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Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47

Structure–activity relationships of synthetic progestins in

a yeast-based in vitro androgen bioassay
L. McRobb a,b , D.J. Handelsman c , R. Kazlauskas d , S. Wilkinson e ,
M.D. McLeod e , A.K. Heather a,b,∗
a Heart Research Institute, Sydney, NSW 2050, Australia
b Discipline of Medicine, University of Sydney, NSW 2006, Australia
c ANZAC Research Institute, University of Sydney, Sydney, NSW 2139, Australia
d National Measurement Institute, Pymble, NSW 2073, Australia
e School of Chemistry, F11, The University of Sydney, NSW 2006, Australia

Received 4 June 2007; accepted 2 October 2007

Abstract
The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable
by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns
about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and
progesterone bioassays to conduct a structure–activity study assessing the intrinsic androgenic potential of commercially available progestins and
their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide
range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17␣-Hydroxyprogesterone
and 19-norprogesterone derivatives with their bulky 17␤-substituents were strong progestins but generally weak androgens. 17␣-Ethynylated
derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had
the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17␣-ethynyl group of
each of these progestins produces 17␣-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic
bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and
identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse
and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17␣-hydroxyprogesterone and
19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.
© 2008 Elsevier Ltd. All rights reserved.

Keywords: Progestin; Androgen receptor; Progesterone receptor; Steroid; Hormone; Androgen; Sports doping

1. Introduction tom manufactured, illicit designer androgens. The banned list of

The recent identification of previously unknown (tetrahydro-
gestrinone or THG) [1–4] or non-marketed (norbolethone and
madol) [5,6] but potent androgens intended for use in sports
doping but previously not detectable by targeted gas chromatog-
raphy mass spectrometry-based urine drug screens has raised
concern regarding the potential for further development of cus-
∗ Corresponding author at: Heart Research Institute, 114 Pyrmont Bridge
Road, Camperdown, NSW 2050, Sydney, Australia. Tel.: +61 2 8208 8900;
fax: +61 2 9565 5584.
E-mail address: heathera@hri.org.au (A.K. Heather).
synthetic androgens however specifies a finite list of known syn-
thetic androgens whereas structural modifications using known
chemistry could produce a wide array of androgenic agonists.
For example, the identification of THG [2–4], a minor one-step
chemical modification of the commercially marketed progestin,
gestrinone, created a potent androgen highlighting that cus-
tom modification of commercially available steroids employing
known chemical transformations may produce novel potent
androgens as sports doping agents that are undetectable by estab-
lished screening tests. One of the most likely sources of starting
material are the synthetic progestins, many marketed for female
contraceptive, hormone replacement therapy or other gyneco-
logical treatments. Although MS-based detection methods can

