Antiandrogenic activity of anthranilic acid ester derivatives as novel lead structures to inhibit prostate cancer cell proliferation

Article abstract of DOI:10.1111/j.1747-0285.2011.01116.x

A plant extract from the fruits of saw palmetto, which is currently used to treat the androgen-dependent benign prostatic hyperplasia and PCa, served as source for new structure variants. We investigated the antiandrogenic potential of an ethanolic total

Full text of DOI:10.1111/j.1747-0285.2011.01116.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01116.x
Chem Biol Drug Des 2011; 77: 450–459
Research Article
Antiandrogenic activity of anthranilic acid ester
derivatives as novel lead structures to inhibit
prostate cancer cell proliferation
2,†
Daniela Roell1,†, Thomas W. Rosler
,
kidney cell line from green monkey, lacking endogenously expressed
functional AR, GR, ER, PR and TR; DBD, DNA-binding domain; DHT, di-
hydrotestosterone; FA, flufenamic acid; Fl, flutamide; GR, human gluco-
corticoid receptor; LBD, ligand-binding domain; LNCaP, lymph node
carcinoma prostate cell line; luc, luciferase; MMTV, mouse mammary
tumor virus; PCa, prostate cancer; PSA, prostate-specific antigen; PR-B,
human progesterone receptor; qRT-PCR, quantitative real-time reverse
transcription polymerase chain reaction; R1881, methyltrienolone; RLU,
relative luciferase units; S. repens, Serenoa repens.
¨
Stephanie Degen1, Rudolf Matusch2 and
Aria Baniahmad1,*
1Institute of Human Genetics, Jena University Hospital, D-07743
Jena, Germany
2Institute for Pharmaceutical Chemistry, Philipps-University Marburg,
D-35037 Marburg, Germany
*Corresponding author: Aria Baniahmad, aban@mti.uni-jena.de
These authors contributed equally to this work and should be
considered co-first authors.
Received 14 July 2010, revised 31 January 2011 and accepted for publi-
cation 6 March 2011
A plant extract from the fruits of saw palmetto,
which is currently used to treat the androgen-
dependent benign prostatic hyperplasia and PCa,
served as source for new structure variants. We
investigated the antiandrogenic potential of an
ethanolic total extract and one of its main aro-
matic components anthranilic acid. An androgen
receptor-antagonistic (antiandrogenic) effect of
the extract was evident, and although anthranilic
acid itself revealed no remarkable effect, its
methyl ester, methyl anthranilate, exhibited an-
tiandrogenic potential. Based on this chemical
structure, we synthesized and investigated the an-
tiandrogenic activity of four AnA ester derivatives,
which were either novel or only little described in
literature. These AnA esters inhibit the androgen-
dependent transactivation of both the wild-type
(wt) androgen receptor and the androgen receptor
point mutant T877A, which often occurs in refrac-
tory PCa. Moreover, they inhibit the androgen
receptor-induced expression of the endogenous
prostate-specific antigen. Importantly, AnA esters
repress the growth of human PCa cells. Deletion
analyses of androgen receptor propose that the
antiandrogenic effect of anthranilic acid esters is
mediated by the ligand-binding domain, most
likely through direct binding, without affecting
androgen receptor protein levels. Taken together,
the data suggest antiandrogenic potential of
anthranilic acid ester derivatives, which can serve
as lead structures for novel antiandrogens.
The risk of men to develop androgen-dependent diseases as benign
prostatic hyperplasia (BPH) and prostate cancer (PCa) increases rap-
idly with rising age. Among men, PCa is, meanwhile, the second
leading cause of cancer death in western countries (1). The andro-
gen receptor (AR), which is the most important drug target for PCa
hormone therapies, is activated by androgens, mainly by 5a-dihyd-
rotestosterone (DHT). The AR is a member of the nuclear hormone
receptor (NHR) superfamily, a large group of ligand-dependent tran-
scription factors (2), and mainly structured into four functional
domains: (I) the N-terminal domain with the major transactivation
function, (II) a highly conserved DNA-binding domain, (III) a less
conserved hinge domain, and (VI) a ligand-binding domain (LBD) at
the C-terminus (3). Androgen binding results in an activated AR that
subsequently translocates into the nucleus, homodimerizes, and
binds to androgen-response elements (AREs) of several target genes
(4). Androgens promote the growth of both normal prostate and of
PCa. In case of PCa, hormone therapy using AR antiandrogens (AR
antagonists) is an efficient manner to block AR-mediated transacti-
vation and tumor growth. Interestingly, mutations of the AR such as
the T877A mutant, which often occurs in refractory PCa (5), turn
the active metabolite 2-hydroxyflutamide of the commonly used
complete antiandrogen flutamide into a potent AR agonist, resulting
in enhanced tumor growth (6). In general, it is important to identify
and synthesize new antiandrogens with other characteristics than
the currently used. Plant extracts from the fruits of saw palmetto
are one possible source to search for new antiandrogen scaffolds.
Key words: anthranilic acid, AR antagonism, ester derivative,
methyl anthranilate, prostate cancer, Sabal serrulata, Serenoa repens
Saw palmetto fruit extracts are successfully used since more than
100 years for the treatment of problems of the urogenital tract (7).
The fruits originate from a scrubby palm Serenoa repens (W. Bar-
tram) (Arecacea) found growing in sandy soil along costal areas in
the southeast of the United States (8). Anti-inflammatory (9),
Abbreviations
AA, atraric acid; AnA, anthranilic acid; AR, androgen receptor; ARE,
androgen response element; BPH, benign prostatic hyperplasia; CV1,
450

Antiandrogenic Activity of Anthranilic Acid Ester Derivatives
antiproliferative (10), and antiandrogenic effects (11) of saw pal-
till gas evolution had ceased. The mixture was cooled and diluted
with water (120 mL). The product settled out as immiscible oil,
which was separated and purified by vacuum distillation. The prod-
uct, a yellow oil (2.92 g, 50 %), passed over at 110 ꢀC and
metto fruit extracts have been shown. Many investigations have
been made concerning the antiandrogenic effect of the extracts'
main component, fatty acids (12), but so far antiandrogenic activity
of aromatic ingredients has not yet been described.
