Synthesis and biological evaluation of cyclopropyl analogs of the antiestrogen MER 25

Article abstract of DOI:10.1006/bioo.1998.1081

In an effort to prepare effective nonsteroidal antiestrogens without intrinsic estrogenicity and with greater antagonism than the triarylethylenes (tamoxifen), four (E)- and (Z)-1,1 dichloro-2-phenyl-2-[4-(2- diethylaminoethoxy)phenyl]-3-(4-methoxyphenyl)cyclopropane analogs of the antiestrogen MER 25, of which two of the compounds had a 4-heptafluorotolyl group in the α-ring, were prepared. The (E)- and (Z)-gem- dichlorotriarylcyclopropanes were tested for their ability to inhibit the growth of estrogen receptor (ER)-positive MCF-7E3 and ER-negative MDA-MB-231 human breast cancer cells in culture. All compounds, except 18E, exhibited a statistically significant (P < 0.01) reduction in estradiol-stimulated growth (antiestrogenic activity) at 1.0 μM concentration in the MCF-7E3 cells: 11Z (88%), 11E (106%), 18Z (65%), and the test compounds 7A(Z) (85%), 7A(E) (53%), MRL 37 (91%), MER 25 (71%), and ICI 182,780 (102%). Inhibition of estradiol-stimulated growth at concentrations lower than 1.0 μM was demonstrated by 11E, MER 25, and ICI 182,780. Compound 11E produced weak inhibition at 0.1 nM (19%) and nearly complete inhibition (79-112%) over a concentration range of 1.0 to 100 nM. MER 25 produced inhibition of estradiol-stimulated growth at 1.0 (39%), 10 (102%), and 100 nM (100%) concentrations. ICI 182,780 completely inhibited estrogen-stimulated growth from 0.1 nM to 1.0 μM concentrations. Two compounds exhibited estrogenic activity: 18E (from 1.0 nM to 1.0 μM concentrations) and MER 25, which had antiestrogenic action at the lower concentration ranges, but exhibited estrogenic properties at 100 nM to 1.0 μM concentrations. None of the test compounds or standards were active in the MDA-MB-231 cell line at the concentrations studied (0.01 nM to 1.0 μM). In addition, none of the compounds inhibited cell growth below control in the MCF-7E3 cell line. The results from both cell lines suggest that the test compounds are devoid of any antitumor properties, which is thought to be mediated through a nonreceptor mechanism. Analog 11E has the potential to be useful in the treatment of hormone-responsive breast cancer.

Full text of DOI:10.1006/bioo.1998.1081

BIOORGANIC CHEMISTRY 26, 15–31 (1998)
ARTICLE NO. BH981081
Synthesis and Biological Evaluation of Cyclopropyl Analogs of
the Antiestrogen MER 25
Lynette B. Overacre and Robert A. Magarian1
Medicinal Chemistry/Pharmaceutics Department, College of Pharmacy, University of Oklahoma
Health Sciences Center, Oklahoma City, Oklahoma 73190
Received August 25, 1997
In an effort to prepare effective nonsteroidal antiestrogens without intrinsic estrogenicity
and with greater antagonism than the triarylethylenes (tamoxifen), four (E)- and (Z)-1,1-
dichloro-2-phenyl-2-[4-(2-diethylaminoethoxy)phenyl]-3-(4-methoxyphenyl)cyclopropane
analogs of the antiestrogen MER 25, of which two of the compounds had a 4-heptafluorotolyl
group in the Ͱ-ring, were prepared. The (E)- and (Z)-gem-dichlorotriarylcyclopropanes were
tested for their ability to inhibit the growth of estrogen receptor (ER)-positive MCF-7E3 and
ER-negative MDA-MB-231 human breast cancer cells in culture. All compounds, except
18E, exhibited a statistically significant (P Ͻ 0.01) reduction in estradiol-stimulated growth
(antiestrogenic activity) at 1.0 ȐM concentration in the MCF-7E3 cells: 11Z (88%), 11E
(106%), 18Z (65%), and the test compounds 7A(Z) (85%), 7A(E) (53%), MRL 37 (91%),
MER 25 (71%), and ICI 182,780 (102%). Inhibition of estradiol-stimulated growth at
concentrations lower than 1.0 ȐM was demonstrated by 11E, MER 25, and ICI 182,780.
Compound 11E produced weak inhibition at 0.1 nM (19%) and nearly complete inhibition
(79–112%) over a concentration range of 1.0 to 100 nM. MER 25 produced inhibition of
estradiol-stimulated growth at 1.0 (39%), 10 (102%), and 100 nM (100%) concentrations. ICI
182,780 completely inhibited estrogen-stimulated growth from 0.1 nM to 1.0 ȐM
concentrations. Two compounds exhibited estrogenic activity: 18E (from 1.0 nM to 1.0 ȐM
concentrations) and MER 25, which had antiestrogenic action at the lower concentration
ranges, but exhibited estrogenic properties at 100 nM to 1.0 ȐM concentrations. None of the
test compounds or standards were active in the MDA-MB-231 cell line at the concentrations
studied (0.01 nM to 1.0 ȐM). In addition, none of the compounds inhibited cell growth below
control in the MCF-7E3 cell line. The results from both cell lines suggest that the test
compounds are devoid of any antitumor properties, which is thought to be mediated through
a nonreceptor mechanism. Analog 11E has the potential to be useful in the treatment of
hormone-responsive breast cancer.
1998 Academic Press
Key Words: synthesis; biological evaluation; gem-dichlorocyclopropanes; MCF-7E3; MDA-
MB-231; antiestrogen; breast cancer.
INTRODUCTION
Nonsteroidal antiestrogens (competitive estrogen antagonists) represent a major
advance in the treatment of hormone-dependent breast cancer in postmenopausal
females and in the prevention and therapy of the disease in premenopausal women.
1 To whom correspondence should be addressed at the Department of Medicinal Chemistry, College
of Pharmacy, University of Oklahoma HSC, 1110 N. Stonewall, Oklahoma City, Oklahoma 73190. Fax:
(405) 271-3830. E-mail: robert-magarian@uokhsc.edu.
15
0045-2068/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.

