Synthesis and chiral separation of some antitumor agents

Article abstract of DOI:10.1006/bioo.1996.0009

Four Z-isomers of 1,1-dichloro-2,2,3-triarylcyclopropane (DTACs), designed as potent antitumor agents, were synthesized from their appropriately substituted ethenes, which were prepared from the Grignard reaction followed by the dehydration of their inter

Full text of DOI:10.1006/bioo.1996.0009

BIOORGANIC CHEMISTRY 24, 81–94 (1996)
ARTICLE NO. 0009
Synthesis and Chiral Separation of Some Antitumor Agents
1
2
SATENDRA
SINGH, KAREN L. MEYER
,
AND
ROBERT A. MAGARIAN
Department of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of
Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
Received June 19, 1995
Four Z-isomers of 1,1-dichloro-2,2,3-triarylcyclopropane (DTACs), designed as potent
antitumor agents, were synthesized from their appropriately substituted ethenes, which were
prepared from the Grignard reaction followed by the dehydration of their intermediate
carbinols. The stereospecific addition of dichlorocarbene to the ethenes followed by fractional
crystallization afforded (Z)-1,1-dichloro-2-(4-benzyloxyphenyl)-2-(4-methoxyphenyl)-3-
phenylcyclopropane
and
(Z)-1,1-dichloro-2,3-diphenyl-2-(4-methoxyphenyl)cyclopro-
pane. Displacement of the bromo group from the ethoxy side chain intermediates
with dimethylamine gave the desired basic side chain compounds, (Z)-1,1-dichloro-2,3-di-
phenyl-2-[4-(2-dimethylaminoethoxy)phenyl]cyclopropane and (Z)-1,1-dichloro-2-[4-(2-
dimethylaminoethoxy)phenyl]-2-(4-methoxyphenyl)-3-phenylcyclopropane.
While
both
E- and Z-stereoisomers of the DTACs were isolated using fractional crystallization, only
the Z-compounds were resolved on a chiral stationary phase consisting of amylose tris-3,5-
dimethylphenyl carbamate coated on silica gel. Complete resolution of the E-compounds was
not observed with this system.
1996 Academic Press, Inc.
INTRODUCTION
Breast cancer is one of the most common and serious diseases affecting women.
Current estimates indicate that one in eight American women who reach age 95
will develop breast cancer (1). It is generally believed that estrogen can directly
stimulate the growth of breast cancer, therefore, its inhibition with antiestrogens
at the estrogen receptor (ER) can provide a particularly useful strategy for the
treatment of hormone-dependent breast tumors in postmenopausal females and
possibly in premenopausal women and can act as a chemosuppressant in women
at high risk of developing breast cancer. Nonsteroidal antiestrogens which exhibit
potent antitumor effects represent a major advance in the management of breast
cancer. The representative of this class is tamoxifen, (Z)-1-[ p-(2-dimethylaminoeth-
oxy)phenyl]-1,2-diphenylbut-1-ene (Chart 1), the only antiestrogen clinically avail-
able for the adjunctive treatment of primary breast cancer in postmenopausal
women (2, 3). Its principal action is ascribed to its competition with natural hormone
estradiol (Chart 1) for binding to the estrogen receptor protein, thereby reducing
the ability of estradiol to stimulate nuclear transcription and consequent cell growth
1 Present address: Department of Botany and Microbiology, University of Oklahoma, Norman, OK
73019.
2 To whom correspondence should be addressed.
81
0045-2068/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

82
SINGH, MEYER, AND MAGARIAN
C
HART 1
(2). Tamoxifen, however, has partial agonist activity which can lead to thromboem-
bolitic events and secondary endometrial tumors, and virtually all patients with
metastatic disease develop tamoxifen resistance (4–6). Because of the partial agonist
activity of tamoxifen, researchers have been synthesizing both nonsteroidal and
steroidal antiestrogens in an attempt to find pure antiestrogens (those devoid of
any uterotropic activity) (7–12). The reasoning behind the search for pure antiestro-
gens is based on the recognition that antiestrogens generally possess partial estro-
genic activity and complete blockade of estrogen action cannot be achieved by
agents like tamoxifen. Novel pure antiestrogens should be more effective than
partial agonists in reducing the mitogenic action of estrogen on the growth of
breast tumors and complete ablation of hormonal-dependent tumor growth is very
desirable since it could provide a more rapid and longer-lasting remission. In addi-
tion, pure antiestrogens could prove more effective in patients who experience
relapse during tamoxifen therapy, serving as second-line treatment, and could dem-
C
HART 2

