D
-84°, c ) 4, H2O) as a chiral auxiliary according to the
procedure described for preparation of (R)-1. The yield was
70%: mp 102-103 °C (lit.32 mp 103-104.5 °C); [R]D -81.2°
25
(c ) 1, MeOH) (lit.32 [R]D -79.2° (c ) 1, MeOH)).
N-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-br om o-
2-h yd r oxy-2-m eth ylp r op a n a m id e ((R)-2). Bromolactone
(R)-2 was prepared from prolineamide (R)-1 according to the
slightly modified procedure described by Tucker.21 We used 2
equiv of NBS instead of 1 equiv as in the original procedure
and obtained the title compound with a better yield (80% vs
49%): mp 152-154 °C (lit.21 mp 157-159 °C for the (S)-
As a whole, these studies demonstrate that chiral
nonsteroidal ligands demonstrate potent, stereoselec-
tive, regioselective, and irreversible binding to the AR.
These compounds will provide valuable tools for the
1
bromolactone); H NMR (DMSO-d6) δ 4.69 (dd, J ) 9.6 Hz, J
) 6.7 Hz, 1H, CH at the chiral center), 4.02 (d, J ) 11.4 Hz,

586 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Kirkovsky et al.
1H, CH(1)), 3.86 (d, J ) 11.4 Hz, 1H, CH(2)), 2.30-2.20 (m,
1H, aliphatic CH), 2.04-1.72 (m, 3H, aliphatic CH2 and CH),
1.56 (s, 3H, Me); 13C NMR (DMSO-d6) δ 167.26, 163.08, 83.89,
57.23, 45.42, 37.82, 29.03, 22.93, 21.58; [R]D26 +124.5° (c ) 1.3,
chloroform) (for (S)-2 lit.21 [R]D -126.5° (c ) 1.19, chloroform)).
Anal. (C9H12BrNO3) C, H, N.
compound was obtained as the mixture of (S)-4 and another
compound (the most likely, corresponding chloroanilide N1-
[4-cya n o-3-(tr iflu or om eth yl)p h en yl]-(2S)-3-ch lor o-2-h y-
d r oxy-2-m eth yl p r op a n a m id e ((S)-4a )) with the ratio of 85:
15 and 80% yield: mp 134.5-135.5 °C (lit.21 mp 106-107 °C
for the (S)-isomer); [R]D +47.0° (c ) 1.0, MeOH) (lit.21 [R]D
27
+50.73° (c ) 1.1, MeOH)); for (S)-4 1H NMR (DMSO-d6) δ
10.51 (s, 1H, NH), 8.53 (d, J ) 1.5 Hz, 1H, ArH), 8.29 (dd, J
) 8.5 Hz, J ) 1.9 Hz, 1H, ArH), 8.10 (d, J ) 8.6 Hz, 1H, ArH),
6.39 (s, 1H, OH), 3.81 (d, J ) 10.4 Hz, 1H, CH(1)), 3.57 (d, J
) 10.4 Hz, 1H, CH(2)), 1.47 (s, 3H, Me).
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2S)-3-ch lor o-
2-h yd r oxy-2-m et h yl p r op a n a m id e ((R)-4). Chloroanilide
(S)-4a was not isolated from the mixture with corresponding
bromoanilide (S)-4 (see Chemistry section): 1H NMR (DMSO-
d6) δ 10.53 (s, 1H, NH), 8.53 (d, J ) 1.5 Hz, 1H, ArH), 8.29
(dd, J ) 8.5 Hz, J ) 1.9 Hz, 1H, ArH), 8.10 (d, J ) 8.6 Hz, 1H,
ArH), 6.38 (s, 1H, OH), 3.90 (d, J ) 11.1 Hz, 1H, CH(1)), 3.67
(d, J ) 11.2 Hz, 1H, CH(2)), 1.42 (s, 3H, Me).
N-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2S)-3-br om o-
2-h yd r oxy-2-m eth ylp r op a n a m id e ((S)-2). Bromolactone
(S)-2 was prepared from prolineamide (S)-1 by the same
method used for preparation of (R)-2. The title compound was
obtained with 64% yield: mp 157-159 °C (lit.21 mp 157-159
25
°C); [R]D -123.6° (c ) 1.3, chloroform) (lit.21 [R]D -126.5° (c
) 1.19, chloroform)).
(2R)-3-Br om o-2-h yd r oxy-2-m eth ylp r op a n oic Acid ((R)-
3). Bromoacid (R)-3 was prepared by acid-catalyzed hydrolysis
of bromolactone (R)-2 in refluxing concentrated HCl according
to the procedure described by Tucker et al.21 The title
compound was obtained as the mixture of (R)-3 and another
compound (corresponding chloroacid (2R)-3-ch lor o-2-h ydr oxy-
2-m eth ylp r op a n oic a cid , (R)-3a ) with the ratio of 85:15 and
79% yield: mp 106.5-109 °C (lit.21 mp 109-113 °C for the
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2R)-3-[(4-am i-
n op h en yl)su lfa n yl]-2-h yd r oxy-2-m et h ylp r op a n a m id e
((R)-5). A solution of freshly distilled 4-aminothiophenol
(short-path distillation on Kugelrohr at approximately 150 °C)
(1.38 g, 11.0 mmol) in anhydrous THF (10 mL) was added
under argon atmosphere to preliminary washed 60% oil
dispersion of NaH (0.45 g, 11.2 mmol) in 10 mL of anhydrous
THF. The reaction mixture was stirred over 36 h until forming
a white suspension. A solution of anilides (R)-4 and (R)-4a
(85:15 ratio) (3.00 g, 8.7 mmol) in 15 mL of anhydrous THF
was injected dropwise. The reaction mixture was stirred for
26 h. The final suspension was filtered out, and the filtrate
was completely evaporated to give an oil. The oil was solidified
after addition of small amount of methanol. This solid was
recrystallized from methanol to give 2.53 g (73%) of the title
compound as white crystals: mp 166.5-167 °C; 1H NMR
(DMSO-d6) δ 10.44 (s, 1H, NH), 8.48 (d, J ) 1.9 Hz, 1H, ArH),
8.23 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.06 (d, J ) 8.6
Hz, 1H, ArH), 7.05-7.01 (m, 2H, ArH), 6.43-6.48 (m, 2H,
ArH), 6.08 (s, 1H, OH), 5.14 (br s, 2H, NH2), 3.17 (d, J ) 13.0
Hz, 1H, CH(1)), 3.07 (d, J ) 13.0 Hz, 1H, CH(2)), 1.40 (s, 3H,
Me); 13C NMR (DMSO-d6) δ 174.97, 148.18, 143.18, 136.13,
(S)-bromoacid (S)-3); [R]D +10.5° (c ) 2.6, MeOH) (lit.21 [R]D
25
-11.78° (c ) 1.16, MeOH) for (S)-3); for (R)-3 1H NMR
(DMSO-d6) δ 3.63 (d, J ) 10.1 Hz, 1H, CH(1)), 3.52 (d, J )
10.1 Hz, 1H, CH(2)), 1.35 (s, 3H, Me).
(2R)-3-Ch lor o-2-h yd r oxy-2-m eth ylp r op a n oic Acid ((R)-
3a ). Chloroacid (R)-3a was not isolated from the mixture with
corresponding bromoacid (R)-3 (see Chemistry section): 1H
NMR (DMSO-d6) δ 3.73 (d, J ) 10.9 Hz, 1H, CH(1)), 3.61 (d,
J ) 10.9 Hz, 1H, CH(2)), 1.30 (s, 3H, Me).