0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2007.10.008
40 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47
be rapidly and readily developed once novel designer androgens
are structurally identified, it would be helpful to identify which
progestins with highest intrinsic androgenic bioactivity are of
most concern for such conversion to potent androgens.
Currently marketed progestins fall into two main classes
according to their structural relatedness to progesterone or
19-nortestosterone [7]. Historically, this split arose from the
finding that, whereas 19-nortestosterone (nandrolone) was a
more potent androgen than testosterone, it was orally inac-
tive and the introduction of a 17␣-ethynyl group to render
19-nortestosterone orally active, as it does for estradiol, pro-
duced unexpected but highly potent progestin activity [8].
Subsequently 19-norprogesterone derivatives were developed
to reduce androgenic activity (as a female contraceptive side-
effect) while enhancing progestin potency [9].
Both androgen and progestin effects are mediated by
interaction with their cognate receptors, members of the ligand-
activated nuclear receptor superfamily of genes and proteins
[10]. Among the classical steroid receptor members of this
class, the androgen and progesterone receptors have the highest
homology at both the gene and protein level, and the great-
est structural homology between their respective ligand-binding
domains (LBDs) which dictate ligand specificity [11,12]. The
effects of synthetic substituents in steroids frequently broaden
specificity but are not strictly predictable. Thus although 19-
nortestosterone derivatives would be expected to have some
androgenic activity, this varies between synthetic steroids, and
conversely even those derived from 17-hydroxyprogesterone
may have androgenic activity. To date, gestrinone is the only
marketed progestin on the World Anti-Doping Agency (WADA)
prohibited list [13]. Yet among commercially marketed pro-
gestins, others have similar or greater androgenic potential than
gestrinone [14] and therefore could serve as starting materi-
als for conversion into potent, undetectable designer androgens.
Therefore it is desirable to characterize the available progestins
according to their potential for development into illicit designer
androgens.
While previous studies have systematically examined andro-
gen receptor (AR) binding for a variety of natural and synthetic
androgens and related steroids, such binding assays do not eval-
uate post-binding biological activity nor can they distinguish
whether a steroid binding to the AR is an agonist or antagonist.
Measurement of receptor interaction by yeast assay has proven
robust and reproducible in a wide variety of pharmacological
and toxicological contexts and is a rapid and inexpensive first
pass screen to determine relative relationships between struc-
ture and bioactivity [15–20]. Yeast lack homologous steroid
hormone receptors yet contain enough conserved basal tran-
scriptional machinery to allow a recombinant human receptor
to facilitate the transcription of a co-transformed reporter vector
(␤-galactosidase) linked to a steroid responsive promoter [21].
In this study, we have used yeast-based in vitro androgen
receptor and progesterone receptor (PR) bioassays to conduct
a systematic structure–activity study of a comprehensive set
of commercially available progestins and related structures, to
identify those compounds or structures with the highest intrinsic
androgenic potency and greatest potential as a synthesis starting
point for further modification and development of illicit sports
doping agents.
2. Materials and methods
2.1. Materials
All steroids used in this study were obtained from Steraloids,
Sigma or were custom synthesized. 13␤-Ethyl-17␤-hydro-
xygon-4-en-3-one (18-methylnandrolone) and 13␤-ethyl-
17␤-hydroxy-18,19-dinorpregn-4-en-3-one (norbolethone)
were synthesized by Dr. R. Davey and 17␤-hydroxy
-17␣-(2-propenyl)-estr-4-en-3-one (17␣-allylnandrolone),
13␤-ethyl-17␤-hydroxy-17␣-(2-propenyl)-gon-4-en-3-one
(17␣-allyl-18-methylnandrolone) and 13␤-ethyl-17␤-hydroxy-
17␣-methylgon-4-en-3-one (17␣,18-dimethylnandrolone) by
Mr. S. Wilkinson at the School of Chemistry, University of
Sydney, Australia. Steroids were dissolved in 100% (v/v)
ethanol.
2.2. Plasmids and reporter gene constructs
The full-length hPRA-cDNA expression plasmid that
encodes the human PR receptor fused to the CUP1 metalloth-
ionein promoter and the PRE-B-galactosidase reporter plasmid
that has steroid responsive elements upstream of lacZ, the ␤-
galactosidase reporter gene, were kindly provided by Dr. D.P.
McDonnell. Yeast strain YPH500 (Mat␣, ura3-52, lys2-801,
ade2-101, trp1-63, his3-200, leu2-1) was co-transformed
with both plasmids by standard alkali-transformation (Alkali
Cation yeast transformation kit, BIO101 systems, Qbiogene
Inc., Carlsbad, CA, USA). Co-transformed yeast cells were
selected by tryptophan and uracil auxotrophy. Yeast strain
YPH500 transformed with YEPAR and YppG2 was also kindly
provided by Dr. D.P. McDonnell as described previously [3].
2.3. Yeast culture
Yeast transformants were grown overnight at 30 ◦ C with vig-
orous orbital shaking at 300 rpm in CSM-leu-ura (AR, BIO101)
or CSM-trp-ura (PR, BIO101). Following overnight culture, the
yeast culture was subcultured in fresh medium and allowed to
grow until early mid-log phase (OD600 ∼1.0).
2.4. Yeast-based AR and PR bioassays
For the AR bioassay, yeast from mid-log phase growth was
diluted to OD600 = 1.0 in selective medium (CSM-leu-ura) plus
100 ␮M CuSO4 to induce receptor production via the CUP1
promoter. For the PR bioassay, yeast was diluted to OD600 = 0.7
in selective medium (CSM-trp-ura). Diluted yeast cultures
were aliquoted into 24-well culture plates (500 ␮l/well) and
then treated with steroid concentrations ranging from 10−11 M
to10−2 M (in a final volume of 5 ␮l). Following incubation yeast
was lysed and assayed for ␤-galactosidase activity as previously
described [3].
 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47 41

