4-(4-Alkylpiperazin-1-yl)phenyl group: A novel class of basic side chains for selective estrogen receptor modulators

Article abstract of DOI:10.1016/j.bmcl.2003.09.049

In the structure-activity relationships of spiro[indene-1,1′-indane] series, the 4-(4-alkylpiperazin-1-yl)phenyl group was found to be functionally equipotent to the 4-(1-piperidinoethoxy)phenyl group, the most widely used basic side chain in selective estrogen receptor modulators.

Full text of DOI:10.1016/j.bmcl.2003.09.049

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4317–4320
4-(4-Alkylpiperazin-1-yl)phenyl Group: A Novel Class of Basic
Side Chains for Selective Estrogen Receptor Modulators
Nobuhide Watanabe,a,* Hiroshi Nakagawa,a Akihisa Ikeno,b Hisao Minato,b
Chie Kohayakawab and Jun-ichi Tsujib
aChemistry Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Enoki-cho 33-94, Suita, Osaka 564-0053, Japan
bPharmacology & Microbiology Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Enoki-cho 33-94,
Suita, Osaka 564-0053, Japan
Received 2 September 2003; revised 20 September 2003; accepted 22 September 2003
Abstract—In the structure–activity relationships of spiro[indene-1,10-indane] series, the 4-(4-alkylpiperazin-1-yl)phenyl group was
found to be functionally equipotent to the 4-(1-piperidinoethoxy)phenyl group, the most widely used basic side chain in selective
estrogen receptor modulators.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
crystal structure analysis of ERa ligand binding domain
complexed with RAL or 4-hydroxyTAM, an active
metabolite of TAM, seems to elucidate the mechanism
of these SERMs antagonist activity.8 Thus, BSC has
been demonstrated to protrude out of the ligand bind-
ing pocket formed by helices 3,5/11, thereby inducing
With the advent of tamoxifen (TAM) and raloxifene
(RAL), selective estrogen receptor modulators (SERMs)
have shown high therapeutic potential for estrogen-
related diseases.1 Thus, TAM, the first SERM to be
approved for the prevention of breast cancer in high-
risk women, is effective for hormone-dependent breast
cancer, whereas RAL is prescribed for the prevention
and treatment of osteoporosis in postmenopausal
women.2 Along this line, several other SERMs such as
lasofoxifene,3 EM-652,4 and ERA-9235 are enjoying the
confidence of good efficacy in clinical trials, and further
investigations for additional diversities and new scaf-
folds that confer improved pharmacological profiles are
continuing (Fig. 1).
A key to identifying new SERMs is to design a phar-
macophore that can be responsible for estrogen
antagonist activity in reproductive tissues, as is evident
from clinical data showing that HRT and TAM have
increased risks of breast and endometrial cancers
respectively.2b,6 It has been reported that the basic side
chain (BSC) in RAL plays a critical role in RAL’s
estrogen antagonist activity in the uterus and that the
nature of substituents on the nitrogen atom of BSC
considerably affects this antagonism.7 Moreover, X-ray
*Corresponding author. Tel.: +81-663-375-906; fax: +81-663-387656;
e-mail: nobuhide-watanabe@dainippon-pharm.co.jp
Figure 1. Structure of 17b-estradiol and SERMs.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.049

