Coumarins as novel 17β-hydroxysteroid dehydrogenase type 3 inhibitors for potential treatment of prostate cancer

Article abstract of DOI:10.1016/j.bmcl.2009.10.111

The synthesis and SAR studies of 3- and 4-substituted 7-hydroxycoumarins as novel 17β-HSD3 inhibitors are discussed. The most potent compounds from this series exhibited low nanomolar inhibitory activity with acceptable selectivity versus other 17β-HSD isoenzymes and nuclear receptors.

Full text of DOI:10.1016/j.bmcl.2009.10.111

Bioorganic & Medicinal Chemistry Letters 20 (2010) 272–275
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Coumarins as novel 17b-hydroxysteroid dehydrogenase type 3 inhibitors
for potential treatment of prostate cancer
a
b
a
c
Koichiro Harada a, , Hideki Kubo , Yoshitaka Tomigahara , Kazuhiko Nishioka , Junya Takahashi ,
*
Mio Momose c, Shinichi Inoue c, Atsuyuki Kojima c
a Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd, 1-98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-8558, Japan
b Corporate Planning & Coordination Office, Sumitomo Chemical Co., Ltd, 27-1, Shinkawa 2-chome, Chuo-ku, Tokyo 104-8260, Japan
c Organic Synthesis Division, Sumika Technoservice Corporation, 2-1, Takatsukasa 4-chome, Takarazuka, Hyogo 665-0051, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR studies of 3- and 4-substituted 7-hydroxycoumarins as novel 17b-HSD3 inhibitors
are discussed. The most potent compounds from this series exhibited low nanomolar inhibitory activity
with acceptable selectivity versus other 17b-HSD isoenzymes and nuclear receptors.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 25 September 2009
Revised 22 October 2009
Accepted 27 October 2009
Available online 10 November 2009
Prostate cancer is among the most common causes of death
from cancer in men, and accounts for 10% of all new cases of cancer
in males worldwide. Between 10% and 60% of the patients experi-
ence biochemical recurrence associated with treatment failure. No
consensus exists on the optimal therapy for advanced prostate can-
cer. The androgens testosterone (T) and dihydrotestosterone (DHT)
are hormones that play an important role in the development of
prostate cancer. The regulation of androgen biosynthesis or its ac-
tion on the androgen receptor is central to the management of
prostate cancer.1 The production of androgens is controlled at
two levels within the central nervous system; in addition, it is con-
trolled locally in peripheral organs that are targeted by the hor-
mones. The active androgens T and DHT can be synthesized
directly through the conversion of the inactive precursor andro-
Me
O
HO
O
1
Figure 1. Structure of 4-methylumbelliferone (4-MU).