2.5. Standard curves and determination of EC50 values

A testosterone or progesterone standard dose–response curve

was included in triplicate in every AR and PR bioassay,
respectively. AR and PR standard curves were calculated by
transforming the raw enzymatic read-out to a percentage of max-
imal activity of testosterone (AR assay) or progesterone (PR
assay) and fitted to a 4-parameter sigmoidal curve with variable
slope using Sigmaplot version 8.0. The EC50 values from the
fitted curves for each test steroid were used to determine the
relative potency of the test steroid with respect to testosterone
(AR bioactivity) or progesterone (PR bioactivity). AR bioac-
tivity of steroid relative to testosterone was determined using
the equation: 100 × EC50 [steroid]/EC50 [T]. PR bioactivity of
steroid relative to progesterone was determined using the follow-
ing: 100 × EC50 [steroid]/EC50 [P]. AR/PR potency ratio was
determined by dividing AR bioactivity by PR bioactivity.

3. Results

A total of 45 commercially available progestins and related

steroids were evaluated using a yeast bioassay for their AR and
PR bioactivity. The mean EC50 , relative bioactivity and AR/PR
potency ratio were determined for each steroid and are shown in
Table 1 (progesterone derivatives) and Table 2 (testosterone and
19-nortestosterone derivatives). Representative dose–response
curves for both the AR and PR bioassays are shown in Fig. 1A
and B, respectively. All steroids showed full agonist activity in
the AR bioassay, reaching close to 100% of maximal T apart
from nilutamide which was a partial agonist, reaching a plateau
of 30% of maximal bioactivity (not shown). Significant deviation
from maximum values in the PR bioassay was seen only with
the PR antagonist, mifepristone (53% of maximal, Fig. 1B) and
the prodrug, ethylestrenol (63% of maximal, not shown).
3.1. AR bioassay
The mean EC50 for testosterone was 4.4 ± 0.47 nM (n = 45)
in the AR bioassay (AR bioactivity of 100). A compari-
son of the AR bioactivity of synthetic progestins and related
steroids in the androgen bioassay ranged from 0.006 for the 19-
norprogesterone derivative nestorone (Table 1) to 688 for the
19-nortestosterone derivative altrenogest (Table 2). In general,
testosterone and 19-nortestosterone derivatives, with the typi-
cal 3-keto-4-ene structure of the steroid nucleus and a hydroxyl
substitution at the 17␤-position (17␤-OH), showed particularly
strong AR activation (Table 2, Fig. 1A). Removal or substitu-
tion of the 3-keto group or replacement of the 4-ene structure
with a 5-10-ene (such as those steroids labeled “prodrugs” in
Table 2) resulted in mid-range AR bioactivity that was reduced
between 10- and 100-fold compared to T. Substitution of the
17␤-position with an acetyl group led to substantially reduced
AR bioactivity as seen for the progesterone, 19-norprogesterone
and 17␣-hydroxyprogesterone derivatives (Table 1, Fig. 1A).
With an AR bioactivity of 10, progesterone showed the
highest androgenic potency compared to its related deriva-
tives. Addition of a 17␣-hydroxy group to the progesterone
Fig. 1. Representative dose–response curves for various steroid classes com-
paring androgen (A) and progestin (B) bioactivity levels in the yeast bioassays.
Yeast cultures were treated with serial dilutions of each steroid: (䊉) proges-
terone; () testosterone; () altrenogest (A) or 19-nortestosterone (B); ()
17␣-hydroxyprogesterone; () 19-norprogesterone; () mifepristone. Standard
curves (dotted lines) for testosterone (AR bioassay) and progesterone (PR bioas-
say) were included in each experiment and relative activity was calculated as
a percentage of maximal activity of each in the respective assays. Each point
represents the mean of at least three independent experiments ±S.E.M.
backbone (17␣-hydroxyprogesterone) decreased AR bioactiv-
ity from 500- to 0.21-fold. 17␣-Hydroxyprogesterone (OHP),
a naturally occurring precursor of bioactive steroids, is inactive
with respect to AR and PR binding unless esterified [22,23].
Commonly marketed OHP derivatives included in this study
(chlormadinone, cyproterone, cyproterone acetate and delmadi-
none acetate) which have a 6-chloro-6-ene substitution, with
or without acetylation of the 17␣-hydroxyl group, increased
AR bioactivity relative to OHP but were still weak agonists
in this yeast assay (0.45–2.5). These steroids are reportedly
potent anti-androgens in vivo [24], while weaker androgen
antagonism is also a feature of megestrol acetate, a 6-methyl-
6-ene derivative [7] which in this assay showed a potency
value of 3.3. The non-steroidal antagonists, hydroxyflutamide
and nilutamide, also showed transactivation in this yeast assay
42 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47