SYNTHESIS AND SEPARATION OF ANTITUMOR AGENTS
83
onstrate a greater efficacy in first-line treatment of advanced breast cancer (11,
13, 14).
The purpose of this earlier research was to search for antiestrogens that were
devoid of estrogen agonist activity. Efforts in this area have led to several interesting
experimental compounds (15–23). Reports from our laboratory (15, 16) and others
(20, 23) have demonstrated that the introduction of a dichlorocyclopropyl or dihy-
droclopropyl moiety in place of the olefinic bridge in estrogenic stilbenes greatly
reduces or abolishes their estrogenic action. One compound, analog II (Chart 1),
has antiestrogenic properties without estrogen agonist activity in the mouse and is
comparable in activity to tamoxifen against the hormone-dependent 7,12-dimethyl
benz[a]anthracene-induced rat mammary tumor model (24, 25). Structure–activity
relationship studies of some of the analog II derivatives (15–17, 26) led us to
design more effective cyclopropyl antiestrogens. Starting with analog II as the lead
compound, we synthesized a series Z-1,1-dichloro-2,2,3-triarylcyclopropanes (Z-
DTACs, 4–8) by introducing a third phenyl ring and a polar substituent in analog
II so that the compounds possess the structural features of both analog II and the
clinically useful Z-triarylethylenes (Chart 2). These Z-diastereomers were found to
be antiestrogenic without any estrogenic activity in the mouse and inhibited the
growth of ER positive MCF-7 human breast cancer cells in culture (19). As stereoiso-
mers often exhibit distinct bioactivities, separating the enantiomers was necessary
to fully evaluate the antitumor activity of the Z-DTACs. Unfortunately the synthetic
methods used to prepare these compounds did not yield substantial amounts of the
pure diastereomers required for further testing. In addition, unlike tamoxifen, the
DTAC stereoisomers consist of enantiomeric pairs. In this study, we report a
modified and improved synthetic method for the Z-DTACs, and the chiral separa-
tion of Z-isomers (4–7) into their (ϩ) and (Ϫ) enantiomers for biological evaluation.
The E-diastereomers of compounds 6 and 7 were also obtained for testing.
EXPERIMENTAL
Materials and Methods
Unless otherwise stated, all the starting materials were obtained from Aldrich
Chemical Co. (Milwaukee, WI) and were used without further purification. Aqueous
dimethylamine was obtained from Fluka Chemical Corp. (Ronkonkoma, NY).
HPLC grade solvents, which were purchased from Fisher Scientific (Houston, TX)
were filtered through a 0.45-Ȑm nylon-66 membrane filter (Ranin Instrument Co.,
Inc., Woburn, MA) and manually mixed by volume prior to use. Anhydrous THF
was freshly distilled from CaH2.
Melting points were determined on a Thomas Hoover capillary melting point
apparatus and are uncorrected. Elemental analysis was performed by Midwest
1
Microlab Ltd. (Indianapolis, IN). H NMR spectra were recorded on a Varian
EM-360A or XL-300 spectrometer. Ultraviolet-visible spectra were recorded on a
Shimadzu UV 160U spectrophotometer. Optical rotations were determined with a
Rudolph Autopol III autonomic polarimeter.