known to exhibit weak inhibitory activity against 17b-HSD3. We
studied new 17b-HSD3 inhibitors with high activity and selectivity
around 4-MU as a seed compound. This report mainly discusses the
SAR of the 3- and 4-positions of 7-hydroxycoumarin derivatives.
4-MU analogs were prepared by the synthetic procedure out-
lined in Scheme 1. The condensation of resorcinol 2 and b-ketoest-
ers in concentrated sulfuric acid at room temperature afforded the
corresponding 7-hydroxycoumarins 3a–h.6 In the case of the syn-
thesis of coumarin derivatives that afforded a complex reaction
mixture under this condition, trifluoroacetic acid as weaker acid
was used to improve the yields. The 4-thioether (4c, 4f–p), 4-ether
(4b,r), and 4-amino derivatives (4d,e) were synthesized by the
nucleophilic substitution of various thiols, alcohols, or amines with
4-chloromethyl and 4-chloroethyl coumarins (3g and 3h) that
were obtained from 4-chloroacetoacetic acid ester and 5-chloro-
3-oxo-pentanoic acid ester.7 Moreover, the coupling of acetoacetic
acid ester 5 with alkyl halides in the presence of 2 equiv base and
stenedione
(D
4-dione) by 17b-hydroxysteroid dehydrogenases
(17b-HSDs) that mediate the final steps in the conversion of sex
steroids in peripheral target tissues.2 17b-Hydroxysteroid dehy-
drogenase type 3 (17b-HSD3) catalyzes the final step in the biosyn-
thesis of the potent androgen T by selectively reducing the C17
ketone of
D
4-dione with NADPH as a cofactor; in addition, it is ex-
pressed at high levels in testes and prostate tissue of some prostate
tumors, suggesting its potential involvement in both gonadal and
nongonadal T biosynthesis. The expression of 17b-HSD3 mRNA in
cancerous prostate biopsies was found to be 30-fold higher than
in normal tissue.3 The role of 17b-HSD3 in T biosynthesis makes
this enzyme an attractive molecular target of a small-molecule
inhibitor for the treatment of prostate cancer.4
ammonium salt (Aliquat 336) afforded c-alkylated-b-ketoesters 6
in moderate yield, and subsequent treatment with resorcinol 2
provided the desired coumarins 4a and 4q. 3-Substituted couma-
rins 9b–e were obtained from a-alkylated-b-ketoesters 8 that were
A high-throughput screening (HTS) of a compound library
(ꢀ220,000 compounds) led to the identification of 4-methylumbel-
liferone (1, Fig. 1)5 as an inhibitor of 17b-HSD3. 4-MU was already
prepared by using b-ketoesters 7 with alkyl halides in the presence
of one equivalent sodium hydride. Other 3-substituted coumarins
9f–i was synthesized from commercially available 2-halo or
* Corresponding author.
E-mail address: haradak@sc.sumitomo-chem.co.jp (K. Harada).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.111