1
0.35 Torr. H NMR (500 MHz, CDCl3) d 0.96 (t, 3H), d 1.31 (d, 3H),
d 1.78-1.59 (m, 2H), d 2.90 (d, 3H), d 5.03 (sextet, 1H), d 6.58 (t,
1H), d 6.66 (d, 1H), d 7.37 (t, 1H), d 7.69 (br s, 1H), d 7.92 (d, 1H);
13C NMR (125 MHz, CDCl3) d (ppm) 9.8, 19.6, 29.0, 29.5, 71.9,
110.66, 110.72, 114.2, 131.5, 134.4, 152.0, 168.4; EI-MS (70 eV):
m ⁄ z (rel.int.) = 207 [M]+ (100), 151 (97), 134 (68), 132 (55), 105 (78),
77 (30); HR-EI-MS: m ⁄ z = 207.1260; C12H17NO2 [M]+ requires
207.1259; IR (plain): kmax = 3370, 2980, 1670, 1580, 1430, 1230;
nD20 = 1.5442.
Materials and Methods
The investigated plant extract was an ethanolic total extract from
the fruits of Serenoa repens (96 %). It was supplied by Finzelberg
GmbH & Co. KG, Andernach (batch: 03120256).
Dexamethasone, progesterone, dihydrotestosterone (DHT), methyl
anthranilate (MA), anthranilic acid (AnA), and flufenamic acid (FA)
were obtained from Sigma. Methyltrienolone (R1881) was obtained
from Perkin Elmer and from LKT Laboratories, Inc. All test com-
pounds were dissolved in ethanol and ⁄ or dimethyl sulfoxide
(DMSO). These compounds were added to the culture medium such
that the final concentration of ethanol and ⁄ or DMSO did not
exceed 0.1%. Control incubations (no test compounds) were per-
formed in the presence of only 0.1% ethanol and ⁄ or DMSO.
Prop-2-enyl-2-methylaminobenzoate (E3)
To a solution of N-methylisatoic anhydride (5.0 g, 28 mmol) in allyl
alcohol (40 mL), sodium hydroxide (0.1 g, 2.5 mmol) was added. The
reaction mixture was stirred and heated until moderate evolution of
CO2 occurred (85 ꢀC). This temperature was maintained till gas evo-
lution had ceased. The mixture was cooled and diluted with water
(120 mL). The product settled out as immiscible oil, which was sep-
arated and purified by vacuum distillation. The product, a yellowish
1
NMR spectra were recorded on a JEOL ECA-500 (500 MHz) spec-
trometer and referenced to the solvent resonances in CDCl3. Chemi-
cal shifts are reported in ppm and are assigned as singlets (s),
doublets (d), triplets (t), quartets (q), sextets, and multiplets (m). The
abbreviation br stands for broad signal. EI-MS and HR-EI-MS spec-
tra were obtained with a Micromass VG 7070H and a Micromass
AutoSpec mass spectrometer employing an ionizing energy of
70 eV. Infrared spectra of the plain substances were measured with
a Bruker Alpha-P IR spectrometer. The refraction indexes were
obtained on a Zeiss refractometer type 92. The syntheses were car-
ried out with a previously described modified procedure (13).
oil (3.40 g, 64%), passed over at 98-99 ꢀC and 0.22 Torr. H NMR
(500 MHz, CDCl3) d 2.91 (d, 3H), d 4.77 (d, 2H), d 5.27 (d, 1H), d
5.40 (d, 1H), d 6.03 (m, 1H), d 6.59 (t, 1H), d 6.67 (d, 1H), d 7.39 (t,
1H), d 7.64 (br s, 1H), d 7.95 (d, 1H); 13C NMR (125 MHz, CDCl3) d
(ppm) 29.5, 64.7, 109.7, 110.9, 114.3, 117.7, 131.5, 132.6, 134.7,
152.1, 168.2; EI-MS (70eV): m ⁄ z (rel.int.) = 191 [M]+ (100), 150 (56),
132 (80), 116 (29), 105 (73), 91 (30), 77 (43); HR-EI-MS:
m ⁄ z = 191.0946; C11H13NO2 [M]+ requires 191.0946; IR (plain):
kmax = 3380, 2900, 1680, 1580, 1430, 1230; nD20 = 1.5699.
1-Methyoxypropan-2-yl-2-methylaminobenzoate
(E4)
Butan-2-yl-2-aminobenzoate (E1)
To a solution of isatoic anhydride (4.08 g, 25 mmol) in racemic 2-
butanol (40 mL), sodium hydroxide (0.05 g, 1.25 mmol) was added.
The reaction mixture was stirred and heated until moderate evolu-
tion of CO2 occurred (90 ꢀC). This temperature was maintained till
gas evolution had ceased. The mixture was cooled and diluted with
water (120 mL). The product settled out as immiscible oil, which
was separated and purified by vacuum distillation. The product, a
colorless oil (2.53 g, 52%), passed over at 126 ꢀC and 5 Torr. 1H
NMR (500 MHz, CDCl3) d 0.97 (t, 3H), d 1.32 (d, 3H), d 1.79-1.61
(m, 2H), d 5.06 (sextet, 1H), d 5.72 (br s, 2H), d 6.64 (t, 2H), d 7.25
(t, 1H), d 7.87 (d, 1H); 13C NMR (125 MHz, CDCl3) d (ppm) 9.7, 19.6,
28.9, 72.1, 111.5, 116.1, 116.6, 131.2, 133.8, 150.4, 167.8; EI-MS
(70 eV): m ⁄ z (rel.int.) = 193 [M]+ (100), 137 (87), 119 (86), 92 (29);
HR-EI-MS: m ⁄ z = 193.1108; C11H15NO2 [M]+ requires 238.1226; IR
(plain): kmax = 3480, 3370, 2970, 1680, 1240, 1090; nD20 = 1.5411.
To a solution of N-methylisatoic anhydride (5.0 g, 28 mmol) and 1-
methoxy-2-propanol (2.52 g, 28 mmol) in 1,4-dioxane (40 mL),
sodium hydroxide (0.1 g, 2.5 mmol) was added. The reaction mix-
ture was stirred and heated until moderate evolution of CO2
occurred (90 ꢀC). This temperature was maintained till gas evolution
had ceased. The mixture was cooled and diluted with water
(120 mL). The product settled out as immiscible oil which was sepa-
rated. Purification was carried out by vacuum distillation to give a
yellow oil (2.9 g, 46 %) passed over at 117–118 ꢀC and 0.25 Torr.
1H NMR (500 MHz, CDCl3) d 1.33 (d, 3H), d 2.90 (d, 3H), d 3.39 (s,
3H), d 3.53 (m, 2H), d 5.27 (m, 1H), d 6.58 (t, 1H), d 6.65 (d, 1H), d
7.37 (t, 1H), d 7.64 (br s, 1H), d 7.92 (d, 1H); 13C NMR (125 MHz,
CDCl3) d (ppm) 16.8, 29.5, 59.2, 69.0, 75.2, 110.2, 110.6 114.2,
131.6 134.5, 152.0, 168.0; EI-MS (70 eV): m ⁄ z (rel.int.) = 223 [M]+
(100), 165 (25), 151 (79), 134 (66), 132 (45), 105 (62), 73 (32); HR-EI-
MS: m ⁄ z = 223.1199; C12H17NO3 [M]+ requires 223.1208; IR (plain):
kmax = 3380, 2930, 1670, 1580, 1520, 1230; nD20 = 1.5538.
