Discovery of a novel hybrid from finasteride and epristeride as 5α-reductase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2010.10.112

Finasteride and epristeride both inhibit 5α-reductase with high potency via competitive and non-competitive mechanism, respectively. A new hybrid of finasteride and epristeride was designed as a new 5α-reductase inhibitor based on combination principles in medicinal chemistry. Human 5β-reductase was chosen as a plausible surrogate of 5α-reductase type II and the results indicate that although the hybrid compound possesses the main bulk of epristeride, its inhibitory mechanism is same as of finasteride. The hybrid turned out to be a potent 5α-reductase inhibitor in low IC ₅₀ ranges.

Full text of DOI:10.1016/j.bmcl.2010.10.112

Bioorganic & Medicinal Chemistry Letters 21 (2011) 475–478
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel hybrid from finasteride and epristeride
as 5a-reductase inhibitor
Zhiyi Yao a, Yingjun Xu a, Minmin Zhang a, Sheng Jiang a, Marc C. Nicklaus b, Chenzhong Liao b,c,
⇑
a College of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 210032, China
b Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, DHHS, Frederick, MD 21702, USA
c College of Pharmacy, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Finasteride and epristeride both inhibit 5
a
-reductase with high potency via competitive and non-com-
Received 1 September 2010
Revised 20 October 2010
Accepted 22 October 2010
Available online 28 October 2010
petitive mechanism, respectively. A new hybrid of finasteride and epristeride was designed as a new
-reductase inhibitor based on combination principles in medicinal chemistry. Human 5b-reductase
was chosen as a plausible surrogate of 5 -reductase type II and the results indicate that although the
hybrid compound possesses the main bulk of epristeride, its inhibitory mechanism is same as of finaste-
ride. The hybrid turned out to be a potent 5 -reductase inhibitor in low IC50 ranges.
Published by Elsevier Ltd.
5a
a
a
Keywords:
Hybrid
Finasteride
Epristeride
5a-reductase inhibitor
Combination principles
Benign Prostatic Hyperplasia (BPH) is an andron-related disor-
der common in old male. Typical symptoms include increased fre-
quency or urgency to urinate, which results from increased
pressure on the urethra. It has been well established that growth
of the prostate is stimulated by androgens, and it now appears that
ring of epristeride, and moving the A ring of finasteride to replace
the D ring of epristeride would increase the inhibition activity of
5-AR (Fig. 1). This article reports the synthesis of this new series
of compounds and the results we have obtained using this
approach.
5
a
-dihydrotestosterone (DHT) plays primary role in the trophic
The hybrid 3 was synthesized by using the procedure described
in Figure 2. Oppenauer oxidation of trans-dehydroandrosterone 4
led to yield compound 5, which was sequentially sulfanilated to
support of this organ.1,2 Steroid 5
a-reductase (5-AR) is an enzyme
responsible for the conversion of testosterone (T) into DHT.3 Thus
selective inhibition of steroid 5-AR could offer an alternative ther-
apy for BPH. There are two isoforms of human 5-AR identified as
type I and type II.4–6 In normal human skin the type I reductase
is the predominant type expressed, whereas 5-AR type II is the ma-
jor form found in the prostate.7
O
O
H
N
H
N
C
D
D
C
Finasteride 1 is a selective inhibitor of the type I and type II iso-
forms of steroid 5-AR. It is currently marketed worldwide as a drug
for BPH, and is also used in the treatment of hair loss8,9 and in the
prevention of prostate cancer.10 A few years later, epristeride,
behaving as an uncompetitive inhibitor, was also launched in
2000 as a therapy for BPH.11 On the basis of their different modes
of action, a combination of finasteride and epristeride may prove
similarly beneficial. It is worthwhile to design and synthesize a no-
vel hybrid which not only combine important pharmacophore
structures of finasteride and epristeride, but also retain both inhi-
bition activities of 5-AR. We hypothesized that keeping the A and B
B
A
B
A
HO
O
N
H
H
O
Epristeride 2
Finasteride 1
H
N
O
HO
O
⇑
Corresponding author. Tel.: +1 301 846 5993/+86 22 23506290; fax: +1 301 846
6033/+86 22 23507760.
Hybrid 3
E-mail addresses: chenzhongliao@yahoo.com, czliao@helix.nih.gov (C. Liao).
Figure 1. Structures of finasteride 1, epristeride 2 and their hybrid 3.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.10.112