16
OVERACRE AND MAGARIAN
SCHEME 1
The representative of this class is tamoxifen (TAM), the only clinically available
adjuvant with surgery in the treatment of primary breast cancer in postmenopausal
women (1, 2). Because of the partial agonist activity found in TAM and other
antiestrogens, workers have synthesized both nonsteroidal (3–7) and steroidal
(8–15) antiestrogens in an attempt to find pure antiestrogens which would compete
effectively with estradiol or act as effective agents in the treatment of estrogen
receptor (ER)-negative breast tumors and in TAM-resistant disease.
A common structural feature in the triphenylethylenes (TPE), such as TAM, is
the presence of three aryl rings with a central ethylenic bridge. Structure–activity
relationships and X-ray crystallographic data of tamoxifen and its analogs (16, 17)
have demonstrated that the Z arrangement of the ͰЈ and ͱ rings is essential for
antiestrogenic activity. In addition, only the Z diastereomers of cyclopropane deriva-
tives prepared in our laboratory have exhibited antiestrogenic activity (Scheme 1)
(6, 7).
MER 25, also known as ethamoxytriphetol, the first nonsteroidal antiestrogen,
was found to inhibit estrogen in all species of animals tested (18). Crystal structure
determination (19) and conformational studies of MER 25 (20) were performed to
identify the preferred conformation of the molecule in various energy states. Molec-

NOVEL ANTIESTROGEN ANALOGS OF MER 25
17
FIG. 1. Newman projection formulas of MER 25.
ular mechanics calculations and steric energy profile searches identified three low-
energy conformers: anti, gauche1 , and gauche2 (Fig. 1), with the anti conformation
as the global minimum. The second-lowest energy conformer of MER 25 is the
gauche1 conformation, which closely resembles the antiestrogenic Z isomer of ta-
moxifen. Difference decoupling and NOE NMR studies of MER 25 in CD3OD at
temperatures ranging from Ϫ60 to 50ЊC at 300 MHz likewise demonstrated that
the molecule existed in the anti conformation. Since the molecular mechanics calcu-
lations have shown that the gauche conformation is the second most stable con-
former of MER 25, it is possible that the anti conformation could be perturbed
into the gauche conformation when approaching and attaching to the receptor,
thereby producing antiestrogenic action.
Previous reports from our laboratory (3, 6, 21) have demonstrated that the
introduction of a dichlorocyclopropyl moiety in place of an olefinic double bond
prevents the isomerization seen with the TPE antiestrogens. The incorporation of
this moiety into a structure resembling MER 25 provided diastereomers which
effectively fixed the anti and gauche conformations of the molecule into nonisomeri-
zable Z and E diastereomers. The biological activity of each diastereomer was
evaluated to discern which isomer was associated with the antiestrogenic activity.
The p-methoxy group in the ͱ ring of MER 25 distinguishes the antiestrogen
from the many tamoxifen-related compounds, which possess various substitutions
only on the Ͱ and ͰЈ rings. In addition to the stereochemistry of MER 25, the
p-methoxy group was of interest to us in the development of more effective antiestro-
gens. Computer-aided molecular studies (20) revealed that the ͱ ring of MER 25
was directed toward the D ring in estradiol such that the p-methoxyphenyl group
was in the vicinity of the 17-ͱ-hydroxy group when MER 25 was in the gauche1
conformer, but not when in the anti conformer (Fig. 1). To evaluate the effect of
the ͱ-ring p-methoxy group on antiestrogenic activity, two compounds previously
synthesized in our laboratory, 7A(Z) and 7A(E) (Fig. 2), which resemble 11Z and
11E (Fig. 2), but without the methoxy group, were evaluated in the in vitro biological

18
OVERACRE AND MAGARIAN
FIG. 2. MRL 37, 7A(Z), 7A(E), and gem-dichlorocyclopropyl analogs of MER 25.
assays with the test compounds. Comparison of the biological activity between Z
and E isomers of compounds, with and without the p-methoxy group in the ͱ ring,
should demonstrate the influence of this moiety on biological activity.
Inclusion of the dichlorocyclopropyl group into the structure provided diastereo-
mers without the ethanolic hydroxyl group found in MER 25. To determine the
importance of the OH group in MER 25, des-hydroxy MER 25 (Fig. 2), also known
as MRL 37, was prepared and tested in conjunction with MER 25 to confirm if
its previously reported in vivo antiestrogenic activity was comparable to an in
vitro system.
This report describes the synthesis and biological evaluation of four cyclopropyl
analogs of MER 25. Two structures have the ͰЈ and ͱ rings in the E (11E) and Z
(11Z) conformation, and two have a heptafluorotolyl ether substituent in the
4-position of the Ͱ ring in the E (18E) and Z (18Z) analogs of MER 25. A fifth
compound known as MRL 37 (des-hydroxy MER 25) was included in this study.
Their in vitro antiestrogenic effects in the ER-positive MCF-7E3 and ER-negative
MDA-MB-231 human breast cancer cells are reported.
EXPERIMENTAL
Materials and Methods
The melting points were determined in open capillary tubes with a Thomas–
Hoover capillary melting-point apparatus and are uncorrected. Elemental analyses
were performed by Midwest Microlab Ltd. (Indianapolis, IN). The structures of all

NOVEL ANTIESTROGEN ANALOGS OF MER 25
19
compounds were supported by their proton NMR spectra of intermediate com-
pounds obtained with a Varian EM-360A spectrometer; spectra of final compounds
were recorded on a Varian XL-300 spectrometer. The chemical shifts are reported
in parts per million (ͳ) with reference to tetramethylsilane as the internal standard.
All starting reagents were used without further purification and were obtained from
Aldrich Chemical Co. (Milwaukee, WI 53233), with the exception of octafluoroto-
luene, which was purchased from PCR, Inc. (Gainesville, FL 32602). Solvents were
either HPLC or ACS grade and were obtained from Fisher Scientific (Fair Lawn,
NJ 07410).
Synthesis
4-Methoxydesoxy benzoin (6). Aluminum chloride (85.66 g, 0.642 mol) and dry
benzene (60.0 ml) were anhydrously combined at 0ЊC, followed by the dropwise
addition of p-methoxyphenylacetyl chloride (5) (53.94 g, 0.292 mol) [1H NMR
(CDCl3 , 60 MHz) ͳ 3.74 (s, 3H, OCH3), 4.02 (s, 2H, ArCH2), 6.88 (d, J ϭ 8 Hz,
2H, ArH (m to OCH3)), 7.18 (d, J ϭ 8 Hz, 2H, ArH (o to OCH3))] dissolved in
30.0 ml benzene. The reaction was then allowed to sit at room temperature overnight
(14 h). The reaction vessel was subsequently cooled on ice and 100 ml H2O was
added. Complete dissociation of the AlCl3 (detectable by the change in color of
the reaction mixture from dark red to yellow) was accomplished by the dropwise
addition of concentrated HCl. The mixture was transferred to a separation funnel
and the organic phase was extracted with three 50-ml portions of benzene. The
combined organic layers were washed with 10% sodium hydroxide (to remove any
unreacted acid chloride) and dried over magnesium sulfate. Following filtration of
the drying agent and removal of the solvent, 20.0 g of yellow-brown solid was
obtained. The crude ketone was purified via column chromatography (silica gel;
CH2Cl2) and recrystallized with petroleum ether: CH2Cl2 to yield 53.91 g (82%) of
off-white solid, mp ϭ 92.5–93.5ЊC. 1H NMR (CDCl3 , 60 MHz) ͳ 3.80 (s, 3H, OCH3),
4.23 (s, 2H, ArCH2), 6.96 (d, J ϭ 8 Hz, 2H, ArH (m to OCH3)), 7.30 (d, J ϭ 8 Hz,
2H, ArH (o to OCH3)), d 7.51 (m, 3H, ArH (m to CuO)), d 8.05 (m, 2H, ArH
(o to CuO)).
(Z and E)-1-͕4-[2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenoxy]phenyl͖-1-phenyl-
2-(4-methoxyphenyl)ethene (7). A modified version of the procedure used by
McCague (22) was employed. Magnesium (3.76 g, 154.61 mmol), 4-(perfluorotoly-
loxy)phenyl bromide (2) (40.1 g, 103.07 mmol) [1H NMR (CDCl3 , 60 MHz) ͳ 6.91
(d, J ϭ 8 Hz, 2H, ArH (o to Br)), 7.53 (d, J ϭ 8 Hz, 2H, ArH (m to Br))], and
100 ml of THF (freshly distilled from Ca2H) were combined. Dibromoethane (9.68
g, 51.54 mmol) dissolved in 50 ml THF was then added dropwise over a 4-h period.
Following the complete addition of the dibromoethane and the disappearance of
the magnesium, 6, dissolved in 50 ml THF, was injected into the reaction. The
reaction was stirred at room temperature for 60 h. The reaction mixture was subse-
quently transferred to a separatory funnel and 100 ml of 0.5 M HCl was added.
The organic phase was removed and the aqueous phase was extracted with three
25-ml portions of Et2O. The combined organic layers were dried over MgSO4 and
filtered, and the solvent was removed, yielding 28.43 g of brown oil. The oil was