Butan-2-yl-2-methylaminobenzoate (E2)
To a solution of N-methylisatoic anhydride (5.0 g, 28 mmol) in race-
mic 2-butanol (40 mL), sodium hydroxide (0.1 g, 2.5 mmol) was
added. The reaction mixture was stirred and heated until moderate
evolution of CO2 occurred (85 ꢀC). This temperature was maintained
Plasmids
The plasmid pMMTV-luc, which contains a luciferase reporter gene
driven by the mouse mammary tumor virus long terminal repeats
Chem Biol Drug Des 2011; 77: 450–459
451

Roell et al.
responsive to androgens, was described previously (11,14). The
expression vectors for the human AR or AR-T877A, pSG5-hAR or
pSG5-hAR T877A were described previously (15). The plasmid for
the expression of human GR is described in reference (16). Human
PR-B expression vector was kindly provided by P. Chambon (Stras-
bourg, France).
Whole cell binding assay
The competitive whole cell binding assay was described elsewhere
(18) with the modification that Cos-7 cells were transfected with
the pSG5-hARwt plasmid and for normalization control together
with the pCMV-lacZ expression vector.
Results
Reporter assays
Reporter assays were described elsewhere (13).
Antiandrogenic effect of an ethanolic total
extract from saw palmetto fruits
We first investigated the antiandrogenic activity of an ethanolic
total extract from saw palmetto fruits. Reporter gene assays were
used to measure the antiandrogenic effect. For that purpose, CV1
cells that do not express endogenous steroid receptors such as AR,
progesterone receptor (PR), glucocorticoid receptor (GR), and estro-
gen receptor, which may interfere with the assay system, were
transiently cotransfected with the human AR-expression plasmid, a
luciferase reporter gene with an androgen-sensitive MMTV pro-
moter (19), and a constitutive active lacZ gene (pCMV-lacZ) for
internal normalization. The slight repression of the AR transactiva-
tion in the absence of androgens is probably due to the inhibitory
effect of S. repens on the 5a-reductase that has been shown in for-
mer studies (20). Therefore, cells were treated with a stable syn-
thetic androgen (R1881) to avoid side-effects of metabolites of DHT
together with the plant extract. Cotreatment of the cells with
androgen and the S. repens extract resulted in about 50 % inhibi-
tion of the AR transactivation (Figure 1), which suggests an antian-
drogenic activity.
Cell growth assays
Human prostate carcinoma lymph node carcinoma prostate cell line
(LNCaP) cells (17) were cultured in RPMI-1640 medium (Invitrogen),
supplemented with 10% (v ⁄ v) fetal calf serum (FCS) (Invitrogen),
1% (v ⁄ v) penicillin and streptomycin (Invitrogen), 1% (v ⁄ v), and 1%
(v ⁄ v) sodium pyruvate (Sigma). For cell growth assays, cells were
seeded onto 24-well tissue culture plates (Greiner) at 5 · 103 cells
per well in appropriate medium containing 5% FCS in triplicates.
After 48 h, cells were fed with fresh medium and treated with eth-
anol ⁄ DMSO or with the indicated compounds. Every second day,
the media were replaced with fresh media together with freshly
added compounds. The cells were trypsinized and counted using a
counting cell chamber (Double Neubauer, Brand, Germany) at the
indicated times.
Protein detection
A total of 25 · 103 LNCaP cells were seeded on 10-cm cell cul-
ture dishes into RPMI medium containing 5% FCS. After 24 h,
cells were incubated with the indicated compounds at 37ꢀC for
additionally 3 days. Proteins were extracted by scrapping cells in
ice cold PBS and pelleting cells by centrifugation at 1000 g for
5 min at 4ꢀC. The cell pellet was resuspended in 500 lL NP-40
lysis buffer (0.1% SDS, 1% NP-40, 50 mM Tris, pH 8, 150 mM
NaCl, 5 mM ETDA, 1 mM PMSF). The lysis was followed by three
cycles of freezing (with liquid N2) and thawing (37ꢀC). The cell
debris were removed by centrifugation at 12 000 · g for 15 min
at 4ꢀC. Equal amounts of protein (10 lg) were separated on SDS-
PAGE and blotted onto a PVDF membrane (Millipore, Billerica, NA,
USA). Western blot analysis was performed by using a mouse
anti-AR antibody [F39.4.1 (BioGenex Laboratories, Fremont, CA,
USA)] and the enhanced chemoluminescence detection method
(Amersham Pharmacia Biotech). As secondary antibody, an anti-
mouse IgG HRP-linked antibody was used (Santa Cruz, Santa Cruz,
CA, USA).
Methyl anthranilate (MA) inhibits the wt AR and
the T877A mutant without affecting the AR
protein level
Investigations into compositions of saw palmetto fruits led to the
identification of AnA (21), which we could also detect in the ethan-
olic total extract. Because natural compounds can exhibit stronger
effects when the carboxylic acid group is esterified (22), we tested
additionally the methyl ester of AnA, methyl anthranilate (MA).
Interestingly, although AnA shows no remarkable inhibition of the
AR-mediated transactivation, MA has inhibitory effects (Figure 2A).
Performed dose curve experiments with the natural androgen DHT
(Figure 2B) or the synthetic androgen R1881 (Figure 2C) confirmed
the effect of MA and revealed a dose-dependent inhibition pattern.
MA reduced androgen-induced AR-mediated transactivation at a
concentration of 3 · 10)4 M significantly and slightly at a concen-
tration of 10)4 M.
Real-time RT-PCR
Effects of MA on the AR mutant T877A
The real-time RT-PCR (qRT-PCR) was performed essentially as
described previously (18) with specific primers for the detection of
prostate-specific antigen (PSA) mRNA and b-actin mRNA for nor-
malization. As modifications, 1.28 · 106 cells were seeded out
directly in RPMI medium containing 10% charcoal-stripped FCS.
After 2 days, cells were treated with the indicated compounds or
solvent (ethanol) and, if indicated, with androgen (R1881) (final con-
centration of 1 nM) for 36 h.