Table 1
Relative potency of progesterone derivatives on the human androgen and progesterone receptors in yeast

Steroid CAS number Androgen receptor bioactivity Progesterone receptor bioactivity AR/PR potency ratiod

EC50 (nM)a Relative potencyb EC50 (nM)a Relative potencyc

Progesterone derivatives
Progesterone 57-83-0 46 9.6 3.9 100 0.096
Dydrogesterone 152-62-5 572 0.77 7.4 53 0.015
17␣-Hydroxyprogesterone derivatives
17␣-Hydroxyprogesterone 68-96-2 2137 0.21 1221 0.32 0.7
Medroxyprogesterone acetate 71-58-9 92 4.8 5.4 72 0.07
Megestrol acetate 595-33-5 132 3.3 4.8 81 0.04
Chlormadinone 1961-77-9 177 2.5 5.1 76 0.03
Cyproterone acetate 427-51-0 431 1.0 52 7.5 0.01
Cyproterone 2098-66-0 925 0.48 59 6.6 0.007
Delmadinone acetate 13698-49-2 972 0.45 39 10 0.045
19-Norprogesterone derivatives
Norprogesterone 472-54-8 115 3.8 2.2 177 0.02
Gestonorone caproate 1253-28-7 131 3.4 12 33 0.1
ZK 187 226 264186-52-9 228 1.9 2.5 156 0.01
Promegestone (R5020) 34184-77-5 1197 0.37 1.8 216 0.002
Org 2058 24320-06-7 4435 0.10 2.4 163 0.0006
Nestorone 7759-35-5 77900 0.006 1.6 244 0.00002
a EC50 (concentration yielding half of the maximal effect) determined from sigmoidal dose response curves.
b Bioactivity of steroid relative to testosterone (100 × EC50 [steroid]/EC50 [T]).
c Bioactivity of steroid relative to progesterone (100 × EC50 [steroid]/EC50 [P]).
d AR potency divided by PR potency.

although their AR bioactivity was reduced over 40,000-fold 3.2. PR bioassay

compared to testosterone (EC50 ’s ∼17,000 nM, AR potency
of 0.026; not shown). Similarly, the two 19-nortestosterone
derivatives which show AR antagonism [25,26] exhibited very
little AR bioactivity (dienogest, 0.15; mifepristone, 0.015). The
19-norprogesterone derivatives also showed very weak ago-
nist activity with relative potencies in the range of 0.006–
3.8 (Table 1).
Although 17␤-substitution reduced AR bioactivity in the
above steroids, 17␣-substitution on the testosterone, 19-
nortestosterone, or 18-methyl-19-nortestosterone backbone or
in combination with the 4,9,11-triene structure resulted in a
series of steroids with strong AR bioactivity (Table 2). Fig. 2
shows the chemical structures of the 17␣-alkylated testosterone
and 19-nortestosterone derivatives and their associated AR and
PR potencies to allow easy comparison of trends between the
groups. Within those shown in Fig. 2 the commercially marketed
progestins include the 17␣-ethynylated derivatives, ethisterone,
gestrinone, norgestrel and norethisterone. Ethynylation resulted
in decreased AR bioactivity in all steroid backbones (potency
values from 19 to 49). Other short 17␣-alkyl substitutions
however showed increased AR bioactivity in comparison. AR
bioactivity for structures with 17␣-methyl substitutions ranged
from 40 (17␣-methyltestosterone) to 244 (methyltrienolone),
while structures with 17␣-ethyl substitutions included THG
(102), norbolethone (222) and norethandrolone (105). The
most potent androgen tested in this study, altrenogest, had
a 17␣-allyl substitution in the 4,9,11-triene backbone. The
potency of this substitution was not maintained however in the
4-ene derivatives of 17␣-allylnandrolone (16) and 17␣-allyl-18-
methylnandrolone (19) (Fig. 2).
The EC50 of progesterone was 3.9 ± 0.39 nM (n = 38) in the
PR bioassay (Table 1, Fig. 1B). The results in Table 1 show
that there was less distinct division of activity between the 17␣-
hydroxyprogesterone and 19-nortestosterone structural classes
of progestins for PR, compared to AR, bioactivity. The most
potent progestins included both 19-norprogesterone and 19-
nortestosterone derivatives. The 19-norprogesterone derivatives
showed the most consistent level of PR bioactivity with six out of
seven steroids tested having potencies greater than progesterone.
While the most potent progestin was found amongst the 19-
nortestosterone derivatives, the weakest progestin also lay within
this structural group. Altrenogest showed a 13-fold greater activ-
ity than progesterone in this bioassay while mifepristone, a
PR antagonist, showed 500-fold decreased activity (Table 2,
Fig. 1B). The 17␣-hydroxyprogesterone derivatives, although
antagonistic with regard to AR bioactivity, remain PR agonists
and in this assay with values in the range of 6.6–8.1 (Table 1).
In the PR bioassay, the classical androgens T (0.2) and DHT
(0.7) showed little bioactivity whereas removal of the 19-methyl
group (as in 19-nortestosterone/nandrolone) increased PR bioac-
tivity 50-fold relative to T. Many commercially available
progestins are ethynylated derivatives of 19-nortestosterone.
In contrast to the effect on AR bioactivity, ethynylation of
the 17␣-position increased PR bioactivity as seen between
T (0.2) and ethisterone (17) or 19-nortestosterone (12) and
norethisterone (80) (Table 2, Fig. 2). Most of the 17␣-
alkylated 19-nortestosterone derivatives with the alkyl side
chains (methyl, ethyl and allyl) in the 4-ene backgrounds showed
an increase in PR bioactivity (range of 28–122) compared to
 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47 43