(2S)-3-Br om o-2-h yd r oxy-2-m eth ylp r op a n oic Acid ((S)-
3). Bromoacid (S)-3 was prepared by acid-catalyzed hydrolysis
of bromolactone (S)-2 in refluxing concentrated HCl according
to the procedure described by Tucker.21 The title compound
was obtained as the mixture of (S)-3 and another compound
(the most likely, corresponding chloroacid (2S)-3-b r om o-2-
h yd r oxy-2-m eth ylp r op a n oic a cid ((S)-3a )) with the ratio
of 85:15 and 64% yield: mp 112-113.5 °C (lit.21 mp 109-113
27
°C for the (S)-bromoacid (S)-3); [R]D -11.3° (c ) 2.6, MeOH)
(lit.21 [R]D -11.78° (c ) 1.16, MeOH)); for (S)-3 1H NMR
(DMSO-d6) δ 3.63 (d, J ) 10.1 Hz, 1H, CH(1)), 3.52 (d, J )
10.1 Hz, 1H, CH(2)), 1.35 (s, 3H, Me).
2
1
133.36, 131.42 (q, J C-F ) 31.7 Hz), 122.49 (q, J C-F ) 273.6
Hz), 122.64, 119.83, 117.36 (q, 3J C-F ) 5.0 Hz), 115.83, 114.22,
(2S)-3-Ch lor o-2-h yd r oxy-2-m eth ylp r op a n oic Acid ((S)-
3a ). Chloroacid (S)-3a was not isolated form the mixture with
corresponding bromoacid (S)-3 (see Chemistry section): 1H
NMR (DMSO-d6) δ 3.73 (d, J ) 10.9 Hz, 1H, CH(1)), 3.61 (d,
J ) 10.9 Hz, 1H, CH(2)), 1.31 (s, 3H, Me).
3
101.74 (q, J C-F ) 2 Hz), 75.32, 47.26, 25.64; IR (KBr) 3486,
3447, 3386, 3363 (NH, NH2, OH), 2234 (CN), 1681 (CO), 1623,
1596, 1583, 1513, 1331 cm-1; [R]D +33.0° (c ) 2, acetone).
27
Anal. (C18H16F3N3O2S) C, H, N.
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-br om o-
2-h yd r oxy-2-m et h ylp r op a n a m id e ((R)-4). Bromoanilide
(R)-4 was prepared from the mixture of the acids (R)-3 and
(R)-3a according to the procedure described by Tucker.21 The
title compound was obtained as the mixture of (R)-4 and
another compound (the most likely, corresponding chloroanil-
ide N1-[4-cyan o-3-(tr iflu or om eth yl)ph en yl]-(2R)-3-ch lor o-
2-h yd r oxy-2-m eth yl p r op a n a m id e, (R)-4a ) with the ratio
of 85:15 and 59% yield: mp 132-134 °C (lit.21 mp 106-107
°C for the (S)-isomer); [R]D27 -48.5° (c ) 1.0, MeOH) (lit.21 [R]D
N 1-[4-Cya n o-3-(t r iflu or om e t h yl)p h e n yl]-(2S )-3-[(4-
a m in op h e n yl)su lfa n yl]-2-h yd r oxy-2-m e t h ylp r op a n a -
m id e ((S)-5). Title compound (S)-5 was obtained as white
crystals (71% yield) from the mixture of anilides (S)-4 and (S)-
4a according to the procedure used for preparation of (R)-5:
1
mp 166.5-167 °C; H NMR (DMSO-d6) δ 10.41 (s, 1H, NH),
8.47 (d, J ) 1.9 Hz, 1H, ArH), 8.22 (dd, J ) 8.6 Hz, J ) 2.0
Hz, 1H, ArH), 8.05 (d, J ) 8.6 Hz, 1H, ArH), 7.06-7.01 (m,
2H, ArH), 6.44-6.48 (m, 2H, ArH), 6.05 (s, 1H, OH), 5.22 (br
s, 2H, NH2), 3.17 (d, J ) 13.0 Hz, 1H, CH(1)), 3.07 (d, J )
13.0 Hz, 1H, CH(2)), 1.39 (s, 3H, Me); 13C NMR (DMSO-d6) δ
174.95, 147.80, 143.15, 136.11, 133.27, 131.42 (q, 2J C-F ) 31.7
1
+50.73° (c ) 1.1, MeOH) for (S)-4); for (R)-4 H NMR (DMSO-
d6) δ 10.51 (s, 1H, NH), 8.53 (d, J ) 1.8 Hz, 1H, ArH), 8.28
(dd, J ) 8.4 Hz, J ) 1.8 Hz, 1H, ArH), 8.09 (d, J ) 8.7 Hz, 1H,
ArH), 6.39 (s, 1H, OH), 3.81 (d, J ) 10.4 Hz, 1H, CH(1)), 3.57
(d, J ) 10.4 Hz, 1H, CH(2)), 1.47 (s, 3H, Me).
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-ch lor o-
2-h yd r oxy-2-m eth ylp r op a n a m id e ((R)-4a ). Chloroanilide
(R)-4a was not isolated from the mixture with corresponding
bromoanilide (R)-4 (see Chemistry section): 1H NMR (DMSO-
d6) δ 10.52 (s, 1H, NH), 8.53 (d, J ) 1.8 Hz, 1H, ArH), 8.28
(dd, J ) 8.4 Hz, J ) 1.8 Hz, 1H, ArH), 8.09 (d, J ) 8.7 Hz, 1H,
ArH), 6.37 (s, 1H, OH), 3.90 (d, J ) 11.1 Hz, 1H, CH(1)), 3.67
(d, J ) 11.2 Hz, 1H, CH(2)), 1.42 (s, 3H, Me).