4318
N. Watanabe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4317–4320
conformational changes that block interaction of ER
with coactivator proteins. Despite the importance of
BSC, efforts for the development of new SERMs, have
only been focused on finding scaffolds such as indoles
and tetrahydronaphthalenes.3ꢀ5,9
Binding assay
Compounds binding affinity for ERwas determined by
a competitive radiometric binding assay in human
breast cancer (MCF-7) cytosol, using [3H]-17b-estradiol
as tracer. Each binding affinity value is expressed as
relative binding affinity (RBA), where the affinity of the
parent compound 1 (Ki=25.3 nM) is considered to be
100%.
We have recently found (+)-3-[4-(1-piperidinoethoxy)
phenyl]-spiro[indene-1,10-indane]-5,50-diol hydrochlo-
ride [(+)-1] as a promising non-steroidal estrogen
receptor agonist for the treatment of hot flush.10 In
order to enhance the antagonist activity of (+)-1 while
maintaining its unique pharmacological profile, logical
extension of our structure–activity relationship (SAR)
studies was centered on modification of the 4-(1-piper-
idinoethoxy)phenyl moiety, the most widely used BSC.
Herein, we report the results of our SARstudies and a
novel class of BSCs, modification of which showed wide
tolerance for ERin terms of in vivo potency as well as
binding affinity.
Cholesterol-lowering effect
Compounds cholesterol-lowering effects were estimated
from the mean percent decrease of plasma cholesterol
level relative to vehicle as follows: Sprague–Dawley
(SD) rats (6–8 weeks old) were ovariectomized and
orally treated for 4 days with test compounds at a dose
of 1 mg/kg/day.
Uterotrophic effect
Compounds uterotrophic effects were estimated from
the mean percent increase in uterine wet weight relative
to vehicle (method 1) or sham (method 2) in two ways:
(1) SD rats (14 weeks old) were ovariectomized and
orally treated for 4 days with test compounds at a dose
of 1 mg/kg/day (Tables 1 and 2). (2) SD rats (21 day
old) were orally treated for 4 days with test compounds
at the indicated doses. Uteri were removed and weighed
(Fig. 2).
Results and Discussion
Chemical synthesis
The starting ketone 2, 1 and (+)-1 were prepared as
described previously.10 BSCs were incorporated by
addition of the corresponding aryllithium reagents 3a–r
and subsequent dehydration with TsOH followed by
demethylation with Ph2PLi to give 4a–r in yields rang-
ing from 32 to 56% in three steps (Scheme 1).
Among the complex biological activities of SERMs,
their cholesterol-lowering ability seems to be the pri-
mary criterion for compounds selection not only
because SERMs are expected to have beneficial effects
on lipid metabolism but also because ED50 values for
RAL analogues activity have been reported to highly
correlate with RBA for ER.11 Therefore, in this study,
we used cholesterol-lowering ability to evaluate in vivo
beneficial effects of compounds. As can been seen from
Table 1, an alkyl group on the nitrogen atom and a
methylene tether in the BSC moiety were well tolerated
in terms of binding affinity (1, 4a–f). In particular,
replacement of the oxygen atom by a methylene led to
approximately 10-fold increase in binding affinity (4f).
However, as was not anticipated from SARs of other
SERMs,12 these subtle modifications resulted in sig-
nificant reduction in cholesterol-lowering effect com-
pared to the prototype 1. As for the pendant phenyl
group, additional substitution by a methyl or fluoro
group and alternate placement of the 1-piper-
idinoethoxy moiety at the R2 position led to reduction
in cholesterol-lowering effect (4g–k). The inconsistent
correlation between binding affinity and in vivo activity
for this series prompted us to identify a new class of
BSCs that have distinct physical properties to improve
pharmacokinetic profiles of compounds.
Given the significance of the tertiary basic nitrogen
atom and the ethoxy tether length in the BSC,13 we
became interested in piperazine structure as a con-
strained aminoethoxy moiety, and then tested 4l–r for
binding affinity (Table 2). As expected, all compounds
Scheme 1. Synthesis of 4a–r: (i) THF, ꢀ78 ꢁC; (ii) TsOH, toluene,
reflux; (iii) Ph2PH, n-BuLi, THF, reflux.

