Benzothiophenes containing a piperazine side chain as selective ligands for the estrogen receptor α and their bioactivities in vivo

Article abstract of DOI:10.1016/j.bmcl.2004.12.074

The synthesis of benzothiophenes containing a piperazine side chain and their binding affinities for estrogen receptors are described. These compounds bearing piperazine side chains were identified to be high-affinity ligands with high selectivity for ER

Full text of DOI:10.1016/j.bmcl.2004.12.074

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1505–1507
Benzothiophenes containing a piperazine side chain as selective
ligands for the estrogen receptor a and their bioactivities in vivo
Chunhao Yang,a,* Guangyu Xu,a Jia Li,b Xihan Wu,a Bo Liu,a Xueming Yan,a
Mingwei Wangb,* and Yuyuan Xiea
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China
bThe National Center for Drug Screening, 189 Guoshoujing Road, Shanghai 201203, PR China
Received 20 June 2004; revised 21 December 2004; accepted 22 December 2004
Available online 22 January 2005
Abstract—The synthesis of benzothiophenes containing a piperazine side chain and their binding affinities for estrogen receptors are
described. These compounds bearing piperazine side chains were identified to be high-affinity ligands with high selectivity for ER a
subtype. They were also potent agonists in bone tissue.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Due to the side effects of estrogen replacement therapy
(ERT), selective estrogen receptor modulators (SERMs)
which potentially antagonize the proliferative effects of
estrogen on uterine and mammary tissue, while mimic-
king the effects of estrogen on the bone and cardiovascu-
lar system, provide an advantageous alternative
approach.1 Raloxifene, the represented SERM, has been
marketed as Evistaꢁ for the prevention and treatment of
osteoprosis in postmenopausal women in Europe and
the United States. Extensive structure activity relation-
ship (SAR) studies on benzothiophene-based SERMs
have been undertaken,2 and the basic side chain (BSC)
of raloxifene is reported to be crucial for its antagonist
activity.3 In order to enhance the selectivity between
the ER a and ER b of raloxifene while maintaining its
unique pharmacological profile, our studies was cen-
tered on modification of the aminoethoxy side chain,
the most widely used BSC. It is hoped that compounds
containing rigid piperazine side chain can potentially
impact the selectivity for estrogen receptor subtype in
comparison with those with flexible basic side chain.
While our program was completed, there appeared one
report about a class of SERMs containing the piper-
azine side chain.4 Herein, we also report the results of
our studies on a novel class of selective ligands for the
ER a (Fig. 1).
The benzothiophenes were synthesized using standard
chemical methods, starting from 2-(4-methoxy-phenyl)-
6-methoxy-benzothiophene 3.(Scheme 1).5 Compound
4 was synthesized by reacting 4-fluoro-benzoyl chloride
with benzothiophene 3 under general Friedel–Crafts
acylation conditions,2c then compound 4 was reacted
with 1-benzylpiperazine to afford compound 5 via nucleo-
philic aromatic substitution (SNAr). The previous de-
scribed conditions with poisonous KF/Al2O3, DMSO,
and harmful 18-C-6 for SNAr substitution in the case
of aza nucleophiles were not employed.2c,6 In this case
N-methyl-2-pyrrolidone was used as solvent in the pres-
ence of excess benzylpiperazine. Debenzylation of com-
pound 5 afforded the common intermediate 6 under
transfer hydrogenation condition (Pd/C–HCOONH4).7
The compounds 7 were obtained via acylation or alkyl-
ation of the key intermediate 6, and then treatment of 7
with AlCl3/EtSH or BBr3 afforded the desired com-
pounds 2 with free phenol. When R is benzoyl (substi-
tuted benzoyl) or benzyl (substituted benzyl), benzyl
halide or benzoyl chloride, triethylamine was used as
the base. When R is alkyl or cycloalkyl, alkyl halide
(chloride/bromide) did not work well while alkyl meth-
anesulfonate or toluenesulfonate were used as alkylation
reagent with potassium carbonate as the base, reaction
proceeded smoothly and the yield is 70–85%.
Keywords: Benzothiophenes; Piperazine; High selectivity; Estrogen
receptor.
*
Corresponding authors. Tel./fax: +86 21 50806770; e-mail:
chyang@mail.shcnc.ac.cn
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.074