1
Hz), 122.63, 122.48 (q, J C-F ) 273.5 Hz), 120.17, 117.36 (q,
3J C-F ) 5.0 Hz), 115.80, 114.38, 101.74 (q, 3J C-F ) 2 Hz), 75.30,
47.17, 25.62; IR (KBr) 3485, 3447, 3385, 3365 (NH, NH2, OH),
2234 (CN), 1681 (CO), 1623, 1596, 1583, 1517, 1330 cm-1; [R]D
-33.7° (c ) 2, acetone). Anal. (C18H16F3N3O2S) C, H, N.
27
N 1-[4-Cya n o-3-(t r iflu or om e t h yl)p h e n yl]-(2R )-3-[(3-
a m in op h e n yl)su lfa n yl]-2-h yd r oxy-2-m e t h ylp r op a n a -
m id e ((R)-6). Neat 3-aminothiophenol (1.30 mL, d 1.179, 12.2
mmol) was added dropwise under argon atmosphere to pre-
liminary washed 60% oil dispersion of NaH (0.46 g, 11.50
mmol) in 10 mL of anhydrous THF. The reaction mixture was
stirred for 30 min until bubbles stopped to eliminate and a
white suspension formed. The solution of anilides (R)-4 and
(R)-4a (85:15 ratio) (3.00 g, 8.7 mmol) in 20 mL of anhydrous
THF was injected dropwise. The reaction mixture was stirred
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2S)-3-br om o-
2-h ydr oxy-2-m eth ylpr opan am ide ((S)-4). Bromoanilide (S)-4
was prepared from the mixture of the acids (S)-3 and (S)-3a
according to the procedure described by Tucker.21 The title

Chiral Nonsteroidal Affinity Ligands for AR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 587
for 15 h. Final suspension was diluted with 50 mL of water
and extracted with EtOAc (2 × 150 mL). The combined EtOAc
extracts were dried over MgSO4, filtered, and evaporated to
give an oil. The oil was applied to a flush column (stationary
phase silica gel, chloroform as mobile phase). Title compound
was obtained as a white solid (1.95 g, 58%): mp 123-125 °C;
1H NMR (DMSO-d6) δ 10.47 (s, 1H, NH), 8.49 (d, J ) 1.7 Hz,
1H, ArH), 8.25 (dd, J ) 8.6 Hz, J ) 1.8 Hz, 1H, ArH), 8.06 (d,
J ) 8.6 Hz, 1H, ArH), 6.86 (t, J ) 7.8 Hz, 1H, ArH), 6.52 (t, J
) 1.9 Hz, 1H, ArH), 6.44 (dm, J ) 8 Hz, 1H, ArH), 6.32 (dm,
J ) 8 Hz, 1H, ArH), 6.22 (s, 1H, OH), 5.06 (br s, 2H, NH2),
3.33 (d, J ) 12.5 Hz, 1H, CH(1), overlapped with water peak),
3.22 (d, J ) 12.6 Hz, 1H, CH(2)), 1.46 (s, 3H, Me); 13C NMR
(DMSO-d6) δ 174.91, 149.00, 143.12, 136.80, 136.16, 131.47
as for compound (R)-7 1H NMR (DMSO-d6) δ 10.47 (s, 1H,
NH), 8.41 (d, J ) 1.7 Hz, 1H, ArH), 8.19 (dd, J ) 8.6 Hz, J )
1.7 Hz, 1H, ArH), 8.06 (d, J ) 8.6 Hz, 1H, ArH), 7.38-7.34
(m, 2H, ArH), 7.26-7.23 (m, 2H, ArH), 6.30 (s, 1H, OH), 3.47
(d, J ) 13.3 Hz, 1H, CH(1)), ‚3.28 (d, J ) 13.4 Hz, 1H, CH(2),
overlapped with water peak), 1.46 (s, 3H, Me); 13C NMR
(DMSO-d6) δ 174.67, 143.00, 136.98, 136.13, 133.43, 131.38
2
(q, J C-F ) 31.3 Hz), 129.27, 127.09, 126.14, 122.71, 122.46
(q, 1J C-F ) 273.7 Hz), 117.31 (q, 3J C-F ) 5.1 Hz), 115.77, 101.90
(q, 3J C-F ) 2 Hz), 75.07, 43.05, 25.76; IR (KBr) 3415, 3376 (NH,
OH), 2240 (CN), 2097 (NCS), 1709 (CO), 1582, 1521, 1431,
27
1328, 1181, 1135 cm-1; [R]D -54.8° (c ) 2, acetone). Anal.
(C19H14F3N3O2S2) C, H, N.
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2R)-2-h ydr oxy-
3-[(3-isoth iocya n a top h en yl)su lfa n yl]-2-m eth ylp r op a n a -
m id e ((R)-8). Title compound was obtained from (R)-6 with
99% yield by the same method used for preparation of (R)-7:
mp 100-102 °C; 1H NMR (DMSO-d6) δ 10.47 (s, 1H, NH), 8.41
(d, J ) 1.8 Hz, 1H, ArH), 8.18 (dd, J ) 8.6 Hz, J ) 2.0 Hz, 1H,
ArH), 8.05 (d, J ) 8.6 Hz, 1H, ArH), 7.36 (t, J ) 1.8 Hz, 1H,
ArH), 7.31 (dt, J ) 7.9 Hz, J ) 1.5 Hz, 1H, ArH), 7.25 (t, J )
7.8 Hz, 1H, ArH), 7.11 (dt, J ) 7.6 Hz, J ) 1.6 Hz, 1H, ArH),
6.28 (s, 1H, OH), 3.49 (d, J ) 13.4 Hz, 1H, CH(1)), 3.28 (d, J
) 13.5 Hz, 1H, CH(2), overlapped with water peak), 1.47 (s,
3H, Me); 13C NMR (DMSO-d6) δ 174.61, 142.96, 138.80, 136.09,
2
1
(q, J C-F ) 31.7 Hz), 129.17, 122.71, 122.48 (q, J C-F ) 273.7
Hz), 117.42 (q, 3J C-F ) 5.1 Hz), 115.80, 115.71, 113.44, 111.55,
3
101.86 (q, J C-F ) 2 Hz), 75.12, 43.44, 25.65; IR (KBr) 3440,
3340, 3300 (NH, NH2, OH), 2248 (CN), 1683 (CO), 1612, 1594,
27
1513, 1487, 1428, 1325, 1240, 1182, 1136 cm-1; [R]D -2.8° (c
) 2, acetone). Anal. (C18H16F3N3O2S) C, H, N.