20
OVERACRE AND MAGARIAN
dissolved in 125 ml benzene, and p-toluenesulfonic acid (1.14 g, 5.99 mmol) was
added. The mixture was refluxed for 18 h. Upon cooling to room temperature, 100
ml of H2O was added to the reaction vessel and the solution was transferred to a
separatory funnel. The aqueous phase was removed and the organic phase was
extracted with three 50-ml portions of benzene. The combined benzene layers were
dried over MgSO4 and filtered, and the solvent was removed, yielding 27.3 g of
brown oil. The oil was purified via column chromatography (silica gel, 1 : 1 petroleum
ether : CH2Cl2) to give 11.20 g (78%) of yellow oil.
Crystallization of the oil from petroleum ether resulted in 1.10 g 7E (mp ϭ
108.0–109.5ЊC from petroleum ether 40–60ЊC). 1H NMR (CDCl3 , 300 MHz) ͳ 3.77
(s, 3H, OCH3), d 6.70 (d, J ϭ 4.4 Hz, 2H, ArH (o to OCH3)), d 6.92–6.97 (m, 4H,
ArH), d 7.00 (s, 1H, olefinic H), d 7.19–7.29 (m, 7H, ArH). Crystallization of the
mother liquor with isopropanol/ether provided 932 mg 7Z (mp ϭ 91.5–93.0ЊC). 1H
NMR (CDCl3 , 300 MHz) ͳ 3.75 (s, 3H, OCH3), 6.67 (d, J ϭ 8.7 Hz, 2H, ArH (o
to OCH3)), 6.87 (s, 1H, olefinic H), 6.93–6.96 (m, 4H, ArH), 7.19–7.36 (m, 7H, ArH).
(Z and E)-1-[4-bromoethoxyphenyl]-1-phenyl-2-(4-methoxyphenyl)ethene (9).
Compound 7E (932 mg, 1.80 mmol) in 20 ml dry DMF was anhydrously combined
with sodium methoxide (1.07 g, 19.78 mmol). The reaction was stirred at room
temperature for 16 h. The reaction mixture was transferred to a separatory funnel
and 50 ml of ether and 50 ml of saturated NaHCO3 were added. The organic
phase was extracted with two additional 50-ml portions of saturated NaHCO3 . The
combined organic layers were dried over MgSO4 and filtered, and the solvent was
removed, yielding a yellow oil. The oil was subjected to the identical reaction
conditions (the reaction was stirred for only 2 h) and workup procedure a second
time (the reaction produces an intermediate product which requires the duplication
of the process to obtain the desired product). Purification of the oil was accomplished
with column chromatography (silica gel, 1 : 1 petroleum ether : CH2Cl2 to 100%
CH2Cl2). A total of 322 mg (49%) of 8E was obtained as a clear oil. Due to the
instability of phenolic moiety, the oil was immediately subjected to alkylation. To
a 50-ml round-bottom flask equipped with a reflux condenser and stir bar was added
8E (322 mg, 1.06 mmol), dibromoethane (15.26 g, 81.23 mmol), triethylbenzylam-
monium chloride (TEBA) (34 mg, 0.15 mmol), and 7 ml of 10% NaOH. The reaction
was heated at 45ЊC for 16 h and was subsequently cooled to room temperature.
The reaction mixture was transferred to a separatory funnel and 20 ml CH2Cl2 and
20 ml water were added. The organic phase was removed and the aqueous phase
was extracted with three 20-ml portions of CH2Cl2 . The combined organic layers
were dried over MgSO4 and filtered, and the low boiling solvents were removed.
The excess dibromoethane was removed under vacuum (6 mm Hg). Purification of
the resulting yellow oil was accomplished with column chromatography (silica gel,
1 : 1 petroleum ether : CH2Cl2). A total of 332 mg (76%) of 9E was obtained as a
1
colorless oil. H NMR (CDCl3 , 300 MHz) ͳ 3.70 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.79
(s, 3H, OCH3), 4.35 (t, J ϭ 6.3 Hz, 2H, OCH2), 6.71–7.34 (m, 13 H, ArH), 6.89 (s,
1H, olefinic H).
Compounds 8Z and 9Z were generated in a manner analogous to those used for
the E isomers. Compound 7E (1.10 g, 2.12 mmol) was reacted two times with
NaOMe (1.26 g, 23.34 mmol) in 20 ml dry THF. Following standard workup and