K. Harada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 272–275
273
R1
X
R2
a
b,c,d
OH
HO
O
O
HO
O
O
HO
3a-f
4b(X=O)
2
1
3g(R =CH2Cl)
4c, 4f-p(X=S)
4d(X=NH)
4e(X=NMe)
1
3h(R =CH2CH2Cl)
from 3g
4r(X=CH O) from 3h
2
Me
R1
R1
e
f
O
O
EtO
O
O
O
EtO
O
HO
R3
R2
S
4a,q
5
6
O
O
HO
R1
R1
h,i,j,l
R1
10a from 9f
10b from 9g
R2
R2
g
f
O
O
O
O
EtO
O
HO
CF3
EtO
O
h,i,k,l
S
N
9a-h
9i(R1=CF3, R2=Me)
7
8
O
O
HO
11 from 9i
Scheme 1. Reagents and conditions: (a) R1COCH2CO2Et, concd H2SO4 or TFA, rt–reflux, 3–15 h, 30–50%; (b) Ac2O, reflux or Ac2O, pyridine, rt, 3–8 h, 80–100%; (c) R2SH or
R2OH or R2NHR(R = H or Me), TEA or NaH or pyridine, DMF, rt, 1–8 h, 50–95%; (d) 1 N NaOH, MeOH, rt, 1–3 h, 70–90%; (e) R1CH2Cl, NaH, n-BuLi, THF, À10 °C–rt, 1 h, 40–50%;
(f) 2, MeSO3H or TFA, rt––reflux, 3–15 h, 20–40%; (g) R2Br, NaH, Aliquat 336, toluene, 0 °C–rt, 1–3 h, 50–70%; (h) Ac2O, reflux or Ac2O, pyridine, rt, 3–8 h, 80–100%; (i) NBS,
BPO, CCl4, reflux, 5 h, 70–80%; (j) R3SH, NaH, DMF, 0 °C–rt, 1 h, 75–80%; (k) 2-mercaptopyridine, NaH, DMF, 0 °C–rt, 3 h, 45%; and (l) 1 N NaOH, MeOH, rt, 1–3 h, 75–90%.
Table 1
2-cyano b-ketoesters. Another thioether derivatives 10a,b and 11
Inhibition of 17b-HSD3 by 4-MU analogs in human cellular assay
were prepared from 9f,g and 9i followed by allylic bromination
R1
by treatment with N-bromosuccinimide (NBS) and benzoyl perox-
ide (BPO).8 Moderate yields were obtained by the protection of the
7-hydroxyl group with acetyl group to afford 2-pyridyl compounds
4f, 4p–4r, and 10a,b that required a strong base such as sodium hy-
dride for the substitution reaction.
5
R2
6
HO
O
O
8
1
The initial result for the inhibition of 17b-HSD3 using our 7-
hydroxycoumarin analogs suggested that the nature of the substi-
tuent at the 4-position affects the activity in a cell-based assay9
that SCH-39110 was used as reference inhibitor (Table 1, com-
pounds 3a–f and 4a). The order of potency cannot be easily ex-
plained based on the physical parameters, but the 4-phenethyl
derivative 4a indicated that the steric allowance at the 4-position
preferred more than two carbon chain. The introduction of the al-
kyl group at the 3-position improved the activity (9b–d). When
electron-withdrawing groups were incorporated at the 3-position
(9f and 9h), the activities were similar to 4-MU 1, and 3-chloro
derivative 9g was 10-fold more potent than 4-MU 1. Moreover,
4-trifluoromethyl analog 9e was found to be favorable and resulted
in a sevenfold increase in potency as compare to 9d.
The SAR of the linker part was studied further with an emphasis
on the 4-phenethyl derivative 4a that had potent activity to ex-
plore more potent compounds (Table 2). The replacement of the
methylene with oxygen and nitrogen led to poor 17b-HSD3 inhib-
itory activity (4b and 4d,e); on the other hand, the thioether (4c)
continued to exhibit good potency. Then, heterorings were opti-
mized as follows with thioether as the linker part. As a result, var-
ious heterorings showed good inhibitory activity, in particular, 2-
pyridyl (4f) exhibited potent activity. In addition, the substituents
on a pyridine ring were studied; 6-methyl-2-pyridyl derivative 4p
substituted with electron-donating group exhibited IC50 = 1.5 nM
and was found to be the most potent 17b-HSD3 inhibitor in this
Compounds
R1
R2
HSD IC50 (lM)
1 (4-MU)
3a
3b
3c
3d
3e
3f
4a
9a
9b
9c
9d
9e
9f
9g
Me
Et
n-Pr
CF3
CH2OMe
Ph
CH2Ph
(CH2)2Ph
H
Me
Me
Me
CF3
H
H
H
H
H
H
H
H
Me
Me
n-Pr
CH2Ph
CH2Ph
F
1.0
0.10
0.059
0.19
5.0
1.0
10
0.020
1.0
0.21
0.24
0.20
0.030
1.0
0.10
1.0
0.10
Me
Me
Me
Cl
CN
9h
SCH-391a
a
See Ref. 10 for reference inhibitor.
series. The basicity of nitrogen at a suitable position was consid-
ered to be important for high inhibitory activity (as revealed by a
comparison between 4f and 4p). On combining the aryl part with
the linker part, ethylene derivative 4q similarly exhibited low
nanomolar activity. However, the introduction of a halogen atom
at the third position (10a,b) reduced the activity to one-tenth of
the original. Substitution of the 4-trifluoromethyl derivative with