N 1-[4-Cya n o-3-(t r iflu or om e t h yl)p h e n yl]-(2S )-3-[(3-
a m in op h e n yl)su lfa n yl]-2-h yd r oxy-2-m e t h ylp r op a n a -
m id e ((S)-6). Title compound (S)-6 was obtained as a white
solid (75% yield) from the mixture of the anilides (S)-4 and
(S)-4a according to the procedure used for preparation of (R)-
6: mp 123-124.5 °C; 1H NMR (DMSO-d6) δ 10.47 (s, 1H, NH),
8.49 (d, J ) 1.6 Hz, 1H, ArH), 8.25 (dd, J ) 8.6 Hz, J ) 1.8
Hz, 1H, ArH), 8.06 (d, J ) 8.6 Hz, 1H, ArH), 6.86 (t, J ) 7.8
Hz, 1H, ArH), 6.52 (t, J ) 1.8 Hz, 1H, ArH), 6.43 (dm, J ) 7.7
Hz, 1H, ArH), 6.31 (dm, J ) 7.9 Hz, 1H, ArH), 6.21 (s, 1H,
OH), 5.06 (br s, 2H, NH2), 3.33 (d, J ) 12.3 Hz, 1H, CH(1),
overlapped with water peak), 3.22 (d, J ) 12.6 Hz, 1H, CH-
(2)), 1.46 (s, 3H, Me); 13C NMR (DMSO-d6) δ 174.95, 149.03,
2
133.62, 131.40 (q, J C-F ) 31.7 Hz), 130.37, 129.88, 127.81,
1
125.10, 122.94, 122.63, 122.44 (q, J C-F ) 274.0 Hz), 117.24
3
3
(q, J C-F ) 5.3 Hz), 115.76, 101.94 (q, J C-F ) 2 Hz), 75.05,
42.96, 25.75; IR (KBr) 3448, 3316 (NH, OH), 2243 (CN), 2209,
2124 (NCS), 1683 (CO), 1581, 1511, 1491, 1429, 1330, 1237,
1174, 1135 cm-1; [R]D -18.3° (c ) 2, acetone). Anal.
(C19H14F3N3O2S2) C, H, N.
27
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2S)-2-h ydr oxy-
3-[(3-isoth iocya n a top h en yl)su lfa n yl]-2-m eth ylp r op a n a -
m id e ((S)-8). Title compound was obtained from (S)-6 with
60% yield by the same method used for preparation of (R)-7:
mp 100-102 °C; 1H NMR (DMSO-d6) δ 10.47 (s, 1H, NH), 8.40
(d, J ) 1.9 Hz, 1H, ArH), 8.18 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H,
ArH), 8.04 (d, J ) 8.6 Hz, 1H, ArH), 7.35 (t, J ) 1.7 Hz, 1H,
ArH), 7.31 (dt, J ) 8.1 Hz, J ) 1.5 Hz, 1H, ArH), 7.24 (t, J )
7.8 Hz, 1H, ArH), 7.10 (dt, J ) 7.7 Hz, J ) 1.6 Hz, 1H, ArH),
6.29 (s, 1H, OH), 3.49 (d, J ) 13.4 Hz, 1H, CH(1)), 3.28 (d, J
) 13.5 Hz, 1H, CH(2), overlapped with water peak), 1.47 (s,
3H, Me); 13C NMR (DMSO-d6) δ 174.61, 142.96, 138.80, 136.09,
2
143.15, 136.82, 136.20, 131.48 (q, J C-F ) 31.8 Hz), 129.20,
1
3
122.72, 122.50 (q, J C-F ) 273.9 Hz), 117.43 (q, J C-F ) 4.9
Hz), 115.84, 115.67, 113.40, 111.55, 101.87 (q, 3J C-F ) 1.8 Hz),
75.13, 43.43, 25.70; IR (KBr) 3440, 3340, 3301 (NH, NH2, OH),
2248 (CN), 1683 (CO), 1614, 1594, 1515, 1487, 1429, 1326,
27
1240, 1183, 1135 cm-1; [R]D +3.2° (c ) 2, acetone). Anal.
(C18H16F3N3O2S) C, H, N.
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2R)-2-h ydr oxy-
3-[(4-isoth iocya n a top h en yl)su lfa n yl]-2-m eth ylp r op a n a -
m id e ((R)-7). Title compound was prepared by general method
proposed by Leclerc.26 Aqueous solutions of NaHCO3 (200 mg,
2.4 mmol) in water (20 mL) were added to a vigorously stirred
solution of (R)-5 (460 mg, 1.16 mmol) in a mixture of 50 mL
of chloroform, 20 mL of CH2Cl2, and 10 mL of EtOAc, followed
by thiophosgene (0.15 mL, d 1.508, 1.91 mmol). The reaction
mixture with a yellow precipitate was stirred for 3 h until the
organic layer become transparent. The organic layer was
separated, and the aqueous portion of the reaction mixture
was extracted with CH2Cl2 (10 mL). Both organic fractions
were combined together, dried over Na2SO4, and evaporated
to give a yellow oil which crystallized when standing. This
mixture was triturated with toluene (20 mL) to give the
product (460 mg, 91%) as a light yellow solid: mp 129-130
2
133.67, 131.42 (q, J C-F ) 31.7 Hz), 130.39, 129.89, 127.83,
1
125.11, 122.95, 122.64, 122.46 (q, J C-F ) 273.7 Hz), 117.25
3
3
(q, J C-F ) 5.1 Hz), 115.77, 101.96 (q, J C-F ) 2 Hz), 75.06,
42.98, 25.76; IR (KBr) 3448, 3316 (NH, OH), 2243 (CN), 2209,
2125 (NCS), 1683 (CO), 1581, 1511, 1491, 1429, 1330, 1237,
1174, 1135 cm-1; [R]D +20.4° (c ) 2, acetone). Anal.
(C19H14F3N3O2S2) C, H, N.
27
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2R)-2-h ydr oxy-
3-[(4-isoth iocya n a top h en yl)su lfon yl]-2-m eth ylp r op a n a -
m id e ((R)-9). A solution of (R)-7 (260 mg, 0.59 mmol) in
CH2Cl2 (30 mL) was mixed with solution of m-chloroperbenzoic
acid (620 mg, 3.6 mmol) in CH2Cl2 (50 mL) at room temper-
ature. The reaction mixture was stirred for 80 min and washed
with saturated aqueous solution of Na2SO3 (2 × 50 mL) and
saturated aqueous solution of NaHCO3 (3 × 50 mL). The
organic layer was separated, dried over Na2SO4, reduced in
volume in vacuo, and applied to a flash column (stationary
phase silica gel, mobile phase chloroform/MeOH ) 195:5).