NOVEL ANTIESTROGEN ANALOGS OF MER 25
21
purification, 316 mg (49%) of 8Z was obtained as a clear oil. 8Z (316 mg, 1.05
mmol) was reacted with dibromoethane (13.1 g, 69.62 mmol) and TEBA (30 mg,
0.132 mmol) in 6 ml of 10% NaOH. Following workup and purification, a total of
1
324 mg (76%) of 9Z was obtained as a colorless oil. H NMR (CDCl3 , 300 MHz)
ͳ 3.64 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.74 (s, 3H, OCH3), 4.29 (t, J ϭ 6.3 Hz, 2H,
OCH2), 6.65–7.85 (m, 13 H, ArH), 6.83 (s, 1H, olefinic H).
(Z and E)-1,1,-dichloro-2-[4-(2-bromoethoxy)phenyl]-2-phenyl-3-(4-methoxyphe-
nyl)cyclopropane (10). Compound 9E (332 mg, 0.811 mmol), TEBA (32 mg, 0.140
mmol), 7 ml CHCl3 , and 5 ml 50% NaOH were combined. The reaction was stirred
at room temperature for 72 h. The reaction mixture was transferred to a separatory
funnel and 20 ml water and 20 ml CH2Cl2 were added. The organic phase was
removed and the aqueous layer was extracted with three 20-ml portions of CH2Cl2 .
The combined organic layers were dried over MgSO4 and filtered, and the solvent
was removed, yielding a brown oil. The oil was purified via column chromatography
(silica gel, 1 : 1 petroleum ether : CH2Cl2). A total of 271 mg (68%) of 10E was
obtained as a colorless oil. 1H NMR (CDCl3 , 300 MHz) ͳ 3.52 (s, 1H, cyclopropyl
H), 3.63 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.82 (s, 3H, OCH3), 4.25 (t, J ϭ 6.3 Hz, 2H,
OCH2), 6.79–7.53 (m, 13H, ArH). The same procedure was employed for 10Z.
Compound 9Z (324 mg, 0.792 mmol) was reacted with TEBA (32 mg, 0.140 mmol),
7 ml CHCl3 , and 5 ml 50% NaOH. Following workup and purification, a total of
191 mg (49%) was obtained. 1H NMR (CDCl3 , 300 MHz) ͳ 3.51 (s, 1H, cyclopropyl
H), 3.62 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.8 (s, 3H, OCH3), 4.24 (t, J ϭ 6.3 Hz, 2H,
OCH2), 6.78–7.50 (m, 13H, ArH).
(Z
and
E)-1,1-dichloro-2-[4-(2-diethylaminoethoxy)phenyl]-2-phenyl-3-(4-
methoxyphenyl)cyclopropane (11). Compound 10E (260 mg, 0.528 mmol) was re-
acted with diethylamine (0.35 g, 4.83 mmol) in 5 ml acetonitrile. The reaction was
stirred at room temperature for 16 h. The solvent was subsequently removed and
the resulting orange semisolid was dissolved in 5 ml ether and 5 ml saturated sodium
bicarbonate. The mixture was transferred to a separatory funnel, the aqueous
phase was removed, and the ether layer was extracted with three 25-ml portions
of saturated NaHCO3 . The organic layer was dried over NaSO4 and filtered, and
the solvent was removed, yielding an orange-brown oil. The oil was purified via
column chromatography (silica gel, 9 : 1 petroleum ether : ether). A total of 121 mg
1
(47%) of 11E was collected as a clear oil. H NMR (CDCl3 , 300 MHz) ͳ 1.13 (t,
J ϭ 7.2 Hz, 6H, NCH2CH3), 2.73 (q, J ϭ 7.2 Hz, 4H, NCH2CH3), 2.95 (t, 2H, J ϭ
6.3 Hz, 2H, CH2N), 3.51 (s, 1H, cyclopropyl H), 3.81 (s, 3H, OCH3), 4.08 (t, J ϭ
6.3 Hz, 2H, OCH2), 6.78–7.52 (m, 13H, ArH).
The same procedure was employed to obtain 11Z. Compound 10Z (185 mg, 0.376
mmol) was combined with diethylamine (0.35 g, 4.83 mmol) in 5 ml of acetonitrile
and was stirred for 16 h at room temperature. Following an identical workup
procedure and purification process, a total of 75 mg (41%) 11E was obtained as a
light yellow oil. 1H NMR (CDCl3 , 300 MHz) ͳ 1.09 (t, J ϭ 7.2 Hz, 6H, NCH2CH3),
2.66 (q, J ϭ 7.2 Hz, 4H, NCH2CH3), 2.88 (t, 2H, J ϭ 6.3 Hz, 2H, CH2N), 3.51 (s,
1H, cyclopropyl H), 3.81 (s, 3H, OCH3), 4.04 (t, J ϭ 6.3 Hz, 2H, OCH2), 6.77–7.45
(m, 13H, ArH).
(Z and E)-1,1-dichloro-2-[4-(2-diethylaminoethoxy)phenyl]-2-phenyl-3-(4-me-

22
OVERACRE AND MAGARIAN
thoxyphenyl)cyclopropane citrate salt (11). To the free base of 11E (96 mg, 0.200
mmol) was added 0.5 ml isopropanol. Anhydrous citric acid (38 mg, 0.200 mmol)
was likewise dissolved in 0.5 ml isopropanol. The two solutions were combined and
stirred at 42ЊC for 30 min. The isopropanol was subsequently removed and the
resulting oil was treated with 3 ml ether. The citrate salt of 11E fell out of solution
as a white solid and was recrystallized from isopropanol : ether to yield 100 mg
(74%), mp ϭ 86.5–87.5ЊC. C34H39Cl2NO9 (Found: C 59.87, H 5.94, N 2.07. Calcd:
C 60.36, H 5.81, N 2.07). The citrate salt of 11Z (50 mg, 0.103 mmol) was obtained
in a similar manner using citric acid (20 mg, 0.103 mmol) in isopropanol. Following
recrystallization with isopropanol : ether, 48 mg (69%) of 11Z citrate salt, mp ϭ
91.0–92.0ЊC. C34H39Cl2NO9 (Found: C 60.16, H 5.94, N 2.12. Calcd: C 60.36, H 5.81,
N 2.07).
4-Bromoethoxy-4Ј-methoxybenzyl ketone (13). A modified version of the proce-
dure used by McCague (22) was followed. 4-Methoxyphenylacetic acid (11.2 g,
67.5 mmol) and trifluoroacetic anhydride (15.1 g, 72.0 mmol) were combined. The
reaction was stirred at room temperature until the acid completely dissolved.
ͱ-Bromophenetole (15.0 g, 75 mmol) was subsequently added in one portion. The
reaction was stirred overnight (16 h) at 20ЊC. The reaction underwent several
color changes from yellow to magenta and eventually to brown (at which point a
precipitate had formed). Water (20 ml) and CH2Cl2 (20 ml) were added to the
reaction vessel and the mixture was transferred to a separatory funnel. The organic
phase was extracted with three 50-ml portions of 10% NaHCO3 . The combined
organic phases were dried over MgSO4 and filtered, and the solvent was removed,
yielding a red-brown solid. The addition of 50 ml ether to the solid resulted in the
production of 16.97 g (72%) of the desired compound as a white solid, mp ϭ
129.0–130.5ЊC (from EtOAC : petroleum ether). 1H NMR (CDCl3 , 60 MHz) ͳ 3.51
(t, J ϭ 6 Hz, 2H, CH2Br), 4.11 (s, 3H, OCH3), 4.29 (t, J ϭ 6 Hz, 2H, OCH2),
6.72–7.18 (m, 6H, ArH), 7.96 (d, J ϭ 6, Hz, 2H, o to CuO).
(Z and E)-1-͕4-[2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenoxy]phenyl͖-1-[4-(2-
bromoethoxy)phenyl-2-(4-methoxyphenyl)ethene (16). A modified version of the
procedure used by McCague (22) was followed. Magnesium (3.84 g, 157.94 mmol),
2 (40.72 g, 104.66 mmol), and 100 ml of THF (freshly distilled from Ca2H) were
combined, and dibromoethane (9.8 g, 52.23 mmol) dissolved in 50 ml THF was
then added dropwise over a 4-h period. Following the complete addition of the
dibromoethane and the disappearance of the magnesium, 13, dissolved in 50 ml
THF, was injected into the reaction. The reaction was stirred at room temperature
for 60 h. The reaction mixture was subsequently transferred to a separatory funnel
and 100 ml of 0.5 M HCl was added. The organic phase was removed and the
aqueous phase was extracted with three 25-ml portions of Et2O. The combined
organic layers were dried over MgSO4 and filtered, and the solvent was removed,
yielding 15.26 g of brown oil. The oil was dissolved in 125 ml benzene, and
p-toluenesulfonic acid (0.763 g, 4.00 mmol) was added. The mixture was refluxed
for 18 h. Upon cooling to room temperature, 100 ml of H2O was added to the
reaction vessel and the solution was transferred to a separatory funnel. The organic
phase was removed and the aqueous phase was extracted with three 50-ml portions
of benzene. The combined benzene layers were dried over MgSO4 and filtered, and

