Androsterone 3beta-substituted derivatives as inhibitors of type 3 17beta-hydroxysteroid dehydrogenase.

Article abstract of DOI:10.1016/S0960-894X(00)00517-5

Androsterone derivatives substituted at position 3 were synthesized starting from dihydrotestosterone in a short sequence of reactions. They proved to be potent inhibitors (IC50 = 57-147 nM) of type 3 17beta-hydroxysteroid dehydrogenase, a key enzyme of s

Full text of DOI:10.1016/S0960-894X(00)00517-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2533±2536
Androsterone 3ꢀ-Substituted Derivatives as Inhibitors of
Type 3 17ꢀ-Hydroxysteroid Dehydrogenase
Beatrice Tchedam Ngatcha,a Van Luu-Theb and Donald Poiriera,*
aMedicinal Chemistry Division, Oncology and Molecular Endocrinology Research Center,
Â
Laval University Medical Center (CHUL) and Laval University, 2705 Laurier Blvd., Quebec, Canada G1V 4G2
bMRC Group in Molecular Endocrinology, Oncology and Molecular Endocrinology Research Center,
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Laval University Medical Center (CHUL) and Laval University, 2705 Laurier Blvd., Quebec, Canada G1V 4G2
Received 8 June 2000; accepted 5 September 2000
AbstractÐAndrosterone derivatives substituted at position 3 were synthesized starting from dihydrotestosterone in a short
sequence of reactions. They proved to be potent inhibitors (IC50=57±147 nM) of type 3 17b-hydroxysteroid dehydrogenase, a key
enzyme of steroidogenesis, which catalyzes the transformation of androstenedione to steroid active androgen testosterone. # 2000
Elsevier Science Ltd. All rights reserved.
The type 3 17b-hydroxysteroid dehydrogenase (type 3
17b-HSD), also called testicular 17b-HSD or andro-
genic 17b-HSD, is found principally in the microsomal
fraction of the testis.1 With NADPH as a cofactor, it
catalyzes the reduction of 4-androstene-3,17-dione (Á4-
dione) to testosterone (T),2 4 which is further converted to
dihydrotestosterone (DHT) by 5a-reductase (Scheme 1).5
Thus, type 3 17b-HSD plays an important role in the
formation of active androgens (T and DHT), which are
both involved in the pathophysiology of various andro-
gen-sensitive diseases, such as benign prostatic
hyperplasia, prostate cancer,6 acne,7 hirsutism,8 and
male-pattern baldness.9
In his evaluation of the ability of 20 steroidal com-
pounds to inhibit the type 3 17b-HSD activity in
microsomal preparations of canine testis, Pittaway sug-
gested the requirement of a 17-keto group and a ster-
oidal unaromatized A-ring to inhibit the enzyme.16
More recently, a screening study with 80 steroids of
di€erent classes led us to consider the C19 steroid
androsterone (ADT) as a potential starting nucleus to
develop inhibitors of type 3 17b-HSD.17 Based on our
results obtained during the development of inhibitors of
type 1 17b-HSD,11,12 ADT derivatives substituted at
position 16 were synthesized, but they turned out to be
only weak inhibitors of type 3 17b-HSD.18 We then
decided to experiment with position 3 of the ADT
nucleus to develop new inhibitors. We herein report the
chemical synthesis of 3b-substituted ADT derivatives
and their ability to inhibit the type 3 17b-HSD activity.
During our e€orts to develop inhibitors of type 1 17b-
HSD,10 12 and type 2 17b-HSD,13 15 we became inter-
ested in the development of type 3 17b-HSD inhibitors.
Scheme 1. Enzymatic steps involved in biosynthesis of testosterone (T) and dihydrotestosterone (DHT) from 4-androstene-3,17-dione (Á4-dione).
*Corresponding author. Tel.: +1-418-654-2296; fax: +1-418-654-
2761; e-mail: donald.poirier@crchul.ulaval.ca
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00517-5