N. Watanabe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4317–4320
4319
Table 1. Binding affinity and in vivo activity of compounds 1 and 4a–k
Table 2. Binding affinity, in vivo activity and uterotrophic effect of
compounds 4l–r
Compd R1
R2
R3
R4 RBA Plasma cholesterol
(%decr.)
Compd
R3a
RBA Plasma cholesterolb Uterotropic effectc
1
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
100
428
460
118
85
44a
4l
207
181
186
103
42
NTd
52*
55*
21
NTd
208
223
NTc
185
139e
114
4a
4b
4c
4d
4e
4f
4g
4h
4i
18
4m
4n
4o
4p
4q
4r
H
8.5
17
6.0
18
6.0
4.5
ꢀ12
H
H
87*
51*
32f*
H
153
956
122
70
125
387
H
Me
H
1
220
155
244
RAL
TAM
aR1=R2=R4=H.
Me
F
22
ꢀ9.1
16
bThe values are expressed as mean percent decrease relative to vehicle.
Statistically significant (p<0.001) differences are denoted by an aster-
isk (*).
4j
247
47
cFourteen-week-old SD rats were used in this experiment (see text).
dNT, not tested.
4k
H
NTb
eAn increase dose (10 mg/kg/day) of 4q produced higher increase
(26.5%) in uterine weight than that of 1 (211%).
fNo standard dose–response curve was observed.
astatistically significant difference (p<0.05).
bNT, not tested.
with small alkyl substituents at N(4) showed good RBA
and high potency in cholesterol-lowering effect (4l–n).
Substitution with bulky alkyl groups such as iso-propyl
and iso-butyl led to a modest decrease in binding affinity
(4o–p). Interestingly, in contrast to its RBA, 4p showed
the highest potency in cholesterol-lowering effect with
87% decrease at a dose of 1 mg/kg/day. In addition,
enlargement of the piperazine ring size or replacement
of N(1) nitrogen by a methylene maintained a good
binding affinity (4q–r). With these results in hand, we
next examined estrogen antagonist effects of these com-
pounds on the uterus. In ovariectomized rat model, oral
administration (1 mg/kg/day) of test compounds pro-
duced acceptable estrogen antagonist effects on the
uterus compared to RAL and TAM. Of the new com-
pounds, 4r was the most potent estrogen antagonist
with only 14% increase in uterine weight relative to
controls. However, the cholesterol-lowering effect of 4r
was weaker than what was anticipated from its RBA,
and did not conform to a standard dose–response curve,
which may indicate the involvement of an unidentified
pathway. On the other hand, while 4q showed potent
estrogen antagonist activity at 1 mg/kg/day, an increase
in its dose (10 mg/kg/day) produced higher increase in
uterine weight than that of 4m. Taken together, 4m was
chosen as a promising alternative to (+)-1, an orally
bioavailable enantiomer of 1, and was finally tested in
an immature rat model. While 4m showed no improve-
ment over (+)-1 in terms of intrinsic estrogen antag-
onistic activity (Fig. 2), all data for 4m demonstrated
that the 4-(4-ethylpiperazin-1-ly)phenyl moiety was
Figure 2. Uterotrophic effect of selected compounds in an immature
rat model.
functionally equipotent to the 4-(1-piperidinoethoxy)-
phenyl moiety in the spiro[indene-1,10-indane] series. As
the compounds tested in this study were in a racemic
form, rationalization of the results obtained should be
taken into account for the possibility that each enan-
tiomer has different biological activity. Our studies with
1 and (+)-1 have, thereby, indicated that the racemate 1
displays the same qualitative profile of activity, with one
enantiomer [i.e., (+)-1] possessing the majority of the
biological effects.10 Further studies on the biological
effects of both enantiomers of these compounds will be
reported in due course.