NOVEL ANTIESTROGEN ANALOGS OF MER 25
23
the solvent was removed, yielding 14.69 g of brown oil. The oil was purified via
column chromatography (silica gel, 1 : 1 petroleum ether : CH2Cl2) to give 8.99 g
(27%) of yellow oil. Fractional crystallization of Z/E 16 with petroleum ether gave
562 mg of the E isomer as white crystals, mp ϭ 132.5–133.5ЊC (from petroleum
1
ether 40–60ЊC). H NMR (CDCl3 , 300 MHz) ͳ 3.70 (t, J ϭ 6.3 Hz, 2H, CH2Br),
3.79 (s, 3H, OCH3), 4.35 (t, J ϭ 6.3 Hz, 2H, OCH2), 6.71–7.32 (m, 12 H, ArH),
6.84 (s, 1H, olefinic H). From isopropanol : ether gave 717 mg of the Z isomer as
1
white crystals, mp ϭ 120.0–121.5ЊC (from petroleum ether 40–60ЊC). H NMR
(CDCl3 , 300 MHz) ͳ 3.67 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.79 (s, 3H, OCH3), 4.33 (t,
J ϭ 6.3 Hz, 2H, OCH2), 6.70–7.29 (m, 12H, ArH), 6.87 (s, 1H, olefinic H).
(Z and E)-1,1-dichloro-2-[4-[2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyloxy]-
phenyl͖-2-[4-(2-bromoethoxy)phenyl]-3-(4-methoxyphenyl)cyclopropane
(17).
Compound 16Z (562 mg, 0.876 mmol) was reacted with TEBA (56 mg, 0.25 mmol),
7 ml CHCl3 , and 5 ml 50% NaOH. The reaction was stirred at room temperature
for 72 h. The reaction mixture was transferred to a separatory funnel and 20 ml
water and 20 ml CH2Cl2 were added. The organic phase was removed and the
aqueous layer was extracted with three 10-ml portions of CH2Cl2 . The combined
organic layers were dried over MgSO4 and filtered, and the solvent was removed,
yielding a brown oil. The oil was purified via column chromatography (silica gel,
2 : 1 petroleum ether : CH2Cl2). A total of 455 mg (72%) of 17Z was obtained as a
1
clear oil. H NMR (CDCl3 , 300 MHz) ͳ 3.48 (s, 1H, cyclopropyl H), 3.64 (t, J ϭ
6.3 Hz, 2H, CH2Br), 3.82 (s, 3H, OCH3), 4.27 (t, J ϭ 6.3 Hz, 2H, OCH2), 6.79–7.50
(m, 12H, ArH).
An identical procedure was used to generate 17E. Compound 16E (717 mg, 1.12
mmol) was combined with TEBA (72 mg, 0.32 mmol) in 9 ml CHCl3 and 7 ml 50%
NaOH. The reaction was stirred at room temperature for 72 h and was isolated
and purified according to the procedure used for 17E. A total of 454 mg (56%) of
17E was collected. 1H NMR (CDCl3 , 300 MHz) ͳ 3.52 (s, 1H, cyclopropyl H), 3.64
(t, J ϭ 6 Hz, 2H, CH2Br), 3.82 (s, 3H, OCH3), 4.28 (t, J ϭ 6 Hz, 2H, OCH2),
6.80–7.51 (m, 12 H, ArH).
(Z and E)-1,1-dichloro-2-͕4-[2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyloxy]-
phenyl͖ - 2 - [4 -(2 - diethylaminoethoxy)phenyl] - 3 - (4-methoxyphenyl)cyclopropane
(18). Compound 17Z (450 mg, 0.621 mmol), diethylamine (0.34 g, 4.83 mmol), and
5 ml acetonitrile were combined. The reaction was stirred at room temperature for
16 h. The solvents were subsequently removed and the resulting orange semisolid
was dissolved in 5 ml ether and 5 ml water. The mixture was transferred to a
separatory funnel and the aqueous phase was removed. The organic phase was
extracted with three 10-ml portions of saturated sodium bicarbonate. The ether
layer was dried over magnesium sulfate and filtered, and the solvent was removed,
yielding a light yellow oil. The oil was purified by column chromatography (silica
gel, 1 : 1 petroleum ether : ether). A total of 150 mg (34%) of 18Z was collected,
mp ϭ 38.5–40.0ЊC. 1H NMR (CDCl3 , 300 MHz) ͳ 1.10 (t, J ϭ 7.2 Hz, 6H, NCH2CH3),
2.68 (q, J ϭ 7.2 Hz, 4H, NCH2CH3), 2.90 (t, J ϭ 6 Hz, 2H, CH2N), 3.47 (s, 1H,
cyclopropyl H), 3.81 (s, 3H, OCH3), 4.05 (t, J ϭ 6 Hz, 2H OCH2), 6.79–7.51 (m,
12 H, ArH). C35H30Cl2F7NO3 (Found: C 58.69, H 4.39, N 1.98. Calcd: C 58.67, H
4.22, N 1.96).