476
Z. Yao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 475–478
O
O
O
Oppenauer
Oxidation, 85%
RfSO2F, DBU
Toluene, 50%
RfSO3
O
HO
6
4
5
Rf= HCF2CF2OCF2CF2
NOH
O
.
PdCl2, PPh3, Et3N
NH2OH HCl
DMF/MeOH (1/1),
CO, 80 oC, 66%
EtOH/C5H5N (1/1)
91%
H3CO2C
H3CO2C
7
8
H
H
N
N
O
O
SOCl2, dioxane
10 oC, 83%
K2CO3, MeOH
HO
H2O, reflux, 98%
H3CO
O
O
Hybrid 3
9
Figure 2. The synthesis of hybrid 3.
Table 1
afford 6 in 50% yield. The RfSO3 group of 6 was transformed to car-
boxylic ester via Pd catalyzed CO addition reaction. After reaction
with NH2OH, Beakmann rearrangement12 was performed with
SOCl2 to afford compound 9, which was rapidly saponified with
K2CO3 to give hybrid 3 in 98% yield.
To evaluate the function of A ring of compound 3, we replace
the A ring with the phenyl. The synthesis of compound 13 was de-
scribed in Figure 3. Starting with the estrone, compound 11 was
synthesized in 99% yield. Then Beckmann rearrangement was per-
formed with SOCl2 to afford compound 12, which was rapidly sul-
fation with sulfamic acid to give 13 in 50% yield.
5a-Reductase inhibition by Hybrid 3, 12 and 13
Compound
IC50 value (nM)
Relative inhibition
(finasteride vs compound)
Finasteride
3
12
13
35
71
>1000
>1000
1
0.49
<0.055
<0.055
NADP+Áfinasteride was determined with a high resolution of
1.70 Å (PDB ID 3G1R)14 and was used as a surrogate of 5
-reduc-
tase type II in this modeling study based on the following reasons:
(1) The reduction of the
4-ene in humans, the initial step in
steroid hormone metabolism, is mediated by 5 -reductases or
5b-reductase to yield the subsequent 5 - or 5b-dihydrosteroids,
respectively,15 which means 5
-reductases or 5b-reductase have
same or at least similar enzymatic functions when metabolizing
steroid hormones. (2) The sequence identities between 5 - and
a
The inhibitory effects of compounds 3 and 13 on human pros-
tatic 5a-reductase were investigated with an in vitro incubation
D
technique.13 Finasteride was used as a reference compound. IC50
values and relative inhibitory efficiencies as compared with the
IC50 of finasteride are presented in Table 1. The investigated steroid
a
a
a
3 exhibit promising 5a-reductase inhibitory effect. The IC50 value
of compound 3 is 71 nM. The relative inhibitory effect of the com-
pound 3 is 0.49. As concerns the effects of replacement at A ring in
3, the introduction of phenyl group (12 and 13) caused a drop-off
a
5b-reductase are very low, mainly because these two kinds of
reductases are encoded by completely unrelated genes. This ham-
pers the developing of a reliable homology model. So using 5b-
in inhibition ability of 5
a
-reductase.
-reductase type I and type II, there are
Currently, for both of 5
a
reductase as a surrogate of 5a-reductases is more reasonable than
no crystal structures available in the RCSB Protein Data Bank (PDB).
Conversely, for 5b-reductase, several crystal structures, binding
with different steroids and same cofactors (NADP+ or NADPH),
have been deposited in PDB. The structure of the AKR1D1Á
developing a homology model to investigate how 5
inhibitors to interact with the enzyme.
The crystal structure of 3G1R was downloaded from the PDB
website, and the monomer B was kept and treated using Schrö-
a-reductase
O
NOH
.
SOCl2, dioxane
NH2OH HCl
EtOH/C5H5N (1/1)
rt, 60%
HO
HO
99%
10
11
H
H
O
N
O
N
HO3SNH2, C5H5N
reflux, 4h, 50%
HO3SO
HO
13
12
Figure 3. The synthesis of compound 13.