Table 2
Relative potency of testosterone and 19-nortestosterone derivatives on the human androgen and progesterone receptors in yeast
Steroid CAS number Androgen receptor Progesterone receptor AR/PR potency Relevant
bioactivity bioactivity ratiod substitution
EC50 (nM)a Relative EC50 (nM)a Relative 13␤ 17␣
potencyb potencyc

C19 methyl
Testosterone 58-22-0 4.4 100 1565 0.2 500 –CH3 –
Ethisterone 434-03-7 23.1 21 23 17 1.2 –CH
17-Methyltestosterone 58-18-4 11.0 40 175 2.2 18.2 –CH3
C19 nor
Nandrolone (19-nortestosterone) 434-22-0 4.3 102 34 12 8.5 –CH3 –
Norethisterone 68-22-4 22.6 19 4.9 80 0.24 –CH
17␣-Methylnandrolone 514-61-4 5.6 79 14 28 2.8 –CH3
Norethandrolone 52-78-8 4.2 105 4.2 93 1.1 –C2 H5
17␣-Allylnandrolonee 7538-46-5 27.4 16 8.1 48 0.33 –C3 H5
C19 nor
18-Methylnandrolonee 793-55-5 3.3 133 60 6.5 20.5 –C2 H5 –
Norgestrel 6533-00-2 9 49 2.6 150 0.33 –CH
17␣,18-Dimethylnandrolonee 14115-37-8 6.5 68 3.4 115 5.9 –CH3
Norbolethonee 1235-15-0 2.0 222 3.2 122 1.8 –C2 H5
17␣-Allyl-18-methylnandrolonee 141300-01-8 22.5 19 4.9 80 0.24 –C3 H5
4,9,11-Triene
Trenbolone 10161-33-8 14.3 31 802 0.5 62 –CH3 –
Gestrinone (R2323) 16320-04-0 18.9 23 49 8.0 2.9 –C2 H5 –CH
Methyltrienolone (R1881) 965-93-5 1.8 244 0.9 433 0.56 –CH3 –CH3
Tetrahydrogestrinone (THG)f 618903-56-3 4.3 102 4.1 95 0.34 –C2 H5 –C2 H5
Altrenogest 850-52-2 0.64 688 0.3 1300 0.53 –CH3 –C3 H5
Prodrugs (lack 3-keto or 4-ene)
Ethylestrenol 965-90-2 37.9 12 56 7.0 1.7
Norethynodrel 68-23-5 40.0 11 25 16 0.7
Tibolone 5630-53-5 70.0 6.3 422 0.9 7
Lynestrenol 52-76-6 86.0 5.1 95 4.1 1.2
Ethynodiol 1231-93-2 284 1.5 208 1.9 0.8
Allylestrenol 432-60-0 663 0.7 381 1.0 0.7
Norgestimate 35189-28-7 2496 0.18 11 35 0.005
Miscellaneous
Dihydrotestosterone (DHT) 521-18-6 2.8 157 546 0.7 224
Danazol 17230-88-5 7.8 59 645 0.6 98
3-Keto-desogestrel 54058-10-1 25.0 18 1.2 325 5.5
Steroidal AR antagonists
Dienogest 65928-58-7 2957 0.15 317 1.2 0.13
Mifepristone 84371-65-3 30430 0.015 2243 0.17 0.09
a EC50 (concentration yielding half of the maximal effect) determined from sigmoidal dose response curves.
b Bioactivity of steroid relative to testosterone (100 × EC50 [steroid]/EC50 [T]).
c Bioactivity of steroid relative to progesterone (100 × EC50 [steroid]/EC50 [P]).
d AR potency divided by PR potency.
e Synthesized at the School of Chemistry, University of Sydney, Australia.
f Synthesized at the National Measurement Institute, Sydney, Australia.