24
OVERACRE AND MAGARIAN
The same procedure was employed in generating 18E. Compound 17E (450 mg,
0.621 mmol), diethylamine (0.34 g, 4.83 mmol), and 5 ml accetonitrile were com-
bined. The reaction was stirred at room temperature for 16 h. Following standard
workup and purification, a total of 64 mg (15%) of 18E were collected, mp ϭ
1
39.5–41.5ЊC. H NMR (CDCl3 , 300 MHz) ͳ 1.11 (t, J ϭ 7.2 Hz, 6H, NCH2CH3),
2.70 (q, J ϭ 7.2 Hz, 4H, NCH2CH3), 2.92 (t, J ϭ 6 Hz, 2H, CH2N), 3.51 (s, 1H,
cyclopropyl H), 3.82 (s, 3H, OCH3), 4.08 (t, J ϭ 6 Hz, 2H OCH2), 6.79–7.42 (m,
12 H, ArH). C35H30Cl2F7NO3 (Found: C 58.62, H 4.22, N 1.96. Calcd: C 58.67, H
4.22, N 1.96).
Z and E-1-[4-(2-bromoethoxy)phenyl]-1-phenyl-2-[4-methoxyphenyl]ethene (20).
Compound 13 (5 g, 14.32 mmol) was dissolved in 100 ml of dry THF, and 3.0 M
phenylmagnesium bromide solution (in ether) (8.1 g, 44.46 mmol) in 100 ml THF
was added dropwise over a 1-h period at 0ЊC. Following completion of addition,
the temperature was brought to and maintained at reflux for 40 h. The reaction
was subsequently cooled to room temperature and 100 ml of 0.5 M HCl was added.
The reaction mixture was transferred to a separatory funnel and the aqueous phase
was extracted with three 75-ml portions of diethyl ether. The combined organic
phases were dried over MgSO4 and filtered, and the solvent was removed, yielding
11.6 g of the intermediate carbinol as a dark brown oil. The oil was dissolved in
125 ml benzene and was combined with 0.57 g of p-toluenesulfonic acid. The reaction
was refluxed for 16 h. Following completion of the dehydration reaction, the mixture
was cooled to room temperature and was transferred to a separatory funnel. The
organic layer was removed and the aqueous layer was extracted with three 100-ml
portions of benzene. The combined benzene layers were dried over MgSO4 and
filtered, and the solvent was removed, yielding a dark brown oil. The oil was purified
via column chromatography (silica gel, 1 : 1 petroleum ether : methylene chloride).
A total of 3.86 g (63%) of light yellow oil was obtained. 1H NMR (CDCl3 , 60 MHz)
ͳ 3.67 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.78 (s, 3H, OCH3), 4.32 (t, J ϭ 6.3 Hz, 2H,
OCH2), 6.88–7.34 (m, 14H, ArH and olefinic H).
1-[4-(2-Bromoethoxy)phenyl]-1-phenyl-2-[4-methoxyphenyl]ethane (21). Com-
pound 20 (1.06 g, 2.59 mmol) was combined with 10% Pd/C (100 mg) in 20 ml dry
THF. Following a thorough flushing of the reaction vessel with nitrogen, hydrogen
gas was bubbled through the mixture periodically over 4 h. The reaction vessel was
sealed and was stirred at room temperature for 18 h. The catalyst was subsequently
removed by filtration through Celite and the solvent was evaporated, leaving 878
mg of pale yellow oil. Purification of the oil by column chromatography (silica gel,
2 : 1 petroleum ether : CH2Cl2) resulted in 450 mg (42%) of 21 as a white solid,
mp ϭ 59.0–60.5ЊC. 1H NMR (CDCl3 , 60 MHz) ͳ 3.25 (d, J ϭ 3.6 Hz, 2H Ar–CH2),
3.56 (t, J ϭ 6.3 Hz, 2H, CH2Br), 3.70 (s, 3H, OCH3), 4.2 (overlapping t, 4H, Ar–CH
and CH2O), 6.60–7.20 (m, 13H, ArH).
1-[4-(2-Diethylaminoethoxy)phenyl]-1-phenyl-2-[4-methoxyphenyl]ethane (22).
Compound 21 (450 mg, 0.479 mmol), diethylamine (2.88 g, 39.38 mmol), and 19 ml
of CHCl3 : CH3CN : isopropanol (3 : 5 : 5) were combined. The reaction was stirred
at 40ЊC for 16 h. The solvent was subsequently removed, leaving an orange-brown
solid. The solid was dissolved in 5 ml water and 5 ml ether and the mixture was
transferred to a separatory funnel. The aqueous layer was removed and the organic

NOVEL ANTIESTROGEN ANALOGS OF MER 25
25
layer was extracted with three 50-ml portions of 10% NaOH and one 50-ml portion
of H2O. The combined organic layers were dried over MgSO4 and filtered, and
the solvent was removed, yielding an orange oil. The oil was purified by column
chromatography (silica gel, 9 : 1 CHCl3 : methanol) and yielded 338 mg (77%) of 22
1
as an orange oil. H NMR (CDCl3 300 MHz) ͳ 1.11 (t, J ϭ 6 Hz, 6H, NCH2CH3),
2.70 (q, J ϭ 6 Hz, 4H, NCH2CH3), 2.92 (t, J ϭ 6.3 Hz, 2H, CH2–N), 3.29 (d, J ϭ
7.8 Hz, 2H, Ar–CH2), 3.77 (s, 3H, OCH3), 4.06 (t, J ϭ 6.3 Hz, 2H, OCH2), 4.15 (t,
J ϭ 7.8 Hz, 1H, Ar–CH), 6.72–7.27 (m, 13H, ArH).
The citrate salt of 22 was prepared by dissolving the oil (356 mg, 0.882 mmol)
in 1 ml of warm absolute ethanol and combining it with a commensurate, 1-ml
ethanolic solution of anhydrous citric acid (169 mg, 0.882 mmol). The two solutions
were combined and swirled in a water bath at 47ЊC for 20 min. The solvent was
removed and 5 ml ether was added to the residue. Upon placement of the residue
in the freezer, the oil solidified. The off-white solid was collected and recrystallized
from isopropanol/ether to provide 312 mg of the citrate salt of 22 (59%), mp ϭ
105.5–106.5ЊC. C33H41NO9 (Found: C 58.62, H 4.22, N 1.96. Calcd: C 58.67, H 4.22,
N 1.96).
Biological Evaluation
The biological evaluation of the test compounds consisted of the in vitro suppres-
sion of the proliferation of the ER-positive MCF-7E3 (23) and the ER-negative
MDA-MB-231 (24) human breast cancer cell lines. Estradiol (pure agonist), MER
25 (pure antagonist), MRL 37 (pure antagonist) 7A(Z)/7A(E), and ICI 182,780
(pure antagonist) were used as standards in both MCF-7E3 and MDA-MB-231
biological evaluations of test compounds.
Cell Culture
The hormone-dependent MCF-7E3 (ER-positive) human breast cancer cell line
used in these studies was obtained at passage 171 from Dr. Sam Brooks (Wayne
State University). Cell stocks were grown in improved minimum essential medium
(IMEM) modified (25) without phenol red (Biofluids, Rockville, MD), supple-
mented with 2 mM glutamine, 2 mM gentamicin, 5% (v/v) calf serum (GIBCO,
Grand Island, NY), and 0.01 nM 17ͱ-estradiol. Cells were removed from flasks by
disrupting the monolayer with media (20 ml) forced through a 7-in. 14-gauge can-
nula. Cell stocks were maintained in T75 Costar flasks in a humid atmosphere of
95% air/5% CO2 , at 37ЊC. All experiments were conducted on cells between passages
180 and 200.
The MDA-MB-231 cells, a hormone-independent (ER-negative) breast cancer
cell line, obtained from the American Type Tissue Culture Collection (Rockville,
MD), were also used in parallel experiments with the MCF-7E3 cell line. Cells
were maintained under the same conditions as were the MCF-7E3 cells with the
addition of 10% (in place of 5%) calf serum. All experiments were conducted on
cells between passages 40 and 50.