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B. Tchedam Ngatcha et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2533±2536
2534
Chemistry
17b-alcohol was directly oxidized by Jones' reagent to
a€ord the desired ADT 3b-substituted derivatives 5±
DHT was used as starting material for the synthesis of
the target compounds 5±15 depicted in Scheme 2. The
17b-hydroxy group of DHT was ®rst protected as a tert-
butyldimethylsilyl ether, using TBDMS-Cl and imida-
zole in DMF to a€ord compound 2 in a 91% yield. The
C3-carbonyl group of 17b-TBDMS-DHT (2) was then
subjected to various alkylating reagents (Grignard or
lithium reagents) to give a mixture of 3 and 4. In most
of the cases (methyl, n-propyl, n-hexyl, n-octyl, cyclo-
hexyl, phenyl, and phenylmethyl derivatives), a com-
mercially available Grignard reagent was used and the
reaction was performed at 0 ꢀC in dry THF. For cyclo-
hexylmethyl and phenylethyl derivatives, the Grignard
reagent was generated in situ, by a well known proce-
dure described by Smith,19 using a magnesium amalgam
and the corresponding halide. For cyclohexylethyl deri-
vative, the lithium reagent was generated in situ with t-
BuLi and cyclohexylethyl bromide, in a mixture of die-
thylether:pentane (2:3), according to the procedure
described by Bailey and Puzalan.20 Finally, a commer-
cially available lithium reagent was used for the synth-
esis of s-butyl derivative. Generally, a mixture of the
two stereoisomers at position 3 was obtained, the pro-
portions varying according to the nature of the alkyl
group.21 The two stereoisomers could be well di€er-
entiated on TLC, the 3b-substituted derivative always
being the less polar one. Hence, a separation by ¯ash
chromatography was done, that separated the 3a-sub-
stituted stereoisomer from the 3b-substituted one and
the sequence of reactions was continued with the latter.
Less than 25% of starting material was also recovered
in most cases. The TBDMS protective group of general
compound 3 was then hydrolyzed with a 2% HCl
methanolic solution at room temperature. The resulting
15.22
Inhibition of Type 3 17ꢀ-HSD
The inhibitory properties toward type 3 17b-HSD
activity of target compounds 5±15 were evaluated in
transfected HEK-293 cells by measuring the amount of
labeled T formed from the natural labeled substrate Á4-
dione in the presence of NADPH as cofactor. The
enzymatic assay was performed as described23 and the
results are summarized in Table 1. The concentration of
compound that produced 50% of inhibition (IC50 value)
was determined from the inhibition curves. The ADT
3b-alkylated derivatives 5±15 were good inhibitors of
type 3 17b-HSD; they all showed an inhibitory activity
higher than that of Á4-dione, when this natural sub-
strate of the enzyme was used as inhibitor itself. The
compounds 5±15 were also stronger inhibitors than
ADT, which had been determined as the strongest type
3 17b-HSD inhibitor from our screening study.17 In the
n-alkyl series, the best inhibitory activity was obtained
for n-propyl derivative 6. However, this activity fell as
the longer 3b side chain was made longer (IC50 of
100 nM and 147 nM for 8 and 9, respectively). This
suggested a length limit for this kind of substituent. This
led us to synthesize and test branched substituents, such
as the s-butyl derivative 7. With an IC50 value of 73 nM,
compound 7 was as good an inhibitor as the other ana-
logues in the n-alkyl series (6 and 8). To test a sub-
stituent with a well de®ned shape, we also evaluated
cyclic derivatives. Cyclohexyl-ADT (10) and phenyl-ADT
(13) gave almost the same inhibitory activity, with IC50
values of 97 and 81 nM, respectively. Adding a methylene
Scheme 2. Synthesis of ADT 3b-substituted derivatives 5±15. Reagents: (a) TBDMS-Cl, imidazole DMF, rt; (b) i. RMgBr(Cl) or RLi, THF, 0 ꢀC. ii.
Flash chromatography; (c) i. MeOH±HCl (2%), rt. ii. Jones' reagent (2.7 M), acetone, 0 ꢀC.