the equivalent structures without 17␣-substitutions (0.5–12),
however there was either little change or decreased PR bioac-
tivity compared to their ethynylated derivatives (80–150). In the
4,9,11-triene background, however, the PR bioactivity was sig-
nificantly increased as seen for example between gestrinone (8)
and its three alkylated congeners, methyltrienolone (433), THG
(95) and altrenogest (1300). PR bioactivity was also sensitive
in these structural backgrounds to the replacement of the C13
methyl with a C18 methyl group (Table 2, Fig. 2) which in gen-
eral increased both PR and AR bioactivity in this assay. This
increased potency is consistent with the decreased dissociation
rate after extended incubation in receptor binding studies found
for this substitution [27]. Compounds that dissociate slower tend
to be more potent agonists in vivo as they can sustain a response
in the nucleus.
3.3. Comparison of AR and PR bioactivity
Fig. 3 plots the AR and PR bioactivity of each steroid.
Deviations from the line reflecting proportionate bioactivity are
44 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47

Fig. 2. Chemical structures of 17␣-alkylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives used in this study with their associated
AR and PR potency values as determined as described in Section 2. Structures are grouped in columns according to their basic skeletal structure: (A) C19 methyl,
(B) 4,9,11-triene, (C) C19 nor with C13 ethyl and (D) C19 nor with C18 methyl; in rows according to their 17␣-substitution.

seen mainly with testosterone derivatives possessing dispropor-
tionately high-androgenic activity whereas 19-norporgesterone
derivatives had dissociation of high progestin with low-
androgenic bioactivity. This is also reflected in the very high
AR/PR potency ratios for the former group which include
those structures unencumbered at the 17␣-position, such as
T, nandrolone and trenbolone (Table 2). The 17␣-alkylated
19-nortestosterone derivatives with both potent androgen and
progestin bioactivities showed more proportionate activities
in Fig. 3 but also a subsequent decrease in the AR/PR ratio
(Table 2), not due to decreases in androgenicity per se but sig-
nificant increases in PR bioactivity.