The AR-T877A is an AR point mutant likely occurring in hormone
therapy–resistant PCa (5). The exchange of one amino acid in the
AR-LBD results in a loss of effectiveness of the currently used anti-
androgen flutamide (Fl) and its metabolite hydroxyflutamide (OH-Fl).
More broadly, Fl acts as agonist for AR-T877A and therefore
increases tumor growth (6). In consideration of this drawback of
antagonist treatment, new antiandrogens should exhibit advantages
and not losing their intended effect. Therefore, the antiandrogenic
452
Chem Biol Drug Des 2011; 77: 450–459

Antiandrogenic Activity of Anthranilic Acid Ester Derivatives
FA, the LBD deletion abrogates MA-mediated AR inhibition (Fig-
ure 4B), suggesting that the AR-LBD is required for MA-mediated
inhibition of AR.
wt AR
No hormone
R1881
20
10
In summary, the data suggest that the AnA methyl ester MA inhib-
its the transactivation of both the wt AR and the AR-T877A mutant.
Furthermore, MA reduces the PSA mRNA levels and requires the
AR-LBD to exhibit an antiandrogenic effect.
Syntheses of AnA ester derivatives lead to
more active antiandrogens
Solvent
S.repens
To study the influences of the ester alcohol of MA for antiandro-
genic effect, we synthesized a derivative with an isobutyl group
extract
(E1) as a branched ester residue. We also combined this ester alco-
hol with a single methylated amino group (compare E1 and E2) as
shown in Table 1. In addition, we synthesized ester alcohol deriva-
tives with an unsaturated residue (E3) and an oxygen containing
branched residue (E4), each combined with a single methylated
amino group. The esters were produced by the reaction of isatoic
anhydride or N-methyl isatoic anhydride and the pursuant alcohol
with small quantities of NaOH as catalyst (24). E2-E4 are novel mol-
ecules and not listed in chemical databases.
Figure 1: Antiandrogenic activity of an ethanolic total extract
from saw palmetto fruits (S. repens). The extract was tested for its
potential to inhibit the hormone-activated human wt androgen
receptor (AR) in a reporter gene–based system. CV1 cells lacking
functional endogenous steroid hormone receptors were transiently
transfected with the expression vector for human AR and the
androgen responsive reporter MMTV luciferase and subsequently
treated with the plant extract (300 lg ⁄ lL). It was tested in the
absence (white bars) and presence (black bars) of the synthetic
androgen R1881 (30 pM). Solvent (ethanol) was used as negative
control. The obtained luciferase values were normalized to the co-
transfected internal control pCMV-lacZ, indicated as normalized rel-
ative light units (RLU). Error bars indicate the deviation of the mean
of two wells. The experiments have been repeated at least three
times.
Interestingly, all modifications increase the antiandrogenic activity
compared to MA, at least tenfold (Figure 5). Dose curve experi-
ments suggest that E2 and E4 are potent at a concentration of
3 · 10-5 M and E1 and E3 at a concentration of 10)5 M (Supporting
Information). The calculated IC50 values of the synthesized com-
pounds range from 6 lM (E3) to 24 lM (E4) compared with an IC50
of MA of 180 lM. The IC50 of E1 and E4 is about 18 lM. Compar-
ing the IC50 of E2 with those of currently used AR antagonists for
PCa therapy, the IC50 of E2 is only slightly superior (25,26). Addi-
tionally synthesized esters with prolonged alkyl ester alcohols, e.g.,
the pentyl ester of AnA, exhibited only a similar antiandrogenic
effect to the methyl ester MA (data not shown), intending that long
alkyl side chains are obstructive for AR inhibition.
activity of MA on this AR mutant was analyzed. The data suggest
that MA still maintains its antiandrogenic potency for this mutant
(Figure 2D) by detection of a significant decrease in the AR-T877A-
mediated transactivation.
To investigate the effect of MA on the mRNA levels of the endoge-
nous AR target gene PSA, quantitative real-time PCR (qRT-PCR)
experiments were performed. The human androgen-dependent
LNCaP cells that endogenously express the AR-T877A mutant were
used as test system. The treatment of cells with MA resulted in an
inhibition of the androgen-activated PSA expression (Figure 3). This
suggests further that MA acts as an antiandrogen.
A very important issue for new drugs is their target specificity. To
analyze whether the five AnA esters are functionally AR specific,
the closely AR-related steroid receptors GR and PR-B, the more
active PR isoform, were employed. Notably, none of the AnA esters
affected the GR-mediated transactivation significantly (Figure 6A),
whereas E1 and E3 only slightly inhibited the PR-B-mediated trans-
activation (Figure 6B). Nevertheless, the inhibition of the PR-B
through E1 and E3 is much weaker compared to their inhibitory role
for AR. These findings suggest that MA and E1-E4 are AR
selective.
To test the possibility that the antiandrogenic effect of MA is
achieved by degradation of the AR protein level. Western blot anal-
ysis with LNCaP cell extracts was conducted. AR protein degrada-
tion has been shown to be the subjacent mechanism of the known
antiandrogen flufenamic acid (FA) (23). Therefore, FA treatment of
LNCaP cells served as positive control. The AR protein levels were
detected with an AR-specific antibody, and as loading control, b-
actin antibody was used. In contrast to FA, MA treatment did not
change the AR protein levels (Figure 4A). This indicates that MA
treatment of PCa cells does not induce AR degradation. To analyze
whether MA inhibits the AR through the LBD, an AR mutant lacking
the LBD (ARDLBD) was employed in reporter gene assays. Interest-
ingly, the antiandrogen FA reduces the transactivation of ARDLBD,
suggesting that FA acts independent of the AR-LBD. As opposed to
Furthermore, the inhibitory potency of the four new ester deriva-
tives (E1-E4) was tested for the AR-T877A mutant. Even though the
derivatives were added tenfold less concentrated, they inhibited the
AR-T877A more potently compared to treatment with MA (Fig-
ure 6C). This indicates that the esters E1-E4 also have the advanta-
geous potency to decrease AR-T877A-mediated transactivation.
To investigate the AR inhibition of endogenously expressed AR, E1-
E4 were tested for their effect on the expression of the AR target
Chem Biol Drug Des 2011; 77: 450–459
453

Roell et al.
wt AR
wt AR
No hormone
DHT
A
B
16
140
120
100
80
No hormone
R1881
12
8
60
40
4
20
Solvent AnA
MA
AA
Solvent
MA
wt AR
AR T877A
C
D
No hormone
R1881
140
No hormone
R1881
80
120
100
80
60
40
20
60
40
20
Solvent AA
MA
Solvent
MA
Figure 2: MA inhibits both the human wt androgen receptor (AR) and the T877A mutant in a concentration-dependent manner. For
description of the test system, see Figure 1. Solvent (DMSO) was used as negative and the known antiandrogen atraric acid (AA, 10 lM) (18)
as positive control. Error bars indicate the deviation of the mean of two wells. The experiments have been repeated at least three times. (A)
MA inhibits the human wt AR. The S. repens-derived compound AnA (1000 lM) and its methyl ester MA (300 lM) were analyzed for their
potency to inhibit the human AR activated by R1881 (30 pM). Each of the non-hormone-treated normalized values was set as one representing
the normalized fold hormone induction is shown. (B) Concentration-dependent inhibition of dihydrotestosterone (DHT) activated human wt AR.