Final compound was crystallized from ether/hexane as a white
1
°C; H NMR (DMSO-d6) δ 10.46 (s, 1H, NH), 8.41 (d, J ) 1.8
Hz, 1H, ArH), 8.19 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH),
8.05 (d, J ) 8.6 Hz, 1H, ArH), 7.39-7.35 (m, 2H, ArH), 7.26-
7.22 (m, 2H, ArH), 6.29 (s, 1H, OH), 3.47 (d, J ) 13.3 Hz, 1H,
CH(1)), ‚3.28 (d, J ) 14 Hz, 1H, CH(2), overlapped with water
peak), 1.47 (s, 3H, Me); 13C NMR (DMSO-d6) δ 174.63, 142.96,
2
136.95, 136.09, 133.45, 131.35 (q, J C-F ) 31.7 Hz), 129.26,
1
1
127.08, 126.10, 122.68, 122.44 (q, J C-F ) 273.6 Hz), 117.29
solid: mp 162.5-163 °C; H NMR (DMSO-d6) δ 10.31 (s, 1H,
3
3
(q, J C-F ) 5.0 Hz), 115.73, 101.87 (q, J C-F ) 2 Hz), 75.04,
NH), 8.40 (d, J ) 1.6 Hz, 1H, ArH), 8.18 (dd, J ) 8.6 Hz, J )
1.8 Hz, 1H, ArH), 8.08 (d, J ) 8.6 Hz, 1H, ArH), 7.90-7.86
(m, 2H, ArH), 7.52-7.48 (m, 2H, ArH), 6.44 (s, 1H, OH), 3.97
(d, J ) 14.8 Hz, 1H, CH(1)), 3.72 (d, J ) 14.8 Hz, 1H, CH(2)),
1.39 (s, 3H, Me); 13C NMR (DMSO-d6) δ 173.54, 143.03, 138.97,
43.05, 25.72; IR (KBr) 3428, 3377 (NH, OH), 2243 (CN), 2080
(NCS), 1710 (CO), 1582, 1522, 1430, 1337, 1187, 1139 cm-1
;
27
[R]D + 54.9° (c ) 2, acetone). Anal. (C19H14F3N3O2S2) C, H,
N.
2
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2S)-2-h ydr oxy-
3-[(4-isoth iocya n a top h en yl)su lfa n yl]-2-m eth ylp r op a n a -
m id e ((S)-7). Title compound was obtained with 87% yield
from (S)-5 according to the same method used for preparation
of (R)-7: mp 131.5-132 °C; 1H NMR (DMSO-d6) was the same
136.42, 136.12, 134.82, 131.38 (q, J C-F ) 31.9 Hz), 130.15,
1
3
126.29, 122.77, 122.49 (q, J C-F ) 273.5 Hz), 117.40 (q, J C-F
3
) 5.5 Hz), 115.79, 101.97 (d, J C-F ) 2.5 Hz), 72.99, 63.34,
27.26; IR (KBr) 3431, 3350 (NH, OH), 2232 (CN), 2193, 2042
(NCS), 1702 (CO), 1588, 1526, 1431, 1327 (SO2 asym), 1179,

588 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Kirkovsky et al.
1141 (SO2 sym) cm-1; [R]D -59.1° (c ) 2, acetone). Anal.
under ether/hexane mixture to give 560 mg (94%) of the title
compound: mp 67 °C started melting; H NMR (DMSO-d6) δ
27
1
(C19H14F3N3O4S2) C, H, N.
10.47 (s, 1H, NH), 10.22 (s, 1H, NH), 8.45 (d, J ) 1.8 Hz, 1H,
ArH), 8.21 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.05 (d, J )
8.6 Hz, 1H, ArH), 7.60 (t, J ) 1.8 Hz, 1H, ArH), 7.29 (dm, J )
7 Hz, 1H, ArH), 7.18 (t, J ) 7.9 Hz, 1H, ArH), 7.06 (dt, J )
7.9 Hz, J ) 1.4 Hz, 1H, ArH), 6.27 (s, 1H, OH), 4.21 (s, 2H,
CH2Cl), 3.41 (d, J ) 12.9 Hz, 1H, CH(1)), 3.27 (d, J ) 12.9 Hz,
1H, CH(2)), 1.47 (s, 3H, Me); 13C NMR (DMSO-d6) δ 174.80,
164.68, 143.08, 138.86, 137.29, 136.19, 131.46 (q, 2J C-F ) 31.7
N1-[4-Cyan o-3-(tr iflu or om eth yl)ph en yl]-(2R)-2-h ydr oxy-
3-[(3-isoth iocya n a top h en yl)su lfon yl]-2-m eth ylp r op a n a -
m id e ((R)-10). Title compound was obtained from (R)-8 with
56% yield by the same method used for preparation (R)-9: mp
97-100 °C dec; 1H NMR (DMSO-d6) δ 10.41 (s, 1H, NH), 8.42
(d, J ) 1.5 Hz, 1H, ArH), 8.20 (dd, J ) 8.6 Hz, J ) 1.5 Hz, 1H,
ArH), 8.08 (d, J ) 8.6 Hz, 1H, ArH), 7.85-7.75 (m, 2H, ArH),
7.63-7.61 (m, 2H, ArH), 6.40 (s, 1H, OH), 4.00 (d, J ) 14.9
Hz, 1H, CH(1)), 3.80 (d, J ) 14.9 Hz, 1H, CH(2)), 1.41 (s, 3H,
Me); 13C NMR (DMSO-d6) δ 173.56, 143.03, 132.33, 136.12,
1
Hz), 129.28, 123.82, 122.73, 122.51 (q, J C-F ) 273.7 Hz),
3
118.87, 117.40 (q, J C-F ) 5.1 Hz), 116.60, 115.87, 101.92 (q,
3J C-F ) 2 Hz), 75.09, 43.52, 43.47, 25.75; IR (KBr) 3330 (broad,
2
135.51, 131.39 (q, J C-F ) 31.6 Hz), 130.77, 130.61, 130.56,
1
NH, OH), 2232 (CN), 1683 (CO), 1588, 1523, 1430, 1327, 1181,
127.03, 125.48, 122.81, 122.46 (q, J C-F ) 273.5 Hz), 117.48
1136 cm-1; [R]D -10.1° (c ) 2, acetone); MS (ESI) m/z 472.
27
3
3
(q, J C-F ) 5.3 Hz), 115.77, 102.04 (q, J C-F ) 2 Hz), 73.03,
63.07, 27.09; IR (KBr) 3455, 3355 (NH, OH), 2232 (CN), 2019
(NCS), 1690 (CO), 1588, 1522, 1432, 1331 (SO2 asym), 1295,
1275, 1182, 1137 (SO2 sym) cm-1; [R]D27 -98.1° (c ) 2, acetone).