Z. Yao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 475–478
477
Table 2
Epristeride could be docked into the active site with similar
interactions with the protein: the main hydrophobic bulk forms
hydrophobic interactions in the hydrophobic active pocket; the
negative charged carboxylic acid group forms two hydrogen bonds
with Glu 120 and Tyr 58. Two hydrogen bonds are observed with
the surrounding water molecules. Although similar interactions
as finasteride, the docking score and calculated Prime MM-GBSA
The calculation results of docking and Prime MM-GBSA
Compound
Glide XP GScore
(kcal/mol)
Prime MM-GBSA
(kcal/mol)
DG binding
Finasteride
Epristeride
Hybrid
À15.57
À12.63
À13.11
À34.75
À14.43
À30.32
D
G is worse than those calculated ones of finasteride, indicating
epristeride maybe have a worse binding affinity than finasteride
to 5b-reductase.
dinger’s Protein Preparation Wizard16 to adjust the protein, the
cofactor, and the ligand. Receptor grids were generated for further
docking studies. The three compounds, finasteride, epristeride, and
hybrid, were processed using LigPrep 2.417, a robust collection of
tools designed to prepare 3D structures for drug-like molecules,
and then docked to the active site of 5b-reductase, represented
by the receptor grids using Glide 5.618 extra precision (XP) mode.
The ligand binding energies of the three docked compounds were
calculated using Prime MM-GBSA.19 When calculating, the protein
structure was relaxed by setting the ‘Size of flexible region in Å’ to
‘Small (8)’.
Finasteride mainly interacts with 5b-reductase by hydrophobic
interactions and hydrogen bonds. The active site, composed of Tyr
26, Tyr 58, Trp 89, Glu 120, Tyr 132, Trp 140, Trp 230, Met 313, and
Trp 314, is very hydrophobic and so can easily accommodate
hydrophobic compounds such as finasteride. The C3 carbonyl oxy-
gen of finasteride forms two hydrogen bonds with the phenolic hy-
droxyl group of Tyr 58 and the neutral anti-oriented conformer of
the carboxylic acid side chain of Glu 120. Additionally, three hydro-
gen bonds forming with water molecules are observed. Our dock-
ing studies and Prime MM-GBSA calculations reproduced this
scenario: the best scored docking conformation of finasteride has
a heavy atom RMSD of 0.461 Å relative to its crystallographic ori-
entation (see Table 2 and Fig. 4A).
The simulated conformation of the hybrid compound 3 in the
active site is similar to the crystallographic orientation of finaste-
ride (see Fig. 4C): the C3 carbonyl oxygen forms two hydrogen
bonds with Glu 120 and Tyr 58; the N4 group engages a hydrogen
bond with an environmental water molecule. The difference is the
carboxylic acid group, which is on the rim of the active site and
forms two hydrogen bonds with Arg 134 and a water molecule.
The docking score and calculated binding affinity are a little bit
worse than the ones of finasteride, but are much better than the
ones of epristeride. The overlapping of the simulated conforma-
tions of these three studied compounds shows that the carboxylic
acid group of epristeride sticks out (see Fig. 4D), which may in-
crease the torsion energies of Glu 120 and Tyr 58 of the protein
and then lead to worse binding affinity.
In summary, a new hybrid 3 from finasteride and epristeride
was designed as a 5
a convenient route. 5b-reductase was chosen as a plausible
surrogate of human 5 -reductase type II because of their similar
a-reductase inhibitor, and synthesized through
a
enzymatic functions. Furthermore, finasteride can inhibit both of
these two enzymes, and NADP+ is an identical cofactor of both of
them. The results indicate, although the hybrid compound
possesses the main bulk of compound 2, its inhibitory mechanism
is same as of compound 1.
Figure 4. The binding modes predicted by Glide XP docking and Prime MM-GBSA. (A) Finasteride; (B) epristeride; (C) hybrid; (D) the overlapping of the simulated
conformations of these three compounds. The protein was presented as a thin tube, and the active site was presented as a light blue molecular surface.

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Z. Yao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 475–478
7. Luthe, V.; Sugimoto, Y.; Puy, L.; Labrie, Y.; Solache, I. L.; Singh, M.; Labrie, F. J.
The hybrid 3, closely resembling structure features of finaste-
ride and epristeride, exhibited promising 5 -reductase inhibitory
effect in vitro. The aromatization of the A ring and the replacement
of the carboxylic group of the hybrid compound 3 led to dimin-
ished activities. This fully elaborated hybrid provides us a novel
template for the development of pharmacokinetically improved
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[4-14C] testosterone and the test compound (in four different concentrations)
in the presence of 1 mM NADPH for 20 min at 37 °C. And the enzymatic
reaction was stopped by the addition of ethyl acetate and freezing. The control
experiments were performed without the test substances in every series. After
extraction the radioactive testosterone and dihydrotestosterone were
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Acknowledgments
We thank the Shanghai University Distinguished Professor
(Eastern scholars) Program (DF2009-02), and Pujiang Talent Plan
Project (09PJ1409200) for financial support.
dihydrotestosterone formed and the substrate testosterone, the 5a-reductase
activity was calculated and expressed in terms of pmol dihydrotestosterone
per mg of protein for a 20-min incubation. The inhibitory effects of the
compounds are given in terms of IC50 values.
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