The concentrations of 300 lM, 100, 30, 10, 3, and 1 lM of MA were used for the inhibition of human wt AR without hormone (white bars)
or with DHT (50 nM) (black bars). (C) Concentration-dependent inhibition of R1881 activated human wt AR. The concentrations of 300, 100,
30, 10, 3, and 1 lM of MA were used for the inhibition of human wt AR without hormone (white bars) or activated by R1881 (30 pM) (black
bars). (D) MA inhibits the AR T877A mutant. MA (300 lM) was analyzed for its potency to inhibit the AR T877A mutant without hormone
(white bars) or activated by R1881 (black bars).
gene PSA with qRT-PCR in LNCaP cells. The concentrations used for
E1-E4 were tenfold lower compared with MA. b-actin mRNA levels
were determined and used for internal normalization. In line with
the previous data, all derivatives including MA inhibit the AR-medi-
ated endogenous PSA expression (Figure 6D and Supporting Infor-
mation). Former studies showed that the AR is recruited to the PSA
promoter in LNCaP cells, even if no androgen is present (14,27–29).
Surprisingly, the repression without added hormone was as strong
or even stronger compared to that with hormone, except in case of
E1 (Figure 6D and Supporting Information), which may be attributed
to residual AR activity mediated in a hormone-independent manner
(28,29). When no hormone was added, the most potent derivative
was E2 (Supporting Information), and if hormone was added, com-
pound E1 showed the most potent inhibition of the PSA gene
expression (Figure 6E). Taken together, the synthesized AnA deriva-
tives exhibit more potency compared to MA.
with 3(H)-mibolerone and either increasing concentrations of non-
labeled mibolerone or E4. The AnA derivative E4 also induces the
competition with the 3(H)-mibolerone for AR binding, with a 50%
displacement at a concentration of 10 lM, which confirms the IC50
mentioned above with being only slightly superior of those of cur-
rently therapeutically used AR antagonists (25,26). Thus, this coin-
cides with the results of the reporter assay and suggests a direct
binding of the AnA ester derivative E4 to the AR and simulta-
neously replacing the bound androgen.
AnA ester derivatives inhibit the proliferation
of human androgen-dependent PCa cells
(LNCaP)
The AR is a major regulator of PCa cell proliferation. To investi-
gate whether MA and E1-E4 in fact can exert influence on the
growth of human PCa cells, we performed cell growth assays
with human androgen-dependent LNCaP cells by treating them for
11 days with the derivatives in medium containing 5% non-treated
FCS (14,27,28). Solvent was used as negative control, and the
antiandrogen atraric acid (AA) was taken as positive control (18).
Cells were counted on the indicated days (Figure 7). Interestingly,
inhibition of LNCaP cell proliferation can be seen for all added
Like MA, the derivatives E1-E4 do require the AR-LBD for the inhibi-
tion of the AR transactivation (data not shown). To investigate
whether the AnA ester derivatives bind to the AR, a competitive
whole cell binding assay was performed. As exemplary substance,
E4 was used. Cos-7 cells that do not contain steroidal receptors
were transfected with the human wt AR. The cells were treated
454
Chem Biol Drug Des 2011; 77: 450–459

Antiandrogenic Activity of Anthranilic Acid Ester Derivatives
Extensive analyses of five AnA esters showed that they can act as
potent antiandrogens for both the wt AR and the T877A mutant.
They also inhibit endogenous AR-mediated PSA expression and
reduce LNCaP cell growth. Among the tested AnA derivatives are
three novel molecules (E2-E4). Our data suggest that AnA esters
provide a novel chemical lead structure for new antiandrogens and
inhibitors of human PCa cell growth.
qRT-PCR
R1881
8
6
4
2
Discussion
In our study, we first discovered the antiandrogenic potential of
AnA ester derivatives derived from AnA, which naturally occurs in
saw palmetto fruits. In addition to the methyl ester MA, we synthe-
sized four AnA esters (E1-E4) including three new structures (E2-E4)
and investigated extensively their antiandrogenic potential. MA
inhibits the AR-mediated transactivation at a concentration of
MA
Solvent
Figure 3: Inhibition of endogenous prostate-specific antigen
(PSA) gene expression by the treatment of human LNCaP cells with
MA. Quantitative real-time RT-PCR (qRT-PCR) experiments were per-
formed for PSA mRNA and b-actin mRNA for normalization. The
androgen-dependent LNCaP cells were grown in charcoal-stripped
medium and treated with R1881 (30 pM) and with or without MA
(300 lM) for 2 days. As negative control, solvent (DMSO) alone was
used. Error bars indicate the deviation of the mean of two PCR
samples. The experiments have been repeated four times.
300 lM significantly. We found that the antiandrogenic activity of
MA can be strongly enhanced by side-chain modifications (E1-E4).
An elongation and branching of the ester alcohol (E1) results in a
stronger antiandrogenic effect. Further variation of the ester alcohol
combined with a single methylated amino group resulted in ester
derivatives (E2-E4) with at least equal functional AR inhibition com-
pared to E1. AnA esters E1-E4 are tenfold more potent than MA
and inhibit the AR transactivation still significantly at a concentra-
tion of 30 lM or less. The conclusion that the antiandrogenic effect
only results from increased lipophilicity because of an ester struc-
ture fails as we generated ester derivatives with different ester
alcohols but with similar lipophilicity and observed great differences
in their antiandrogenic potential (data not shown), suggesting that
the ester alcohol residue is involved in antiandrogenic effectiveness.
Previously, investigations in rats and rabbits showed that a consid-
erable proportion of an orally administered AnA ester reaches the
bloodstream unchanged, suggesting that these esters are stable
(30).
ester derivatives comparing cell numbers to negative control. E4
showed the strongest effect on reducing the growth of PCa cells,
suggesting E4 acts as a very potent PCa cell growth inhibitor.
Neither apoptosis nor toxicity could be observed during derivative
treatment.
Summarizing the data, our investigations resulted in the discovery
of AnA ester derivatives as novel antiandrogenic active substances.