26
OVERACRE AND MAGARIAN
Hormones
17ͱ-Estradiol was obtained from Sigma, and ICI 182,780 was a gift from Zeneca
Pharmaceuticals (Cheshire, England). MER 25 was a gift from the Merrell Dow
Research Institute, Division of Merrell Dow Pharmaceuticals, Inc. (Cincinnati, OH).
All other compounds were synthesized in our laboratory. Stock solutions were
prepared in 95% ethanol and were diluted with cell culture medium. The final
ethanol concentration never exceeded 0.01%. Control cells received medium and
ethanol (0.01%) only.
Inhibition Studies
MCF-7E3 cells were harvested from stock flasks on day 0 and were plated into
Costar 24-well tissue culture plates (Corning 24-well tissue culture plates were used
previously for estrogen-responsive cells, but were found to release estrogen-like sub-
stances into the culture medium, which produced spurious results) containing 1 ml of
phenol red-free IMEM supplemented with 5% dextran-coated charcoal-stripped calf
serum (Sigma). The seeding density was dependent upon the cell line: 20 ϫ 103 cells/
well, MCF-7E3; 10 ϫ 103 cells/well, MDA-MB-231. Medium containing compounds
was added on day 2 and was replaced on days 4 and 6. On day 7, the medium was
removed and the cells were freeze-lysed according to the method of Rago (26). Pro-
pidium iodide was then added (100 Ȑl of 200 Ȑg/ml in 1.12% sodium citrate per well).
Following a 1-h incubation period, the arbitrary fluorescence units (AFUs) per well
were determined with a Cytofluor 2300 fluorescence measurement system (Milli-
pore). A standard curve of bovine thymus DNA (R2 ϭ 0.99) was used to convert
AFU/well to DNA/well. The MDA-MB-231 cells were washed three times with PBS
24 h prior to seeding and were placed into IMEM supplemented with 5% dextran-
coated charcoal-stripped calf serum (Sigma). Percentage of inhibition of control
growth was calculated as the ratio of the control mean (DNA/well) minus the treat-
ment group mean (DNA/well) to the control mean (DNA per well), multiplied by
100. The percentage of inhibition of estradiol-stimulated growth was calculated as
the ratio of the estradiol-stimulated (DNA/well) mean minus the compound plus
estradiol (DNA/well) mean to the estradiol-stimulated mean (DNA/well) minus the
control mean (DNA/well), multiplied by 100.
RESULTS
Chemistry
Coe and Scriven (27) reported the separation of the Z and E isomers of heptaflu-
orotoluene-protected ethene leading to 4-hydroxytamoxifen with column chroma-
tography, but this technique was not successful with the diastereomers of ethene
7. Although tedious and time consuming, fractional crystallization was employed
as the method of separation. Combinations of petroleum ether with several polar
solvents (methylene chloride, ether, acetonitrile, ethanol, isopropanol, and ethyl
acetate) were investigated as possible solvent systems before it was discovered that

NOVEL ANTIESTROGEN ANALOGS OF MER 25
27
the addition of petroleum ether alone to the 1 : 1 Z : E mixture of 7 (an oil) resulted
primarily in the precipitation of the E isomer. Removal of the petroleum ether
from the mother liquor followed by the addition of 95% ethanol resulted in the
precipitation of the enhanced Z isomer. Fractional crystallization only allowed for
10% separation of the individual isomers resulting from the Grignard reaction.
Assignment of the conformation of the Z or E isomers was made according to
NMR chemical shifts of the O-methylene protons in the bromoethoxy or diethylami-
noethoxy side chain (para position of the ͰЈ ring). It has been established in both
triphenylethene and triphenylcyclopropane compounds that the methylene protons
of the side chain (OCH2CH2R) appear up field for the E isomer relative to the Z
isomer due to shielding effects caused by the adjacent phenyl ring (22, 28).
Displacement of the bromine in the ͰЈ ring side chain was initially attempted
using conditions frequently employed in our laboratory: diethylamine in a 3 : 5 : 5
mixture of CHCl3 : CH3CN : isopropanol heated at 45ЊC. This procedure resulted
in the production of a rearranged product: a 1 : 1 ratio of cyclopropane to alternate
product was obtained with the E isomer and a 9 : 1 ratio was found with the Z
isomer. Isolation of a minute quantity of the unknown product via column chroma-
tography and subsequent NMR analysis documented the absence of the characteris-
tic methine proton of the cyclopropane (3.6 ppm) and the appearance of a new
singlet at 4.8 ppm. This change in spectral characteristics correlates well with pre-
viously observed (6) ring opening and subsequent indene formation. The reaction
procedure was consequently altered to prevent ring opening from occurring. Com-
pound 10 was stirred with diethylamine in acetonitrile at room temperature for 16
h. Although the yields were 10–20% lower than with the previous method, the
presence of the indene was not detected.
Removal of the heptafluorotolyl protecting group from 17Z and 17E was at-
tempted using the standard procedure of NaOME in DMF. The reaction produced
an assortment of highly colored compounds, none of which possessed the structural
features of the desired compounds. These results suggested that the phenolic analogs
(19Z and 19E) readily decompose following deprotection. Due to these circum-
stances, the penultimate compounds 18Z and 18E (Fig. 2) were subjected to in
vitro biological evaluation.
The reduction of ethene 20 was attempted with the aminoethoxy side chain
already in place since these types of reaction are known to proceed in the presence
of amino groups (29). None of the desired product was collected when this sequence
of reactions was employed. Consequently, reduction of the double bond was per-
formed with the dibromoethane side chain (p-position of the ͰЈ ring) followed by
displacement of the bromine with diethylamine.
Biological
The test compounds (11Z, 11E, 18Z, and 18E; Fig. 2) and standards (MER 25,
MRL 37, 7A(Z), 7A(E), and ICI 182,780) were evaluated for their effects on the
growth of the estrogen receptor-positive MCF-7E3 and estrogen receptor-negative
MDA-MB-231 human breast cancer cell lines. In the MCF-7E3 line, 17ͱ-estradiol
stimulated the cellular proliferation rate 6- to 14-fold over the control growth rate.

28
OVERACRE AND MAGARIAN
TABLE 1
Percentage of Inhibitiona of Estradiol-Stimulated Growth (Antiestrogenic Activity) by Compounds
11Z, 11E, 18Z, 18E, 7A(Z), 7A(E), MER 25, MRL 37, and ICI 182,780 in the MCF-7E3 Cell Line
Compound
ϩ 0.1 nM E2
0.01 nM
0.1 nM
1.0 nM
10 nM
100 nM
1.0 ȐM
11Z
11E
18Z
*
*
*
*
*
*
*
*
75.9
*
19.4
*
*
*
*
*
*
*
85.7
*
*
*
*
78.8
*
*
*
87.7
106.1
65.4
*
85.1
54.7
70.63
90.8
102.0
111.8
*
*
*
*
38.6
*
100.3
18E
7A(Z)
7A(E)
MER 25
MRL 37
ICI 182,780
*
*
102.0
*
105.3
100.3
*
104.3
*
106.9
(E2 stimulated) Ϫ (compound ϩ E2)
(E2 stimulated Ϫ control)
* Indicates no statistically significant inhibition (P Ͻ 0.05).
a Percentage of inhibition of E2-stimulated growth ϭ
100.
ͫ
ͬ
The compounds were tested in the MCF-7E3 cell line in combination with 17ͱ-
estradiol (0.1 nM) to determine their antiestrogenic activity (Table 1) and in the
absence of estradiol to identify any antitumor activity.
At 1.0 ȐM concentrations, statistically significant (P Ͻ 0.01) inhibition of estra-
diol-stimulated growth (antiestrogenic activity) was demonstrated (Table 1) by
compounds 11Z (87.7%), 11E (106%), 18Z (65.4%), 7A(Z) (85.1%), 7A(E) (54.7%),
and MRL 37 (90.8%). Inhibition of estradiol-stimulated growth at concentrations
lower than 1.0 ȐM were demonstrated by compounds 11E, MER 25, and ICI
182,780. Test compound 11E exhibited 19.4% inhibition at 0.1 nM and nearly
complete inhibition (111.8–78.8%) over a concentration range of 1.0 to 100 nM.
MER 25 produced 38.6% reduction in estrogen-stimulated growth at 1.0 nM concen-
tration and complete inhibition at 10 and 100 nM. ICI 182,780 completely inhibited
estrogen-stimulated growth from 0.1 nM to 1.0 ȐM.
Estrogenic activity was observed with two compounds, 18E and MER 25. Test
compound 18E demonstrated strictly estrogenic activity from 1.0 nM to 1.0 ȐM
concentrations. Interestingly, MER 25, which produced promising antiestrogenic
activity at lower concentration ranges, exhibited estrogenic activity at 100 nm and
1.0 ȐM concentrations.
In the MCF-7E3 cell line, no test compound or standard produced statistically
significant (P Ͻ 0.05) inhibition of control growth, which is indicative of antitumor
activity. None of the test compounds or standards were active in the MDA-MB-
231 cell line at the concentrations studied (0.01 nM–1.0 ȐM), indicating a lack of
antitumor activity, also.
DISCUSSION
Based on earlier studies, we predicted that there is probably only one conforma-
tion of MER 25 that is responsible for its antiestrogenic activity. The bioactive