274
K. Harada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 272–275
Table 2
Table 3
Inhibition of 17b-HSD3 by 4-phenethyl-7-hydroxycoumarin derivatives in human
cellular assay
IC50 value of the subtype of 17b-HSD and nuclear receptors for compounds 4p, 4q,
and 9e
R1
Compounds
4p
4q
9e
5
AR antagonista
agonista
50
na
na
50
na
na
na
na
l
l
M
M
na
na
na
na
na
na
na
na
6.0
na
na
1.0
na
na
na
na
l
l
M
M
R2
6
ER antagonista
agonista
HO
O
O
8
1
GR antagonista
agonista
Compounds R1
R2
HSD IC50
M)
17-HSD type 1b
17-HSD type 2b
(
l
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
(CH2)2-Phenyl
CH2O-Phenyl
CH2S-Phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.020
10
0.093
4.0
6.0
0.0030
5.0
1.0
0.42
0.200
0.010
0.096
0.091
0.088
0.23
na = not active.
a
Reporter gene assay.
Human cell-based assay.
b
CH2NH-Phenyl
CH2NMe-Phenyl
CH2S-2-Pyridiyl
CH2S-4-Pyridyl
CH2S-2-Pyrimidinyl
CH2S-2-Thienyl
CH2S-1,3,4-Thiadiazol-2-yl
CH2S-2-Thiazolyl
CH2S-2-Thiazolidinyl
CH2S-1-Methyl-2-imidazolyl
CH2S-5-Nitro-2-pyridyl
CH2S-5-Trifluoromethyl-2-
pyridyl
4p
4q
4r
10a
10b
11
CH2S-6-Methyl-2-pyridyl
(CH2)2-6-Methyl-2-pyridyl
(CH2)2O-6-Methyl-2-pyridyl
CH2S-6-Methyl-2-pyridyl
CH2S-6-Methyl-2-pyridyl
CF3
H
H
H
F
0.0015
0.0080
1.0
0.027
0.047
0.32
Cl
CH2S-2-
pyridyl
2-pyridylthiomethyl at the 3-position resulted in reduced activity
(11).
Next, replacement of the 7-hydroxyl group and substitution at
the 6- and 8-positions were studied; various functional groups
such as hydrogen-bonding donor/acceptor (amino, carboxyl,
amide, cyano, fluoro, etc.) at the 7-positon resulted in a consider-
able decrease in enzymatic activity. A polar group, alkyl, or halogen
at the 6-position decreased the activity or made it disappear
completely, in a manner similar to substitution at the 8-position
(data not shown). The introduction of a substituent near the phe-
nolic hydroxyl group appeared to prevent the access of the hydro-
xyl group to the hydrogen-binding acceptor of the ligand-binding
site in the enzyme.
Another brief SAR of this chemotype was generated. 7-Hydroxy-
4-methylquinoline and the N-methylated was not tolerated (data
not shown). It was suggested that the lactone part had low steric
allowance or that it was unsuitable for a hydrogen-donor moiety.
Selectivity over 17b-HSD isoenzymes and nuclear receptors is
shown in Table 3. The inhibitory activity to types 1 and 2 was
not observed for compounds 4p, 4q, and 9e. In selectivity over
the nuclear receptors, compound 9e exhibited AR antagonist activ-
Figure 2. (A) In silico overlap of low-energy conformation of 4q (brown) with
androstenedione (red). (B) Reverse orientation of upper panel (A): blue: hydrogen-
bonding acceptor; purple: hydrogen-bonding donor/acceptor; gray: hydrophobic
group.
The basicity of the nitrogen of the pyridine ring appeared to affect
the interaction with the enzyme.
7-Hydroxy-4-substituted coumarins represent a potent class of
17b-HSD3 inhibitors. Preliminary SAR of this series led to the dis-
covery of novel and selective 7-hydroxy-4-(2-pyridylethyl) couma-
rin 4q and 7-hydroxy-4-(2-pyridylthiomethyl) coumarin 4p. These
compounds will be considered for further in vitro and in vivo
evaluation.
ity and ER
a agonist activity. Affinity of the nuclear receptors was
not observed in the case of compounds 4p and 4q; 4-substituted
coumarins had better selectivity than the 3-substituted ones.
Superimposition of the 6-methyl-2-pyridylethyl derivative 4q
with 4-androstene-3,17-dione (D
4-dione) which is an endogenous
ligand was performed using Hopfield Neural Network (HNN) meth-
od.11 These compounds were sterically rigid, and therefore, the ac-
tive conformer is nearly equal to the low-energy conformation.
Acknowledgments
Figure 2 shows that 4q overlaps well with
phobic regions and hydrogen-bond functions; the two carbonyl
groups of
4-dione correspond to the 7-hydroxyl group and the
nitrogen atom of the pyridine ring at the 4-position of coumarin.
D
4-dione in the hydro-
The authors are grateful to Sumika Chemical Analysis Service
for their help in compound synthesis and characterization. Dr. Ta-
naka is acknowledged for assistance with the computer chemistry
studies.
D

K. Harada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 272–275
275
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