ARΔLBD
A
B
Solvent FA MA
MW(kDa)
30
20
10
No hormone
R1881
100
70
AR
55
40
100
70
55
40
β-actin
Solvent
FA
MA
Figure 4: The AR-LBD is essential for MA-mediated inhibition of androgen receptor (AR). (A) MA does not affect the AR protein level in
prostate cancer cells. The androgen-dependent prostate cancer cells LNCaP treated with MA for 3 days. Western blotting experiments were
performed to detect the human wt AR. As negative control the treatment with solvent (ethanol) and as positive control flufenamic acid (FA)
are indicated. Detections of b-actin levels served as internal control. (B) The ligand-binding domain is target for MA-mediated inhibition. The
AR deletion mutant lacking the LBD (ARDLBD) was tested for its ability to be repressed by MA in an experimental setup as described in Fig-
ure 1. Cells were treated with MA, as negative control solvent (ethanol) and as positive control FA were used.
Chem Biol Drug Des 2011; 77: 450–459
455

Roell et al.
Table 1: Overview of the investigated compounds
O
O
H
N
R1
R2
R1
R2
Compound Name
Symbol
-H
-CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH2CHCH2
-CH(CH3)CH2OCH3
-H
-H
-H
-CH3
-CH3
-CH3
Anthranilic acid
Methyl anthranilate
Butan-2-yl-2-aminobenzoate
Butan-2-yl-2-methylaminobenzoate
Prop-2-enyl-2-methylaminobenzoate
1-Methyoxypropan-2-yl-2-methylaminobenzoate
AnA
MA
E1
E2
E3
E4
cells with the esters. When no androgen was present, this inhibi-
tion could also be detected, surprisingly mostly higher compared to
the androgen added samples. The reason might be attributed to the
fact that the AR in LNCaP cells can bind to the PSA promoter in
the absence of androgen (27). If so, our ester derivatives are able
to inhibit the AR that is bound to the PSA gene regulatory regions,
resulting in a decrease in the PSA mRNA level. In competitive
whole cell binding studies, E4 indicated AR binding and thus it is
likely that also the other AnA ester derivatives inhibit the AR by
replacing the bound androgen.
wt AR
No hormone
R1881
30
20
10
In accordance with these results, all ester derivatives reduced the
growth of the AR-T877A mutant expressed in LNCaP cells signifi-
cantly. E4 even abrogated the growth of the LNCaP cells and there-
fore appears to be a very potent PCa cell growth inhibitor. The
ester derivatives E1-E3 as well as MA are in the same range of cell
growth inhibition as the positive control AA. We know that AA
inhibits PCa cell growth AR dependently as the proliferation of PCa
cells lacking the AR (PC3) cannot be inhibited by AA treatment (18).
E4, in contrast, shows a very strong inhibition of LNCaP prolifera-
tion in the range of the error bar, resulting in a nearly complete
block of cell proliferation. This suggests that MA as well as the
compounds E1-E3 also inhibits PCa cell growth AR dependently,
whereas E4 may have an additional AR-independent effect on PCa
cell proliferation.
-
300 30 30
3
30
E2
3
30
E3
3
30
E4
3
c(mM)
Solvent
MA
E1
Figure 5: A variety of Anthranilic acid ester derivatives exhibit
enhanced antiandrogenic effects. The indicated concentrations of
compounds E1-E4 were analyzed for their potency to inhibit the
human wt androgen receptor without hormone (white bars) or acti-
vated by R1881 (30 pM) (black bars) in an experimental setup as
described in Figure 1.
Notably, all AnA ester derivatives inhibit the AR-T877A mutant. This
mutant is found in several refractory PCa samples (5) particularly in
hormone refractory PCa, which is very difficult to treat. It is fatal
that the active metabolite of the currently used antiandrogen fluta-
mide acts agonistic upon the AR-T877A resulting in an increased
tumor growth.
Studies investigating the concentration of OH-Fl in the plasma after
oral application of Fl to PCa patients and healthy men revealed
very high plasma concentrations (up to 8.5 lM) (31). The normal
dosage of the therapeutically used antihormone Fl is about 250 mg
(corresponding to 150 lM application for an 80-kg men) several
times per day and the same concentration was also applied in this
study (31).
Importantly, the esters did not impair the transactivation of the
close AR relative to GR. The NHR PR-B is slightly targeted by E1
and E3 but compared with the AR inhibition, it is only an extremely
weak effect.
Furthermore, our data reveal that the AR inhibition of AnA esters is
not because of AR degradation on the protein level. Also corepressor
interaction assays with the known AR corepressor Alien (27) revealed
no measurable recruitment of Alien (Supporting Information).
As a further indication of AR inhibition, we found the mRNA
amount of the AR target gene PSA decreasing by treating LNCaP
456
Chem Biol Drug Des 2011; 77: 450–459

Antiandrogenic Activity of Anthranilic Acid Ester Derivatives
GR
PR-B
A
B
80
60
40
20
No hormone
Progesterone
No hormone
Dexamethasone
30
20
10
Solvent MA
E1
E2
E3
E4
Solvent MA
E1
E2
E3
E4
C
AR T877A
80
60
40
20
No hormone
R1881
Solvent MA
E1
E2
E3
E4
D
E
qRT-PCR
25
20
15
100
80
Mibolerone
E4
No hormone
R1881
60
10
5
40
20
c (M) 0
10–10 10–9 10–8 10–7 10–6 10–5 10–4
MA
Solvent
E1
E2
E3
E4
Figure 6: Anthranilic acid ester derivatives inhibit specifically the wt androgen receptor (AR) and the T877A mutant and endogenous pros-
tate-specific antigen (PSA) gene expression. (A) MA and none of the derivatives E1-E4 inhibit significantly the GR. MA (300 lM) and E1-E4
(30 lM) were tested for their influence on the NHR GR without hormone (white bars) or activated by dexamethasone (1.8 nM) (black bars) in
an experimental setup as described in Figure 1. (B) Influence of MA and E1-E4 on PR-B. The compounds MA (300 lM) and E1-E4 (30 lM)
were tested for their activity on the NHR PR-B without hormone (white bars) or activated by progesterone (1 nM) (black bars) in an experimen-
tal setup as described in Figure 1. (C) The derivatives E1-E4 inhibit the AR T877A mutant. E1-E4 (30 lM) were analyzed for their potency to
inhibit the AR T877A mutant without hormone (white bars) or activated by R1881 (30 pM) (black bars) in the experimental setup described in
Figure 1. (D) Inhibition of endogenous PSA gene expression by MA and E1-E4 in the absence (white bars) and presence (black bars) of R1881
(1 nM). Quantitative real-time RT-PCR experiments were performed for PSA mRNA and b-actin mRNA detection to analyze the potency of MA
and E1-E4 to inhibit PSA expression. The androgen-dependent LNCaP cells were grown for 2 days in charcoal-stripped medium and treated
with the indicated substance for 36 h. The final concentration of MA was 300 and 30 lM for E1-E4. As negative control, solvent (DMSO)
was used solely. Indicated is the fold activation where the RNA expression of the negative control containing solvent and in the absence of
hormone is set as one. b-actin mRNA levels were used for normalization. Error bars indicate the standard deviation of the mean of two PCR
setups. (E) E4 competes with androgens for AR binding. Competitive whole cell binding assays were performed using pSG5-hARwt-transfected
COS-7 cells incubated with 1 nM 3(H)-mibolerone in the absence and presence of increasing concentrations of either unlabeled mibolerone
3
(diamond) or E4 (triangles) for 90 min. Competition for binding is illustrated by the percent of (H)-mibolerone specifically bound to the AR.