84
SINGH, MEYER, AND MAGARIAN
HPLC Conditions
A Beckman Gold HPLC system equipped with dual Model 110 (A and B) pumps
(only one pump was used) and 320SX controller, a 210A loop injector (20- and
500-Ȑl loops were used), a 163 variable wavelength uv detector, and an Analog
Interface Module 406 was used. Chiralpak AD columns, either analytical (250 ϫ
4.6 mm, i.d.) or semipreparative (250 ϫ 10 mm, i.d.) (Diacel Chemical Industries,
CA) were used for chiral separation. These columns consisted of amylose tris-3,5-
dimethylphenyl carbamate as chiral stationary phase coated on a 10-Ȑm silica gel.
A short nitrile guard column (5 Ȑm, 10 ϫ 3 mm, i.d.) (Regis Chemical Company, IL)
was used with both columns. The columns were maintained at ambient temperature
throughout the separation. Dead time was estimated with 1,3,5-tri-t-butylbenzene
(27).
Three solvent systems were used at a flow rate of 1 ml/min for analytical analysis.
Solvent system A consisted of hexane : ethanol (9 : 1; v/v), B consisted of hexane : 2-
propanol (9 : 1; v/v), and C consisted of hexane : 2-propanol : dimethylamine
(9 : 1 : 0.1; v/v). A volume of 5 Ȑl containing 1 mg/ml of compound in the same
solvent mixture was injected into the HPLC system. For semipreparative separation
the same solvent system (A, B, and C) were used at 2.4 ml/min flow rate. A volume
of 100–500 Ȑl containing 6–7 mg/ml of compound dissolved in either hexane : 2-
propanol (2 : 1 or 3 : 2) or hexane : chloroform (4 : 1) was injected.
Synthesis
Benzyl-4-benzyloxyphenyl ketone (10). The method of Iyer and Gopalachari (28)
was used to prepare ketone 10. A white solid crystallized from ethanol/benzene
yielded white needles (7.1 g, 81%), mp 134–136ЊC (lit. (28) 132ЊC). 1H NMR (CDCl3;
60 MHz): ͳ 4.24 (s, 2H, ArCH2O), 5.18 (s, 2H, CH2CO), 7.04 (d, 2H, Ar), 7.28–7.42
(overlapping s, 10H, ArH), 8.07 (d, 2H, ArH).
(Z/E)-1-(4-Benzyloxyphenyl)-1-(4-methoxyphenyl)-2-phenyl ethene (12). A solu-
tion of Grignard reagent was generated from 4-bromoanisole (11; 7.5 ml, 59.6 mmol)
and magnesium turnings (1.1 g, 43.7 mmol) in 280 ml of anhydrous THF which was
refluxed for 2 h. A solution of 10 (12.0 g, 39.7 mmol) in THF (60 ml) was added
and a gentle reflux was maintained for 16 h. The mixture was allowed to cool to
room temperature and a saturated solution of NH4Cl (12 ml) was added. The
mixture was transferred to a separatory funnel and ether (15 ml) and water (5 ml)
were added. The aqueous layer was removed and the ether layer was extracted
with water (3 ϫ 10 ml). The aqueous layers were combined and extracted with
ether (3 ϫ 15 ml). The combined ether layers were dried over anhydrous MgSO4
and filtered, and the solvent was removed under reduced pressure. Without further
purification, the intermediate carbinol was dehydrated with 2N H2SO4 (30 ml) in
95% ethanol (150 ml) at room temperature for 1 h. The mixture was then transferred
to a separatory funnel, extracted with ether (3 ϫ 15 ml), and the combined ether
layers were washed first with a saturated aqueous solution of sodium bicarbonate
followed by water until neutral. The organic layer was dried (anhydrous MgSO4),
filtered, concentrated, and purified over silica gel (pet ether/CH2Cl2, 6 : 1 to 1 : 1)
to yield 9.2 g (63%) of an oil. Fractional crystallization from pet ether/acetone gave

SYNTHESIS AND SEPARATION OF ANTITUMOR AGENTS
85
TABLE 1
Characterization of Z/E Isomers
1H NMR dataa,b
Cyclopropyl H OCH3 OCH2C6H5 OCH2CH2 OCH2CH2 NCH3
Comp.
No.
M.p.
(ЊC)
Isomer
4
5
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
153–156
134–136
128–130
116–120
92–96
113–115
128–129
114–120
111–112
112–114
120
3.53
3.53
3.53
3.53
3.54
3.52
3.50
3.52
3.52
3.53
3.49
3.52
3.79
3.78
3.75
3.76
5.02
5.03
24
25
6
4.23
4.24
4.22
4.25
4.02
4.05
4.01
4.04
3.63
3.59
3.60
3.62
2.69
2.75
2.69
2.72
3.76
3.79
2.31
2.34
2.31
2.33
7
3.76
3.77
108–111
a 1H NMR spectra were run on a Varian XL-300 spectrometer with TMS as internal standard in
CDCl3. The chemical shifts are given in ppm (ͳ).
1
b Compared to the H NMR of the X-ray structure of the (Z)-triarylcyclopropanes (37, 38).
1
E-isomer (2.21 g, 14%) mp 114–115.5ЊC. H NMR (CDCl3; 300 MHz): ͳ 3.85 (s,
3H, OCH3), 5.10 (s, 2H ArCH2O), 6.85 (s, 1H, vinyl H), 6.85–7.00 (m, 4H, ArH),
7.02–7.20 (m, 7H, ArH), 7.23–7.32 (m, 2H, ArH), 7.32–7.52 (m, 5H, ArH).
The Z-isomer was obtained from mother liquor by fractional crystallization from
1
pet ether/acetone (12; 3.41 g, 22%) mp 60–61ЊC. H NMR (CDCl3; 300 MHz): ͳ
3.86 (s, 3H, OCH3), 5.10 (s, 2H, ArCH2O), 6.86 (s, 1H, vinyl H), 6.87–7.00 (m, 4H,
ArH), 7.05–7.20 (m, 7H, ArH), 7.27–7.31 (m, 2H, ArH), 7.35–7.52 (m, 5H, ArH).
(Z)-1,1-Dichloro-2-(4-benzyloxyphenyl)-2-(4-methoxyphenyl)-3-phenylcyclo-
propane (4). The Z-ethane of 12 (3.2 g, 8.2 mmol) was stirred with 50% aq NaOH
solution (13 ml), benzyltriethylammonium chloride (TEBA, 0.3 g, 1.3 mmol) and
chloroform (40 ml) for 72 h at room temperature. The organic and aqueous layers
were separated and the aqeous layer was extracted three times with 25 ml of
dichloromethane. The combined organic layers were dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue was partially purified
over silica gel (pet ether/benzene, 1 : 1) and recrystallized from pet ether/ethyl
acetate to afford 1.9 g (49%) of the Z isomer as a white solid. The E-isomer of 4
was obtained in a similar manner from E-1-(4-benzyloxyphenyl)-1-(4-methoxyphe-
nyl)-2-phenyl ethene in 41% yield after being recrystallized from pet ether/ethanol.
1H NMR data are shown in Table 1.
(Z/E)-1-(4-Methoxyphenyl)-1,2-diphenyl ethene (16). Method A: A solution of
4-bromoanisole (11: 15.3 ml, 122 mmol) in THF (60 ml) was slowly added to a
flame-dried flask containing magnesium turnings (2.69 g, 112 mmol) and THF (65
ml). The Grignard reaction was facilitated by iodine crystals and heat. The mixture
was heated and refluxed for 2 h. Deoxybenzoin (13; 20.0 g, 102 mmol) in THF (65