4. Discussion

In this study, we used in vitro yeast bioassays as a rapid and

Fig. 3. Comparison of AR vs. PR potency of structural groups tested in this
study. Mean values of AR potency are plotted vs. mean PR potency for each
group of structurally related steroids with bidirectional error bars (S.E.M.). The
potency values are relative to testosterone (1 0 0) in the androgen bioassay (AR)
and progesterone (1 0 0) in the progestin bioassay (PR). Open symbols represent
subsets of the testosterone and 19-nortestosterone derivatives as follows: () AR
antagonists, () prodrugs, () androgens, () 17␣-alkylated derivatives and (♦)
4,9,11-trienes. Closed symbols represent derivatives of (䊉) 19-norprogesterone,
() progesterone (P) and 17␣-hydroxyprogesterone (OHP).
inexpensive first pass screen to determine the intrinsic andro-
genic potential of a comprehensive set of commercially available
progestins and related steroid structures. The most potent andro-
gens determined by the yeast bioassay, and hence those most
at risk for abuse in a sports doping context were derived from
either testosterone or 19-nortestosterone and maintained the
basic steroidal structure of the natural androgen, T, with a 3-keto
group and a 17␤-OH substitution. This finding is consistent with
previous studies showing that substitutions at the C3 or C17 posi-
tion of the steroid nucleus are important for androgen receptor
binding [19,22,28,29].
We previously demonstrated using this yeast bioassay that
THG, with its 17␣-ethyl substitution is a potent androgen [3],
 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47
a finding since confirmed by others using micro-array analysis
showing that THG carries the signature of a potent androgen
[4]. We now extend this finding to observe that substitution
at the 17␣-position with alkyl side chains in a testosterone or
19-nortestosterone skeleton or in the presence of three conju-
gated double bonds (the 4,9,11-trienes) resulted in a subset of
molecules with very potent androgenic and progestogenic activ-
ity (Table 2). Commercially marketed progestins in this subset
include the 17␣-ethynylated derivatives, ethisterone, norethis-
terone, norgestrel and the 4,9,11-triene, gestrinone. Their AR
bioactivity was in the range of 19–23 although gestrinone is
reported to have high binding affinity in receptor binding stud-
ies and androgenic effects on the rat ventral prostate although
at 10-fold higher concentrations than T [30]. Both norethis-
terone and norgestrel have equivalent if not greater androgenicity
than gestrinone in these same studies [27,30]. This suggests that
they represent likely starting materials for novel illicit designer
androgens.
Synthetic modification of the 17␣-ethynyl side chains
of gestrinone, norethisterone and norgestrel, can form
either methyl (methyltrienolone, 17␣-methylnandrolone or
17␣,18-dimethylnandrolone) or ethyl (THG, norbolethone and
norethandrolone) substituents, respectively. Saturation of this
side chain significantly increased AR bioactivity between 1.5-
and 10-fold for the methyl-substituted structure and 4- to 5-fold
for the ethyl-substituted structure but had less significant effects
on PR bioactivity. Further increases in potency in vivo would be
expected of these 17␣-alkylated derivatives due to the fact that
metabolism to the inactive 17-keto derivative has been shown
to be eliminated by the alkylation at the 17␣-position, result-
ing in an increased half-life and potency [31,32]. Indeed these
compounds were originally developed in the 1950s to increase
oral bioavailability, however their clinical suitability was limited
by an associated increase in hepatotoxicity [33–35]. The most
potent progestin and androgen in this study however was the
17␣-allyl-substituted 4,9,11-triene, altrenogest, a potent veteri-
nary progestin [36,37] which was 6 times more potent as an
androgen than testosterone and 13 times more potent a pro-
gestin than progesterone. However, when this side chain was
examined in the 4-ene skeleton of 17␣-allylnandrolone and 17␣-
allyl-18-methylnandrolone, the androgenic potential decreased
in comparison to the ethynylated derivatives. It may be deduced
from these results that short, saturated side chain substitutions
increase AR binding and activation while longer chain lengths
reduce receptor fit. This is consistent with AR crystal structure
studies which show that longer side chains in the 17␣-position
appear to interfere with the hydrogen bonding between the 17␤-
OH group and the receptor [38]. Studies have shown the greater
inherent flexibility of the unsaturated triene backbone [27] which
may allow better receptor fit in the presence of longer side chains
and may also explain the increase in cross-reactivity between
steroid receptors of this group.
Gestrinone is the only marketed progestin currently on the
WADA banned list but the results from this study and others
[27,30] show that the other ethynylated steroids have intrinsic
androgenicity equal to or greater than gestrinone and present as
much risk for potential abuse. Apart from their intrinsic andro-
45
genicity, the ability in this study to synthesize norbolethone and
the two allyl containing steroids from norgestrel by relatively
simple substitution of the ethynyl group of norgestrel, and the
previous identification of THG [2] which is derived from gestri-
none by hydrogenation of the 17␣-ethynyl side chain, shows that