Results are averages of triplicates (€SEM).
Chem Biol Drug Des 2011; 77: 450–459
457

Roell et al.
References
12
10
8
Solvent
E3
1. Edwards B.K., Ward E., Kohler B.A., Eheman C., Zauber A.G.,
Anderson R.N., Jemal A., Schymura M.J., Lansdorp-Vogelaar I.,
Seeff L.C., van Ballegooijen M., Goede S.L., Ries L.A.G. (2010)
Annual Report to the Nation on the Status of Cancer, 1975–
2006, Featuring colorectal cancer trends and impact of interven-
tions (risk factors, screening, and treatment) to reduce future
rates. Cancer;116:544–573.
2. Evans R.M. (1988) The steroid and thyroid hormone receptor
superfamily. Science;240:889–895.
3. Tenbaum S., Baniahmad A. (1997) Nuclear receptors: structure,
function and involvement in disease. Int J Biochem Cell
Biol;29:1325–1341.
E2
E1
AA
6
MA
4
2
E4
4. Riegman P.H., Vlietstra R.J., van der Korput J.A., Brinkmann
A.O., Trapman J. (1991) The promoter of the prostate-specific
antigen gene contains a functional androgen responsive ele-
ment. Mol Endocrinol;5:1921–1930.
Day
5
8
11
5. Taplin M.E., Bubley G.J., Ko Y.J., Small E.J., Upton M.,
Rajeshkumar B., Balk S.P. (1999) Selection for androgen receptor
mutations in prostate cancers treated with androgen antagonist.
Cancer Res;59:2511–2515.
6. Bohl C.E., Wu Z., Miller D.D., Bell C.E., Dalton J.T. (2007) Crystal
structure of the T877A human androgen receptor ligand-binding
domain complex to cyproterone acetate provides insight for
ligand-induced conformational changes and Structure based
Drug-design. J Biol Chem;282:13648–13655.
Figure 7: MA and E1-E4 inhibit the growth of the human PCa
cell line LNCaP. LNCaP cells were treated with 300 lM MA or
30 lM E1-E4. Equal amounts of cells were seeded out in 5%
untreated fetal calf serum containing medium and treated with the
indicated derivatives. Solvent (DMSO) alone was used as negative
and AA (10 lM) as positive control. The number of cells was
counted at the indicated days (0, 5, 8, and 11). Error bars indicate
the standard deviation of the mean of four wells.
7. Murray M.T., Pizzorno J. (1998) Encyclopedia of Natural Medi-
cine, 2nd edn. Roseville, USA: Prima Publishing.
8. Kraemer H. (1910) A Text-Book of Botany and Pharmacognosy,
4th edn. Philadelphia, USA: J.B. Lippincott, 578 p.
9. Hiermann A. (1989) ꢀber die Inhaltsstoffe von Sabalfrꢁchten und
deren Prꢁfung auf entzꢁndungshemmende Wirkung. Arch
Pharm;322:111–114.
10. Vacherot F., Azzouz M., Gil-Diez-De-Medina S., Colombel M., De La
Taille A., Belda M.-A.L., Abbou C.C., Raynaund J.-P., Chopin D.K.
(2000) Induction of apoptosis and inhibition of cell proliferation by
the lipido-sterolic extract of Serenoa repens (LSESr, PermixonT) in
benign prostatic hyperplasia. Prostate;45:258–266.
11. Schleich S., Papaioannou M., Baniahmad A., Matusch R. (2006)
Extracts from Pygeum africanum and other ethnobotanical spe-
cies with antiandrogenic activity. Planta Med;72:807–813.
12. Raynaud J.-P., Cousse H., Martin P.-M. (2002) Inhibition of type 1
and type 2 5a-reductase activity by free fatty acids, active ingre-
dients of Permixonꢁ. J Steroid Biochem Mol Biol;82:233–239.
13. Papaioannou M., Schleich S., Roell D., Schubert U., Tanner T.,
Claessens F., Matusch R., Baniahmad A. (2010) NBBS isolated
from Pygeum africanum bark exhibits androgen antagonistic
activity, inhibits AR nuclear translocation and prostate cancer
cell growth. Invest New Drugs;28:729–743.
14. Moehren U., Papaioannou M., Reeb C., Grasselli A., Nanni S.,
Asim M., Roell D., Prade I., Farsetti A., Baniahmad A. (2008)
Wild-type but not mutant androgen receptor inhibits expression
of the hTERT telomerase subunit: a novel role of AR mutation
for prostate cancer development. FASEB J;22:1258–1267.
15. Dotzlaw H., Moehren U., Mink S., Cato A., Iniguez Lluhi J.A.,
Baniahmad A. (2002) The amino terminus of the human AR is
Because MA is currently used as aromatic substance in several
foods, its toxicity has been widely investigated. The WHO deter-
mined an ADI value of 1.5 mg ⁄ kg ⁄ daya. Pharmacological experi-
ments using rats found that doses up to 150 mg ⁄ kg ⁄ day cause no
toxic effects (32,33). The ADME properties (absorption, distribution,
metabolism, and excretion) of propyl anthranilate were investigated
in rats and rabbits in vivo. The biggest part of oral-administered
propyl anthranilate was absorbed in the stomach. Only 7% reaches
the small intestine (34). Besides only a very small part of the ester
is hydrolyzed during the absorption process, whereas the biggest
part (84%) of the ester reaches the blood circulation unchanged
(34). Together, the facts that AnA esters seem to be stable esters
in vivo (5), that MA has already been toxicologically studied with
good tolerance (30), and that AnA esters can be easily synthesized
render them to promising antiandrogenic drug candidates in the
treatment of androgen-dependent diseases especially in PCa.