NOVEL ANTIESTROGEN ANALOGS OF MER 25
29
conformer of MER 25 was thought to be the gauche1 (Fig. 1B), which acts at the
receptor, and not the anti conformer (Fig. 1A) even though nonbiological evidence
pointed to the E isomer. To test this hypothesis, two conformationally restricted
lead compounds (11Z and 11E; Fig. 2) were prepared as probes for antiestrogenic
activity, since 11Z resembles the gauche conformation of MER 25 and 11E resem-
bles the anti conformer. To prove this, the restricted cyclopropyl analog 11Z was
biologically evaluated against the rigid anti conformer of MER 25, 11E. The analogs
were compared for their growth-inhibitory activity on the hormone-responsive
MCF-7E3 and hormone-unresponsive MDA-MB-231 human breast cancer cell
lines.
Analogs 11Z and 11E were expected to exhibit antiestrogenic (antagonist) proper-
ties devoid of estrogenic (agonist) activity, based on MER 25 being a pure antiestro-
gen in vivo in multiple species. However, there have been reports which indicate
MER 25 is estrogenic at high doses (30, 31), which tends to indicate that MER 25
has both high- and low-affinity binding sites on the receptor. The results from the
MCF-7E3 testing clearly demonstrated: (i) antiestrogenic activity of MER 25 (Table
1) at concentrations of 1.0 nM–1.0 ȐM and estrogenic activity at concentrations of
100 nM and 1.0 ȐM and (ii) antiestrogenic activity of the cyclopropyl analogs which
inhibited estrogen-stimulated growth (Table 1) without an estrogen agonist effect.
Diastereomer 11E was found to have a more potent inhibition of estrogen-stimu-
lated growth than 11Z over a range of concentrations (Table 1), which indicates
that it is, indeed, the anti conformer and not the gauche1 conformer that is active.
The des-hydroxy MER 25 (Fig. 2), also known as MRL 37, was prepared and
tested along with MER 25 to determine effectiveness of the ethanolic hydroxyl
group in antiestrogenic activity. MER 25 was a potent antiestrogen over a concentra-
tion range of 1.0 nM–1.0 ȐM (Table 1). MRL 37 exhibited only antiestrogenic
activity at 1.0 ȐM concentration and was a more potent antiestrogen than MER
25 at that concentration. The absence of the hydroxyl moiety in the cyclopropyl
analogs of MER 25 (11Z and 11E; Fig. 2) was thought to have only a minimal
effect on activity. However, 11Z inhibited the estrogen-stimulated growth, without
estrogen agonist effect, only at 1.0 ȐM concentration. The E isomer demonstrated
greater antiestrogenic activity over a concentration range of 1.0 nM–1.0 ȐM (Table
1) and was the strongest antiestrogen of the test compounds. This finding tends to
indicate that the hydroxyl group of MER 25, while not a requirement for antiestro-
genic activity, may be beneficial, since MER 25 was more active than MRL 37 at
lower concentrations.
Earlier work (20) indicated that the presence of a 4-methoxy group in the ͱ ring,
as in 11E and 11Z, might increase their antiestrogenic activity relative to analogs
which are devoid of the methoxy moiety [7A(Z) and 7A(E)] (Fig. 2). In vitro
evaluation of the activity of 11Z and 11E against 7A(Z) and 7A(E), which lack
the 4-methoxy group, demonstrated inhibition of estradiol-stimulated growth by
both compounds at 1.0 ȐM concentration. The lack of statistically significant differ-
ence in the percentage of inhibition of estradiol-stimulated growth observed for
11(Z) (85.1%) and 7A(Z) (87.7%) indicates that the p-methoxy group in the ͱ
ring may not significantly influence antiestrogenic activity in the Z conformation.
However, the greater antiestrogenic activity exhibited by 11E over 7A(E) illustrates
the importance of the methoxy group in potentiating antiestrogenic activity in the

30
OVERACRE AND MAGARIAN
E isomer. It is possible that the positioning of the methoxy group of the ͱ ring in
the anti conformation allows for an interaction of the molecule with an area of the
estrogen receptor which elicits increased antiestrogenic activity.
The 4-hydroxy derivatives (19Z and 19E) of analogs 11Z and 11E were not
isolated due to their decomposition following the removal of the heptafluorotolyl
protecting group from 18Z and 18E (Fig. 2). After repeated attempts to isolate
19Z and 19E, it was decided to test the penultimate heptafluorotolyl isomers (18Z
and 18E; Fig. 2) to learn what effect a large group in the 4-position of the Ͱ ring
would have on activity. Furthermore, some enzymatic hydrolysis of the compounds
in the cell to yield the phenols 19Z and 19E might be possible. The in vitro system
is certainly not as efficient as an in vivo model for the production of metabolites
via enzymatic hydrolysis, and it was not surprising that the inhibition of estrogen-
stimulated growth was seen only at the highest concentration for 18Z (65% at 1.0
ȐM). The inefficient rate of hydrolysis is probably an important factor in the low
antiestrogenic potency of 18Z. In contrast, 18E exhibited estrogen agonist effects
over the control at 100 nM and 1.0 ȐM concentrations and potentiated the estrogen-
stimulated growth at the same concentrations. Its estrogenic activity could have
been directly related to the bulky heptafluorotolyl group on the positioning of
the E isomer in the estrogen receptor. Hence, 18Z is antiestrogenic, while 18E
is estrogenic.
The lack of inhibition of control growth (antitumor activity) in both the MCF-
7E3 and the MDA-MB-231 cell lines demonstrates that all of the cyclopropyl
analogs, which possess antiestrogenic activity, are devoid of any antitumor activity
regulated through a non-estrogen-receptor mechanism.
ACKNOWLEDGMENTS
This article was taken in part from the dissertation submitted by L.B.O. to the Graduate College for
the degree of Doctor of Philosophy. The authors thank the American Foundation for Pharmaceutical
Education for the support of L.B.O. through a Predoctoral Fellowship. MER 25 was a gift from Marion
Merrell Dow.
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