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B. Tchedam Ngatcha et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2533±2536
2535
Table 1. Inhibition of type 3 17b-HSD by ADT 3b-substituted deri-
vatives 5±15
enzyme, type 3 17b-HSD. Interestingly, no inhibition of
other reductive 17b-HSDs (type 1 and type 5) was
observed at a 0.3 mM concentration of compounds 5±15
suggesting a speci®c inhibition. To the best of our
knowledge, these ADT 3b-substituted derivatives con-
stitute the ®rst inhibitors of type 3 17b-HSD ever syn-
thesized. Further experiments are being carried out to
optimize the inhibitory activity of these compounds and
to determine the exact mechanism of inhibition. The
results will be published later in a full paper with a
complete description of the experimental procedure
(chemical synthesis and enzymatic test).
Inhibition of
type 3 17b-HSD (%)a
Compounds
R
0.3 mM
3 mM
IC50 (nM)
Á4-Dione
ADT
5
6
7
3-Keto-4-ene
H
CH3
24
50
72
89
85
93
88
88
93
92
88
90
93
78
88
93
94
90
96
92
93
95
93
95
94
93
758Æ139
330Æ60
ndb
Acknowledgements
CH3(CH2)2
CH3CH2(CH3)CH
CH3(CH2)5
CH3(CH2)7
Cyclohexyl
Cyclohexyl-CH2
Cyclohexyl-CH2CH2
Ph
67Æ6
73Æ5
We thank the Medical Research Council of Canada
(MRC) and Le Fonds de la Recherche en Sante du
Quebec (FRSQ) for operating grants and fellowships.
We are grateful to the Laboratory of Molecular Endo-
crinology (Dr. F. Labrie, Director) for providing che-
mical and biological facilities. We also thank Guy
Reimnitz and Mei Wang for the enzymatic assay.
8
9
100Æ10
147Æ29
97Æ3
10
11
12
13
14
15
87Æ19
60Æ16
81Æ6
PhCH2
Ph CH2CH2
57Æ5
99Æ1
aErrorÆ10%.
bNot determined.
References and Notes
group resulted in a gain of inhibitory activity, which was
more important in the case of phenyl: IC50 value of 87nM
for cyclohexylmethyl-ADT (11) and 57 nM for phe-
nylmethyl-ADT (14). The addition of two methylene
groups led to another gain of inhibitory activity in
cyclohexyl series (IC50 value of 60 nM for cyclohex-
ylethyl-ADT (12)), but led to a loss of inhibitory activity
in the phenyl series (IC50 value of 99 nM for pheny-
lethyl-ADT (15)). With an IC50 value of 57 nM, 3b-
phenylmethyl-ADT (14) was the most potent type 3
17b-HSD inhibitor obtained in this study, it was 6-fold
more powerful than ADT and 13-fold more powerful
than Á4-dione, the natural substrate of the enzyme.
1. Andersson, S.; Geissler, W. M.; Patel, S.; Wu, L. J. Steroid
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3. Penning, T. M. Endocrine-Related Cancer 1996, 3, 41.
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Breton, R. Steroids 1997, 62, 148.
5. Li, X.; Calin, C.; Singh, S. M.; Labrie, F. Steroids 1995, 60,
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6. Labrie, F.; Dupont, A.; Belanger, A. In Important Advances
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P. J. Endocrinol. 1977, 75, 83.
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Biol. 1998, 66, 179.
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15. Sam, K. M.; Labrie, F.; Poirier, D. Eur. J. Med. Chem.
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Â
17. Poirier, D.; Labrie, F.; Luu-The, V. Medecine-Sciences
(Suppl. 2) 1995, 11, 24.
In an attempt to correlate hydrophobicity and inhibi-
tory activity, the LogP values were calculated for all
compounds.24 This logarithm of partition coecient
between n-octanol and water expresses the relative
hydrophobicity of a compound. Á4-Dione and ADT,
which gave the lowest inhibitory activities of tested
compounds, were less hydrophobic (LogP=3.5 and 4.2,
respectively) than inhibitors 5±15 (LogP ranging from
4.4 to 7.4), suggesting that hydrophobicity is required.
On the other hand, compound 9, which happened to be
the most hydrophobic with a LogP value of 7.4, showed
only moderate inhibitory activity (IC50=147 nM).
Thus, the presence of a hydrophobic substituent in
position 3 of ADT is important for good inhibition of
type 3 17b-HSD, but a limitation was also observed for
this hydrophobicity thus implicating important steric
e€ects.
18. Tchedam Ngatcha, B., PhD thesis, University Laval,
Quebec, Canada, 1999.
19. Smith, J. J. Chem. Soc. 1932, 738.
In conclusion, ADT 3b-substituted derivatives 5±15
were synthesized and found to inhibit the steroidogenic

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B. Tchedam Ngatcha et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2533±2536
2536
20. Bailey, W. F.; Punzalan, E. R. J. Org. Chem. 1990, 55, 5404.
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Boston, MA) and indicated concentration of inhibitors. The
reaction was stopped by adding 2 mL of diethylether contain-
ing 10 mM of unlabeled Á4-dione and T. The metabolites were
extracted with diethylether before being applied on silica gel
60 TLC plates. TLCs were developed in a mixture of toluene
and acetone (4:1). Substrates and metabolites were identi®ed
by comparison with reference steroids, revealed by auto-
radiography, and quanti®ed using the phosphorImager
(Molecular Dynamics, Sunny Vale, CA). The percentage of
inhibition and IC50 values were then calculated.
24. The logarithm of partition coecient of n-octanol/water
(LogP values) were calculated by CS ChemDraw Pro (Cam-
bridgeSoft Corporation, Cambridge, MA) using the Crippen's
fragmentation method (Ghose, A. K.; Crippen, G. M. J.
Chem. Inf. Comput. Sci. 1987, 27, 35).
22. All compounds were characterized by 1H NMR, 13C
NMR, FT-IR, MS, and elemental analysis.
23. Enzymatic assay (brie¯y): An expression vector encoding
for type 3 17b-HSD was transfected into human embryonal
kidney (HEK)-293 cells using the calcium phosphate proce-
dure.25 Cells were then sonicated in 50 mM sodium phosphate
bu€er (pH 7.4), containing 20% glycerol and 1 mM EDTA,
and centrifuged at 10,000 g for 1 h to remove the mitochon-
dria, plasma membranes, and cells fragments. The supernatant
was further centrifuged at 100,000 g to separate the micro-
somal fraction and this was used for measurement of type 3
17b-HSD activities. The test was carried out at 37 ꢀC for 1 h in
1 mL of above reported bu€er, containing 2 mM of cofactor
(NADPH) and 0.1 mM of 14C-Á4-dione (New England Nuclear,
25. Luu-The, V.; Zhang, Y.; Poirier, D.; Labrie, F. J. Steroid
Biochem. Mol. Biol. 1995, 55, 581.