Anal. (C19H14F3N3O4S2) C, H, N.
Anal. (C20H17ClF3N3O3S‚0.1H2O) C, H, N.
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({4-[(2-
br om oa cetyl)a m in o]p h en yl}su lfa n yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-13). Title compound was obtained with
95% yield by acylation of (R)-5 with bromoacetyl bromide
according to the same procedure used for preparation of (R)-
11: mp 158-159.5 °C; 1H NMR (DMSO-d6) δ 10.45 (s, 1H,
NH), 10.33 (s, 1H, NH), 8.43 (d, J ) 1.8 Hz, 1H, ArH), 8.20
(dd, J ) 8.6 Hz, J ) 1.8 Hz, 1H, ArH), 8.04 (d, J ) 8.6 Hz, 1H,
ArH), 7.45-7.38 (m, 2H, ArH), 7.31-7.28 (m, 2H, ArH), 6.21
(s, 1H, OH), 3.99 (s, 2H, CH2Br), 3.38 (d, J ) 13.1 Hz, 1H,
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({4-[(2-
ch lor oa cetyl)a m in o]p h en yl}su lfa n yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-11). Neat chloroacetyl chloride (0.1 mL,
d 1.418, 1.2 mmol) was added dropwise under argon over
period of 1-2 min to the vigorously stirred suspension,
containing solution of (R)-5 (410 mg, 1.04 mmol) in anhydrous
CH2Cl2 (50 mL) and dry powder of CaCO3 (0.56 g, 5.6 mmol).
The reaction mixture was stirred overnight and filtered out,
and the filtrate was completely evaporated in vacuo to give a
white solid. The solid was recrystallized from EtOAc/hexane
to give 377 mg (77%) of the title compound in ball-shape
CH(1)), 3.22 (d, J ) 13.1 Hz, 1H, CH(2)), 1.44 (s, 3H, Me); 13
C
NMR (DMSO-d6) δ 174.81, 164.62, 143.08, 136.84, 136.14,
2
131.41 (q, J C-F ) 31.7 Hz), 130.86, 130.08, 122.67, 122.48 (q,
3
1J C-F ) 273.5 Hz), 119.54, 117.35 (q, J C-F ) 5.1 Hz), 115.84,
101.85 (q, 3J C-F ) 1.8 Hz), 75.19, 44.47, 30.28, 25.75; IR (KBr)
1
crystals: mp 134.5-135 °C; H NMR (DMSO-d6) δ 10.44 (s,
3466, 3317, 3263 (NH, OH), 2238 (CN), 1679 (CO), 1667, 1608,
1H, NH), 10.24 (s, 1H, NH), 8.43 (d, J ) 1.8 Hz, 1H, ArH),
8.20 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.03 (d, J ) 8.6
Hz, 1H, ArH), 7.45-7.43 (m, 2H, ArH), 7.31-7.28 (m, 2H,
ArH), 6.20 (s, 1H, OH), 4.20 (s, 2H, CH2Cl), 3.38 (d, J ) 13.1
Hz, 1H, CH(1)), 3.23 (d, J ) 13.1 Hz, 1H, CH(2)), 1.45 (s, 3H,
Me); 13C NMR (DMSO-d6) δ 174.76, 164.43, 143.05, 136.69,
1597, 1510, 1429, 1327, 1173, 1135 cm-1; [R]D +24.0° (c ) 2,
27
acetone). Anal. (C20H17BrF3N3O3S‚0.09CH2Cl2) C, H, N.
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({3-[(2-
br om oa cetyl)a m in o]p h en yl}su lfa n yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-14). Title compound was obtained with
94% yield by acylation of (R)-6 with bromoacetyl bromide
according to the same procedure used for preparation of (R)-
11: mp 60-65 °C started melting; 1H NMR (DMSO-d6) δ 10.47
(s, 1H, NH), 10.36 (s, 1H, NH), 8.45 (d, J ) 1.8 Hz, 1H, ArH),
8.21 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.04 (d, J ) 8.6
Hz, 1H, ArH), 7.59 (t, J ) 1.7 Hz, 1H, ArH), 7.29 (dm, J ) 8.8
Hz, 1H, ArH), 7.18 (t, J ) 7.9 Hz, 1H, ArH), 7.05 (dt, J ) 7.9
Hz, J ) 1.3 Hz, 1H, ArH), 6.27 (s, 1H, OH), 3.99 (s, 2H, CH2-
Br), 3.41 (d, J ) 12.9 Hz, 1H, CH(1)), 3.27 (d, J ) 12.9 Hz,
1H, CH(2)), 1.47 (s, 3H, Me); 13C NMR (DMSO-d6) δ 174.76,
164.78, 143.05, 138.96, 137.28, 136.15, 131.41 (q, 2J C-F ) 31.7
2
136.09, 131.39 (q, J C-F ) 31.7 Hz), 130.85, 130.06, 122.64,
1
3
122.46 (q, J C-F ) 273.7 Hz), 119.67, 117.33 (q, J C-F ) 5.0
Hz), 115.79, 101.85 (q, 3J C-F ) 2 Hz), 75.17, 44.46, 43.44, 25.69.
IR (KBr) 3464, 3321, ‚3265 (NH, OH), 2238 (CN), 1679 (CO),
1609, 1597, 1522, 1429, 1327, 1174, 1135 cm-1; [R]D +23.4°
(c ) 2, acetone). Anal. (C20H17ClF3N3O3S) C, H, N.
27
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2S)-3-({4-[(2-
ch lor oa cetyl)a m in o]p h en yl}su lfa n yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((S)-11). Title compound was obtained with
56% yield from (S)-5 by the same method used for preparation
of (R)-11: mp 132-134 °C; 1H NMR (DMSO-d6) δ 10.44 (s,
1H, NH), 10.23 (s, 1H, NH), 8.43 (d, J ) 1.7 Hz, 1H, ArH),
8.20 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.04 (d, J ) 8.6
Hz, 1H, ArH), 7.45-7.42 (m, 2H, ArH), 7.31-7.28 (m, 2H,
ArH), 6.20 (s, 1H, OH), 4.20 (s, 2H, CH2Cl), 3.38 (d, J ) 13.2
Hz, 1H, CH(1), overlapped with water peak), 3.23 (d, J ) 13.1
Hz, 1H, CH(2)), 1.44 (s, 3H, Me); 13C NMR (DMSO-d6) δ 174.81,
1
Hz), 129.23, 123.75, 122.68, 122.47 (q, J C-F ) 273.7 Hz),
3
118.68, 117.37 (q, J C-F ) 4.9 Hz), 116.43, 115.82, 101.88 (q,
3J C-F ) 2 Hz), 75.06, 43.42, 30.27, 25.70; IR (KBr) 3331 (broad,
NH, OH), 2232 (CN), 1678 (CO), 1587, 1523, 1430, 1327, 1181,
1137 cm-1; MS (ESI) m/z 516, 518; [R]D -14.7° (c ) 2,
27
acetone). Anal. (C20H17BrF3N3O3S‚0.65H2O) C, H, N.