4320
N. Watanabe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4317–4320
In conclusion, we have investigated SARs of 1 centered
on modification of its BSC and devised 4-(4-alkylpiper-
azin-1-yl)phenyl group as a constrained aminoethoxy
side chain. Our SARstudies demonstrated considerable
tolerance for modification around the piperazine ring
region in this series of compounds. Of the compounds
tested, 4m was found to have beneficial effect on lipid
metabolism while maintaining marginal estrogen
antagonist effect on the uterus. These findings imply
that 4-(4-alkylpiperazin-1-yl)phenyl group may serve as
novel BSCs that elicit reasonable pharmacological
profiles in other classes of SERMs.11
7. Grese, T. A.; Sluka, J. P.; Bryant, H. U.; Cullinan, G. J.,;
Glasebrook, A. L.; Jones, C. D.; Matsumoto, K.; Palkowitz,
A. D.; Sato, M.; Termine, J. D.; Winter, M. A.; Yang, N. N.;
Dodge, J. A. Proc. Natl. Acd. Sci. U.S.A. 1997, 94, 14105.
8. (a) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard,
R. E.; Bonn, T.; Engstrom, O.; Ohman , L.; Greene, G. L.;
Gustafsson, J.-A.; Carlquist, M. Nature 1997, 389, 753. (b)
Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner,
P. J.; Agard, D. A.; Greene, G. L. Cell 1998, 95, 927.
9. For recent other examples, see: (a) Jørgensen, A. S.;
Jacobsen, P.; Christiansen, L. B.; Bury, P. S.; Kanstrup, A.;
Thorpe, S. M.; Bain, S.; Nærum, L.; Wassermann, K. Bioorg.
Med. Chem. Lett. 2000, 10, 399. (b) Kim, Y.-W.; Mobley, J. A.;
Brueggemeier, R. B. Bioorg. Med. Chem. Lett. 2003, 13, 1475,
and references cited therein.
10. Watanabe, N.; Ikeno, A.; Minato, H.; Nakagawa, H.;
Kohayakawa, C.; Tsuji, J. J. Med. Chem. 2003, 46, 3961.
11. Kauffman, R. F.; Bensch, W. R.; Roudebush, R. E.; Cole,
H. W.; Bean, J. S.; Phillips, D. L.; Monroe, A.; Cullinan, G. J.;
Glasebrook, A. L.; Bryant, H. U. J. Pharmacol. Exp. Ther.
1997, 280, 146. In addition, most of current SERMs have
similar beneficial effects on lipid and bone metabolism.
Therefore, the potency in cholesterol-lowering effect may cor-
relate to that in preventive effect on osteoporosis; see: Leven-
son, A. S.; Kliakhandler, I. L.; Svoboda, K. M.; Pease, K. M.;
Kaiser, S. A.; Ward, J. E., III; Jordan, V. C. Br. J. Cancer
2002, 87, 449.
12. (a) Grese, T. M.; Cho, S.; Finley, D. R.; Godfrey, A. G.;
Jones, C. D.; Lugar, C. H., III; Martin, M. J.; Matsumoto, K.;
Pennington, L. D.; Winter, M. A.; Adrian, M. D.; Cole, H. W.;
Magee, D. E.; Phillips, D. L.; Rowley, E. R.; Short, L. L.;
Glasebrook, A. L.; Bryant, H. U. J. Med. Chem. 1997, 40, 146.
(b) Grese, T. M.; Pennington, L. D.; Sluka, J. P.; Adrian,
M. D.; Cole, H. W.; Fuson, T. R.; Magee, D. E.; Phillips,
D. L.; Rowley, E. R.; Shetler, P. K.; Short, L. L.; Venugopa-
lan, M.; Yang, N. N.; Sato, M.; Glasebrook, A. L.; Bryant,
H. U. J. Med. Chem. 1998, 41, 1272.
References and Notes
1. (a) Jordan, V. C. J. Med. Chem. 2003, 46, 883. (b) Jordan,
V. C. J. Med. Chem. 2003, 46, 1081. (c) Riggs, B. L.; Hart-
mann, L. C. N. Engl. J. Med. 2003, 348, 618.
2. (a) Fisher, B.; Costantino, J. P.; Wickerham, D. L.; Redmond,
C. K.; Kavanah, M.; Cronin, W. M.; Vogel, V.; Robidoux, A.;
Dimitrov, N.; Atkins, J.; Daly, M.; Wieand, S.; Tan-Chiu, E.;
Ford, L.; Wolmark, N., other National Surgical Adjuvant
Breast and Bowel Project Investigators J. Natl. Cancer Inst.
1998, 90, 1371. (b) Plouffle, L., Jr.; Facog, C. M. J. Soc.
Gynecol. Invest. 2000, 7, S38.
3. Rosati, R. L.; Jardine, P. D. S.; Cameron, K. O.; Thomp-
son, D. D.; Ke, H. Z.; Toler, S. M.; Brown, T. A.; Pan, L. C.;
Ebbinghaus, C. F.; Reinhold, A. R.; Elliot, N. C.; Newhouse,
B. N.; Tjoa, C. M.; Sweetman, P. M.; Cole, M. J.; Arriola,
M. W.; Gauthier, J. W.; Crawford, D. T.; Nickerson, D. F.;
Pirie, C. M.; Qi, H.; Simmons, H. A.; Tkalcevic, G. T. J. Med.
Chem. 1998, 41, 2928.
4. Labrie, F.; Labrie, C.; Belanger, A.; Simard, J.; Giguere,
V.; Tremblay, G. J. Ster. Biochem. Mol. Biol. 2001, 79, 213.
5. Miller, C. P.; Harris, H. A.; Komm, B. S. Drugs 2002, 27, 117.
6. Collaborative group on hormonal factors in breast cancer
and hormone replacement therapy Lancet 1997, 350, 1047.
13. (a) Levenson, A. S.; Jordan, V. C. Cancer Res. 1998, 58,
1872. (b) Jordan, V. C.; Schafer, J. M.; Levenson, A. S.; Liu,
H.; Pease, K. M.; Simons, L. A.; Zapf, J. W. Cancer Res. 2001,
61, 6619. (c) Fahrnow, M.; Egner, A.; Curr, U. Opin. Biotech.
1999, 10, 550.