1506
C. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1505–1507
Figure 1. Designed compounds derived from raloxifene.
ER a and ER b.8 The results are listed in Table 1. As ex-
pected, the benzothiophenes containing piperazines ex-
hibit efficient binding to human estrogen receptors.
Most of the compounds displayed high binding affinity
for both ER a and ER b that is comparable to raloxifene
even with bulky substitutents on piperazines. Several
compounds such as 2e,j,n, and q turned out to be potent
estrogen receptor ligands. Compounds 2j,n,q are even
more potent than raloxifene. In addition, the piperazine
basic side chain not only affect the binding affinity, but
also impact on the selectivity of SERMs. 2j,n have mod-
est selectivity for ER a (up to 40·), and 2q showed high-
est selectivity for ER a (up to 120·). These results
demonstrated that the piperazine basic side chain can
bind reasonably to the ER in the binding pocket and
the steric bulk could also be well tolerated.
According to the binding affinities, we selected three
compounds 2e,n,q to test their effects on bone and uterus
of immature mice.9 The biological data showed that all
three compounds can prevent the lose of bone mineral
and reduce the stimulation to uterus, and the effect of
2q10 on bone mineral is very close to that of raloxifene.
It also reduced the stimulation to mice uterus distinctly
compared to raloxifene. We also found that bulky
Scheme 1. Synthesis of compounds 2a–t. Reagents and conditions: (a)
p-fluoro-benzoyl chloride, AlCl3, CH2Cl2, 5–10 ꢂC; (b) benzylpiper-
azine, NMP, 140 ꢂC, 20 h; (c) MeOH, Pd/C–HCOONH4, reflux, 1.5 h;
(d) benzyl halide/benzoyl chloride, THF, TEA or alkyl methanesulfo-
nate, THF, K2CO3, reflux; (e) AlCl3/EtSH or BBr3, CH2Cl2.
Compounds 2a–t were evaluated for their ability to
compete with [3H] 17b-estradiol for binding to both
Table 1. Estrogen receptor binding affinities (IC50) for compounds
Compd
R
ER a (IC50, nM)a
ER b (IC50, nM)a
Selectivity [b]/[a]
1
—
Benzyl
0.73
0.97
0.86
2.65
2.74
0.46
0.78
1.39
3.05
3.94
0.27
1.72
14.46
2.15
0.22
3.58
0.43
0.28
1.08
2.12
1.43
18.90
5.53
25.9
5.7
29.4
7.6
1.8
8.0
5.2
14.2
2.6
4.5
46.2
1.9
6.5
0.6
41
2a
2b
2c
2d
2e
2f
Benzoyl
Furan-2-carbonyl
p-Toluoyl
25.29
20.09
4.94
3-Chlorobenzoyl
4-Chlorobenzoyl
3-Hydroxybenzoyl
2-Hydroxybenzoyl
o-Toluoyl
3.70
4.02
2g
2h
2i
19.77
7.92
17.67
12.47
3.21
2j
m-Toluoyl
2k
2l
2-Chlorobenzoyl
Isonicotinoyl
4-Nitrobenzoyl
4-Chlorobenzyl
2-Nitrobenzoyl
4-Nitrobenzyl
Isopropyl
93.57
1.27
9.07
2m
2n
2o
2p
2q
2r
2s
2t
12.90
14.22
33.92
18.15
66.52
29.37
3.6
33
121
17
Cyclopentyl
4,6-Dimethyl-pyrimidin-2-yl
n-Propyl
31
20
a Values are means of at least three experiments.

C. Yang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1505–1507
1507
Table 2. Biological data in vivo
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Compd
Bone mineral density
(mg/cm3)
Uterine weight assay
(mg)
2e
2n
432.1 10.6**###
482.5 37.5***#
540.8 77.6***#
549.1 75.2***
386.2 24.8
29.3 1.3*###
30.5 2.3*#
2q
38.8 9.8*##
52.0 6.0***
28.9 1.1
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Raloxifene
OVX + DW
Sham + DW
592.0 51.9***#
203.4 37.6***###
Kun-Ming female mice of two months old which were ovariectomized
were treated (sc) with test compounds for 4 weeks at 4 lM. The bone
mineral density was determined by peripheral quantitative computed
tomography (pQCT, Stratec XCT-Research SA). The uterine weights
were determined on the day the mice were killed (n = 5).
* P > 0.05.
** P < 0.05.
*** P < 0.01 versus distilled water (DW).
# p > 0.05.
## p < 0.05.
### p < 0.01 versus raloxifene.
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substituents on piperazines displayed lower agonist
property in bone and higher antagonist property in uter-
ine tissue than smaller substituents (Table 2).
In summary, the piperazine basic side chain was found
to be functionally equipotent to the aminoethoxy group
in benzothiophenes SERMs. Of all the compounds
tested, 2q was found to have highest binding affinity
and selectivity for ER a. It is also a potent agonist in
bone tissue and an antagonist in uterus. Itꢀs expected
that these findings could provide a valuable hit in devel-
oping new SERMs.
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Acknowledgement
9. Yan, X. M.; Zhang, Z. D. Chin. J. Osteoporosis 2004, 10,
288.
This work was financially supported in part by the
National Natural Science Foundation of China
(20302009).
10. Physical data of 2q: 1H NMR (400 MHz, acetone-d6) d
1.02 (6H, d, J = 6.3), 2.60 (4H, t, J = 5.0), 2.69 (1H, m),
3.33 (4H, t, J = 5.0), 6.74 (2H, q, J = 1.9, J = 6.6), 6.83
(2H, d, J = 9.0), 6.90 (1H, dd, J = 2.2, J = 8.8), 7.26–7.30
(3H, m), 7.35 (1H, d, J = 2.2), 7.63 (2H, q, J = 1.9, J =
7.1). Mp 158–160 ꢂC. MS(EI):472(M+). Anal. Calcd for
C28H28N2O3SÆ2H2O: C, 66.12; H, 6.34; N, 5.51. Found: C,
66.19; H, 6.06; N, 5.39.
References and notes
1. (a) Cummings, S. R.; Eckert, S.; Krueger, K. A.; Gray, D.;
Powles, T. J.; Cauley, J. A.; Norton, L.; Nickelson, T.;