2
164.46, 143.09, 136.71, 136.14, 131.41 (q, J C-F ) 31.7 Hz),
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({4-[(2-
ch lor oa cetyl)a m in o]p h en yl}su lfon yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-15). Aqueous solution of peracetic acid
(4 mL, 32%, d 1.13, 19 mmol) was added to a solution of (R)-
11 (220 mg, 0.47 mmol) in CH2Cl2 (10 mL). After 2.5 h of
stirring at room temperature the reaction mixture was diluted
with 200 mL of EtOAc and washed with saturated aqueous
solution of Na2SO3 (50 mL) and brine (30 mL). The organic
layer was separated, dried over Na2SO4, and completely
evaporated to dryness to give a white solid. Recrystallization
of the solid from EtOAc/hexane gave the title compound in
quantitative yield: mp 174-175 °C; 1H NMR (DMSO-d6) δ
10.61 (s, 1H, NH), 10.33 (s, 1H, NH), 8.40 (d, J ) 1.8 Hz, 1H,
ArH), 8.17 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.05 (d, J )
8.6 Hz, 1H, ArH), 7.81-7.78 (m, 2H, ArH), 7.70-7.67 (m, 2H,
ArH), 6.38 (s, 1H, OH), 4.25 (s, 2H, CH2Cl), 3.89 (d, J ) 14.7
Hz, 1H, CH(1)), 3.62 (d, J ) 14.7 Hz, 1H, CH(2)), 1.39 (s, 3H,
Me); 13C NMR (DMSO-d6) δ 173.67, 165.23, 143.13, 142.93,
1
130.86, 130.07, 122.67, 122.49 (q, J C-F ) 273.8 Hz), 119.67,
3
3
117.34 (q, J C-F ) 5.3 Hz), 115.84, 101.86 (q, J C-F ) 1.8 Hz),
75.19, 44.46, 43.49, 25.75; IR (KBr) 3464, 3320, 3268 (NH, OH),
2239 (CN), 1679 (CO), 1609, 1598, 1521, 1429, 1327, 1174,
1135 cm-1; [R]D -23.4° (c ) 2, acetone). Anal. (C20H17
-
27
ClF3N3O3S) C, H, N.
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({3-[(2-
ch lor oa cetyl)a m in o]p h en yl}su lfa n yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-12). Neat chloroacetyl chloride (0.12
mL, d 1.418, 1.5 mmol) and neat N,N-diisopropylethylamine
(0.26 mL, d 0.742, 1.5 mmol) were simultaneously added
dropwise over a period of 5 min to a solution of (R)-6 (500 mg,
1.26 mmol) in anhydrous CH2Cl2 (50 mL) in the ice bath under
argon. The reaction mixture was stirred for 4 h in the ice bath,
warmed to room temperature, diluted with CH2Cl2 (50 mL),
and washed with water (50 mL), 0.1 N HCl (50 mL), aqueous
saturated NaHCO3 solution (50 mL), and brine (50 mL). The
organic layer was separated, dried over Na2SO4, and evapo-
rated to dryness to give a white solid. The solid was triturated
2
136.12, 143.85, 131.38 (q, J C-F ) 31.6 Hz), 129.53, 122.78,
122.51 (q, 1J C-F ) 273.6 Hz), 118.65, 117.43 (q, 3J C-F ) 5 Hz),

Chiral Nonsteroidal Affinity Ligands for AR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 589
115.85, 101.93 (q, 3J C-F ) 2 Hz), 73.09, 63.56, 43.46, 27.26; IR
(KBr) 3477, 3357, 3325 (NH, OH), 2235 (CN), 1692 (CO), 1592,
1527, 1422, 1403, 1329 (SO2 asym), 1301, 1186, 1144 (SO2 sym)
tissue (about 0.4 g/rat) was minced, weighed, and homogenized
(PRO 200 homogenizer, PRO Scientific, Monroe, CT) with 1
mL of the homogenization buffer per 500 mg of prostate tissue.
The homogenate was then centrifuged at 105000g for 1 h at 0
°C in a Beckman L8-M ultracentrifuge (Beckman Instruments
Inc., Palo Alto, CA).29 The supernatant (cytosol) containing AR
protein was removed and stored at -80 °C until use. Total
protein was determined by Peterson’s modification of the
micro-Lowry method, using a protein assay kit from Sigma
Diagnostics, St. Louis, MO.
Da ta An a lysis. Specific binding for each experiment was
calculated by subtracting the binding of [3H]MIB observed in
the presence of excess unlabeled MIB (nonspecific binding)
from the binding of [3H]MIB observed in the absence of
unlabeled MIB (total binding). Competitive radioligand dis-
placement curves were then constructed with percent specific
binding (specific binding of [3H]MIB at a particular ligand
concentration expressed as a percentage of the specific binding
of [3H]MIB in the absence of ligand) on the vertical axis and
ligand concentration on the horizontal axis. The ligand con-
centration that reduces the percentage of specific binding by
50% (IC50) was determined by computer fitting data for the
competitive binding of each AR ligand to the following equa-
tion, using a FORTRAN subroutine written for WIN-NONLIN
(SCI Software, Lexington, KY):
cm-1; MS (ESI) m/z 516, 518; [R]D -49.4° (c ) 2, acetone).
27
Anal. (C20H17ClF3N3O5S) C, H, N.