these ethynylated compounds have more potential as lead com-
pounds for the production of either more androgenic compounds
or compounds with an equivalent androgenicity but novel struc-
tures that may be undetectable by current screening methods.
The 17␣-ethyl and allyl-substituted derivatives, with or without
18-methyl substitutions, of the ethynylated testosterone deriva-
tive, ethisterone, were not considered in this study but may also
pose a similar risk. Although previous studies have shown that
methyl-substituted 4,9-dienes have lower receptor binding affin-
ity for AR than mono- or tri-enes [27], these compounds still
appear to have intrinsic androgenicity and further 17␣ conver-
sion may create more androgenic but undetectable compounds
that should be further considered for their associated risk.
Progestins classified as progesterone, 19-norprogesterone
and 17␣-hydroxyprogesterone derivatives displayed little andro-
genic activity in the yeast AR bioassay. This suggests that they
possess low risk as sports doping agents. Loss of the 17␤-OH
group in these molecules results in the disruption of hydro-
gen bonding while replacement with a bulky acetyl group is
thought to reduce binding due to an increased steric hindrance
in the hAR binding pocket [28,38–41]. The C16 substitution and
even bulkier substitutions found in the 17␤-position of the 19-
norprogesterone derivatives tested in this study would provide
even further steric hindrance in the binding pocket of the AR
and may explain their very low level of AR bioactivity deter-
mined in this assay. Among the 19-norprogesterone derivatives
are the newer or fourth generation of progestins designed to
further increase relative progestational potency while reducing
androgenicity [9,42]. In the PR bioassay, five of the six steroids
tested in this group were more potent than progesterone while
corresponding androgenic activity was in the range of minimal
activation observed with androgen antagonists.
In general the yeast assay showed good consistency with
published androgen receptor binding studies resulting in a
similar ranking of steroids for progestin and androgenic activ-
ities [7,19,30,43]. One notable discrepancy however was that
medroxyprogesterone acetate (MPA) showed less AR bioactiv-
ity than predicted from previous binding studies. In the latter,
MPA had equivalent binding activity to the more androgenic
progestins, norgestrel and norethisterone, while in our bioassay
MPA had much lower bioactivity compared to the latter two pro-
gestins. This result highlights the essential difference between
receptor binding studies and bioassays where the latter requires
not only receptor binding but subsequent transactivation as well.
Results in our in vitro bioassay are more consistent with the in
vivo assay on rat prostate which showed androgenic activity but
only at very high doses of this steroid [30,44]. It is however
a limitation of in vitro bioassays that they do not evaluate in
vivo activation or inactivation of the parent steroid to metabo-
lites with or without, respectively, bioactivity [45,46]. In this
study, the relatively low activity of the so-called prodrugs, such
as lynestrenol and norethyndrel, which lack intrinsic activity
46 L. McRobb et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 39–47
without further metabolism to the active norethisterone [47],
illustrate the limited metabolic capabilities of yeast with respect
to steroid metabolism [15].
While in vitro assays are no replacement for subsequent
in vivo testing, the yeast assay used in this study provides a
rapid and inexpensive first pass screen for determining the rela-
tionship between structure and androgenicity. Analysis of a
comprehensive list of steroids in this study has given thresh-
olds within which to examine and classify new structures to
determine the potential for in vivo androgenic (and progesto-
genic) activity. This has also allowed us to predict from current
marketed progestins which of those structures are at greatest
risk of abuse or as a synthesis starting point for further mod-
ification. We have shown that while 17␣-hydroxyprogesterone
and 19-norprogesterone derivatives have relatively weak intrin-
sic AR bioactivity and represent little risk of abuse in a sports
doping context, we have identified strong AR agonists among
the 17␣-alkylated-19-nortestosterone derivatives. The intrinsic
androgenicity of the 17␣-ethynylated progestins that are com-
mercially marketed rank them as high risk for use as sports
doping agents, and their relatively simple conversion to more
potent androgens makes them high risk as lead compounds for
the synthesis of undetectable designer androgens. Consideration
should be given to their addition to the WADA banned substances
list and priority given to the development of GC–MS methods
for their potential derivatives.
Acknowledgements
This work was supported by funding from the World Anti-
Doping Agency and the Australian Anti-Doping Research Panel.
L. McRobb was co-funded by a University Postgraduate Award
(UPA), the Department of Medicine, University of Sydney and
the Heart Research Institute, Sydney. The authors are grateful
to Dr. P. Stamford and Mr. S. Wilkinson, School of Chemistry,
University of Sydney for help with steroid synthesis, and to Dr.
R. Sitruk-Ware of the Population Council, New York, for the
supply of Nesterone.
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