Conclusions
Altogether we synthesized potent AR antagonists by taking the nat-
ural compound AnA from saw palmetto fruits extracts as basic
structure. All ester derivatives reveal AR-specific inhibition and
repression of PCa cell growth, whereas they are novel or only less
described compounds. In contrast to the currently used antiandro-
gens, the AnA esters likely do not recruit the AR corepressor Alien,
also inhibit the hormone-therapy-resistant AR mutant, and thus pre-
sumably have another molecular mechanism for AR inhibition.
Therefore, AnA ester derivatives can be a suitable basic structure
to develop potent active substances as AR antagonists.
458
Chem Biol Drug Des 2011; 77: 450–459

Antiandrogenic Activity of Anthranilic Acid Ester Derivatives
target for corepressor action and antihormone agonism. Mol
Endocrinol;16:661–673.
29. Kang Z., Jꢂnne O.A., Palvimo J. (2004) Coregulator recruitment
and histone modifications in transcriptional regulation by the
androgen receptor. Mol Endocrinol;8:2633–2648.
30. Fahelbum I.M.S., James S.P. (1979) Absorption, distribution and
metabolism of propyl anthranilate. Toxicology;12:75–87.
31. Bꢃlanger A., Giasson M., Couture J., Dupont A., Cusan L., Labrie
F. (1988) Plasma levels of hydroxy-flutamide in patients with
prostatic cancer receiving the combined hormonal therapy: an
LHRH agonist and flutamide. Prostate;12:79–84.
32. Jenner P.M., Hagan E.C., Taylor J.M., Cook E.L., Fitzhugh O.G.
(1964) Food flavourings and compounds of related structure I.
Acute oral toxicity. Food Cosmet Toxicol;2:327–343.
33. Charconnet-Harding F., Dalgliesh C.E., Neuberger A. (1953) The
relation between riboflavin and tryptophan metabolism, studied
in the rat. Biochem J;53:513–521.
34. Hagan E.C., Hansen W.H., Fitzhugh O.G., Jenner P.M., Jones
W.I., Taylor M.J., Long E.L., Nelson A.A., Brouwer J.B. (1967)
Food flavourings and compounds of related structure II. Sub-
acute and chronic toxicity. Food Cosmet Toxicol;5:141–157.
16. Schulz M., Eggert M., Baniahmad A., Dostert A., Heinzel T., Ren-
kawitz R. (2002) RU486-induced glucocorticoid receptor agonism
is controlled by the receptor N-terminus and by corepressor
binding. J Biol Chem;277:26238–26243.
17. Protopopov A.I., Li J., Winberg G., Gizatullin R.Z., Kashuba V.I.,
Klein G., Zabarovsky E.R. (2002) Human cell lines engineered for
tetracycline-regulated expression of tumor suppressor candidate
genes from a frequently affected chromosomal region, 3p21. J
Gene Med;4:397–406.
18. Papaioannou M., Schleich S., Prade I., Degen S., Roell D., Schu-
bert U., Tanner T., Claessens F., Matusch R., Baniahmad A.
(2009) The natural compound atraric acid is an antagonist of the
human androgen receptor inhibiting cellular invasiveness and
prostate cancer cell growth. J Cell Mol Med;13:2210–2223.
19. Gast A., Schneikert J., Cato A. (1998) N-terminal sequences of
the human androgen receptor in DNA binding and transrepress-
ing functions. J Steroid Biochem Mol Biol;65:117–123.
20. Habib F.K., Ross M., Ho C.K., Lyons V., Chapman K. (2005)
Serenoa repens (Permixon) inhibits the 5alpha-reductase activity
of human prostate cancer cell lines without interfering with PSA
expression. Int J Cancer;114:190–194.
Notes
aWorld Health Organization: Toxicological Evaluation of Some Fla-
vouring Substances and Non-Nutritive Sweetening Agents. FAO
Nutrition Meetings No. 44A, available at: http: ⁄ ⁄ www.
inchem.org ⁄ documents ⁄ jecfa ⁄ jecmono ⁄ v44aje27.htm.
21. Hꢂnsel R., Schçpflin G., Rimpler H. (1966) Notiz ꢁber das Vork-
ommen von Anthranilsꢂure in Sabalfrꢁchten (Serenoa repens).
Planta Med;14:261–265.
22. Gebauer H. (2006) Use of angelic acid esters as an antiphlogis-
tic active ingredient and antiphlogistic Preparations. Patent De
102006012107.
Supporting Information
23. Zhu W., Smith A., Young C.Y. (1999) A nonsteroidal anti-inflam-
matory drug, flufenamic acid, inhibits the expression of the
androgen receptor in LNCaP cells. Endocrinology;140:5451–5454.
24. Staiger R.P., Miller E.B. (1959) Isatoic anhydride. IV. Reactions
with various nucleophiles. J Org Chem;24:1214–1219.
25. Luo S., Martel C., LeBlanc G., Candas B., Singh S.M., Labrie C.,
Simard J., Bꢃlanger A., Labrie F. (1996) Relative potencies of
flutamide and casodex: preclinical studies. Endocr-relat. Can-
cer;3:229–241.
26. Ayub M., Levell M.J. (1989) The effect of ketoconazole related
imidazole drugs and antiandrogens on [3H]5a-dihydrotestosterone
and [3H]cortisol binding to plasma proteins. J Steroid Bio-
chem;33:251–255.
Additional Supporting Information may be found in the online ver-
sion of this article:
Figure S1. Subjacent reaction mechanism of the synthesized
Esters E1-E4 and an overview of the structures of the analyzed
compounds.
Figure S2. MA, E1-E4 inhibit the human wt AR in a dose depen-
dent manner.
Figure S3. Inhibition of endogenous PSA gene expression by MA
and E1-E4.
27. Moehren U., Papaioannou M., Reeb C., Hong W., Baniahmad A. (2007)
Alien interacts with the human androgen receptor and inhibits
prostate cancer cell growth. Mol Endocrinol;21:1039–1048.
28. Eisold M., Asim M., Eskelinen H., Linke T., Baniahmad A. (2009)
Inhibition of MAPK-signaling pathway promotes the interaction
of the corepressor SMRT with the human androgen receptor and
mediates repression of prostate cancer cell growth in the pres-
ence of antiandrogens. Mol Endocrinol;42:429–435.
Figure S4. The AR inhibition by MA and E1-E4 does not involve
the recruitment of the corepressor Aliena.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
Chem Biol Drug Des 2011; 77: 450–459
459