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({3-[(2-
ch lor oa cetyl)a m in o]p h en yl}su lfon yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-16). Aqueous solution of peracetic acid
(1 mL, 32%, d 1.13, 5 mmol) was added to a solution of (R)-12
(208 mg, 0.44 mmol) in acetone (3 mL). After 10 min of stirring
at room temperature the reaction mixture was diluted with
200 mL of EtOAc, and washed with saturated aqueous solution
of Na2SO3 (50 mL). The organic layer was separated, dried
over Na2SO4, and completely evaporated to dryness to give a
colorless oil which crystallized when triturated under hexane
to give quantitative yield of a creamy solid: mp 130 °C dec,
started melting; 1H NMR (DMSO-d6) δ 10.58 (s, 1H, NH), 10.37
(s, 1H, NH), 8.42 (d, J ) 1.8 Hz, 1H, ArH), 8.20 (dd, J ) 8.6
Hz, J ) 1.9 Hz, 1H, ArH), 8.11 (t, J ) 1.7 Hz, 1H, ArH), 8.07
(d, J ) 8.6 Hz, 1H, ArH), 7.79 (dt, J ) 7.7 Hz, J ) 1.7 Hz, 1H,
ArH), 7.59-7.50 (m, 2H, ArH), 6.40 (s, 1H, OH), 4.24 (s, 2H,
CH2Cl), 3.91 (d, J ) 14.7 Hz, 1H, CH(1)), 3.67 (d, J ) 14.7 Hz,
1H, CH(2)), 1.41 (s, 3H, Me); 13C NMR (DMSO-d6) δ 173.65,
165.01, 143.13, 141.45, 138.90, 136.15, 131.38 (q, 2J C-F ) 31.7
1
Hz), 129.75, 123.68, 123.08, 122.79, 122.51 (q, J C-F ) 273.8
Hz), 118.12, 117.48 (q, 3J C-F ) 5.1 Hz), 115.86, 101.93 (q, 3J C-F
) 2 Hz), 73.17, 63.29, 43.39, 27.16; IR (KBr) 3336 (broad, NH,
OH), 2232 (CN), 1696 (CO), 1598, 1527, 1431, 1328 (SO2 asym),
B ) Bo[1 - C/(IC50 + C)]
27
1304, 1181, 1137 (SO2 sym) cm-1; MS (ESI) m/z 504; [R]D
where B is the specific binding of [3H]MIB in the presence of
a particular concentration of ligand, Bo is the specific binding
of [3H]MIB in the absence of ligand, and C is the ligand
concentration. Binding affinity of the ligand was then com-
pared using the equilibrium dissociation constant (Ki). The Ki
of each ligand was calculated using the equation:
-54.2° (c ) 2, acetone). Anal. (C20H17ClF3N3O5S‚0.25H2O) C,
H, N.
N1-[4-Cya n o-3-(tr iflu or om eth yl)p h en yl]-(2R)-3-({4-[(2-
br om oa cetyl)a m in o]p h en yl}su lfon yl)-2-h yd r oxy-2-m eth -
ylp r op a n a m id e ((R)-17). Title compound was obtained with
83% yield by oxidation of (R)-13 with m-chloroperbenzoic acid
according to the procedure used for preparation of (R)-9: mp
112-114 °C dec, started melting; 1H NMR (DMSO-d6) δ 10.68
(s, 1H, NH), 10.31 (s, 1H, NH), 8.40 (d, J ) 1.7 Hz, 1H, ArH),
8.17 (dd, J ) 8.6 Hz, J ) 1.9 Hz, 1H, ArH), 8.05 (d, J ) 8.6
Hz, 1H, ArH), 7.84-7.75 (m, 2H, ArH), 7.72-7.63 (m, 2H,
ArH), 6.35 (s, 1H, OH), 4.03 (s, 2H, CH2Br), 3.89 (d, J ) 14.7
Hz, 1H, CH(1)), 3.62 (d, J ) 14.7 Hz, 1H, CH(2)), 1.39 (s, 3H,
Me); 13C NMR (DMSO-d6) δ 173.68, 165.42, 143.13, 142.93,
Ki ) (IC50 × Kd)/(L + Kd)
where Kd is the equilibrium dissociation constant of [3H]MIB
and L is the concentration of [3H]MIB (1 nM).
Ack n ow led gm en t. We thank Dr. Howard Tucker
(Zeneca Pharmaceuticals, U.K.) for assistance with
synthetic procedure and providing samples of racemic
bicalutamide and thiobicalutamide. We also thank Dr.
Nina Stourman and graduate student Craig Marhefka
for preparation of some starting materials. This work
was supported in part by a grant from the National
Cancer Institute (1 R29 CA68096-01 to J .D.), The
American Cancer Society (Grant IN.176-C), Assisi Foun-
dation (Memphis, TN), and Van Vleet Foundation
(Memphis, TN).
2
136.12, 143.88, 131.38 (q, J C-F ) 32.0 Hz), 129.53, 122.78,
1
3
122.51 (q, J C-F ) 273.7 Hz), 118.57, 117.44 (q, J C-F ) 5.4
Hz), 115.86, 101.92 (q, 3J C-F ) 2 Hz), 73.09, 63.56, 30.06, 27.24;
IR (KBr) 3442, 3350, 3327 (NH, OH), 2234 (CN), 1704 (CO),
1593, 1527, 1432, 1404, 1329 (SO2 asym), 1181, 1139 (SO2 sym)
cm-1; [R]D -45.4° (c ) 2, acetone). Anal. (C20H17BrF3N3O5S)
27
C, H, N.
Ma ter ia ls for Bin d in g Stu d ies. Chemicals were pur-
chased from Aldrich Chemical Co., Milwaukee, WI (L-proline,
thionyl chloride, 3-aminothiophenol, 4-aminothiophenol, per-
acetic acid, chloroacetyl chloride, bromoacetyl bromide, N,N-
diisopropylethylamine, and anhydrous dichloromethane and
THF in SureSeal bottles), Lancaster Synthesis, Wyndham, NH
(D-proline), Lancaster Synthesis, England (4-amino-2-trifluo-
romethylbenzonitrile), and J anssen Chimica, Geel, Belgium
(methacryloyl chloride and m-chloroperbenzoic acid). [17R-
methyl-3H]Mibolerone ([3H]MIB, 83.5 Ci/mmol) and unlabeled
MIB were purchased from DuPont Research NEN Products,
Boston, MA. Triamcinolone acetonide, phenylmethanesulfonyl
fluoride (PMSF), Tris base, sodium molybdate, and dithio-
threitol were purchased from Sigma Chemical Co., St. Louis,
MO. EcoLite (+) scintillation cocktail was purchased from ICN
Research Products Division, Costa Mesa, CA.
P r ep a r a tion of Cytosolic AR. Male Sprague-Dawley rats
(Harlan Sprague-Dawley, Indianapolis, IN), weighing 248-
290 g, were castrated 24 h prior to the removal of prostates.
Ventral prostates were surgically removed, weighed, and
immersed immediately in ice-cold homogenization buffer.
Homogenization buffer consisted of 10 mM Tris, 1.5 mM
disodium EDTA, 0.25 M sucrose, 10 mM sodium molybdate,
and 1 mM PMSF and was adjusted to pH 7.4.28 The prostate
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