86
SINGH, MEYER, AND MAGARIAN
ml) was then added and a gentle reflux was maintained for 5.5 h, cooled to room
temperature, and treated with a saturated aqueous NH4Cl solution (20 ml) for 30
min, then filtered and the filtrate extracted with ether (3 ϫ 25 ml). Removal of
solvent afforded an oil (8.0 g), which was dissolved in benzene (150 ml) and heated
to reflux for 2.5 h in the presence of p-TSA (1.5 g). The mixture was transferred
to a separatory funnel and extracted two times with 10 ml of saturated sodium
bicarbonate solution. The aqueous extracts were combined and washed with ben-
zene (3 ϫ 20 ml). The benzene layers were combined, dried (anhydrous MgSO4),
filtered, concentrated under vacuum, and the resulting oil was purified over silica
gel (pet ether/CH2Cl2, 1 : 0 to 7 : 1) to afford 23.0 g (79%) of the Z/E ethene mixture.
1H NMR (CDCl3; 60 MHz): ͳ 3.83 (s, 3H, OCH3), 6.70–7.40 (m, 15H, ArH and
vinyl H).
Method B: A solution of benzyl magnesium chloride (15; 2
M
solution, 32 ml, 64
mmol) in THF was added dropwise to the reaction flask contianing 4-methoxy-
benzophenone (14; 12.0 g, 5 mol) and THF (45 ml). The reaction was warmed
slowly and heated to reflux, which was maintained overnight. The reaction mixture
was cooled and a saturated NH4Cl solution (25 ml) was added. The mixture was
transferred to a separatory funnel and other (50 ml) and water (25 ml) were added.
The aqueous layer was separated and the ether layer was extracted with water
(3 ϫ 25 ml). The combined aqueous layers were extracted with ether (3 ϫ 50 ml).
The combined ether layers were dried (anhydrous MgSO4), filtered, and concen-
trated under vacuum. The intermediate carbinol (18.5 g) was obtained and dehy-
drated with 1.4 g p-TSA in 90 ml benzene for 3.5 h at reflux. The mixture was
cooled and the benzene layer was extracted with water until free of acid, dried
(anhydrious MgSO4), and concentraetd under vacuum. The residual oil was purified
over silica gel (pet ether/CH2Cl2; 4 : 1), which afforded 8.91 g (56%) of a clear oil.
(Z)-1,1-Dichloro-2,3-diphenyl-2-(4-methoxyphenyl) cyclopropane (5). Ethene
(16; 4.49 g, 15.7 mmol), 50% aq NaOH (25 ml), chloroform (40 ml), and TEBA
(0.30 g, 1.3 mmol) were mixed together and stirred at room temperature for 72 h.
The two layers were separated and the aqueous layer was extracted with CH2Cl2
(3 ϫ 50 ml). The combined organic layers were dried (anhydrous MgSO4), filtered,
and concentrated under vacuum to afford 5.9 g crude product, which was purified
over silica gel (pet ether/CH2Cl2, 100 : 1 to 4 : 1). Recrystallization of the white solid
from pet ether followed by pet ether/acetone gave the Z-isomer as white crystals
(0.69 g, 12%). The E-isomer (2.2 g) was obtained from the mother liquor. Recrystalli-
zation of the E-1,1-dichloro-2,3-diphenyl-2-(4-methoxyphenyl) cyclopropane from
pet ether and then from pet ether/acetone gave 0.49 g (8.5%) of white crystals (see
1
Table 1 for H NMR data of Z- and E-isomers).
4-(2-Bromoethoxy)benzophenone (20). A mixture of 4-hydroxybenzophenone
(17; 40.0 g, 202 mmol) and 1,2-dibromoethane (140 ml) was added to a flask con-
taining 10% aqueous NaOH solution (140 ml) and TEBA (4.8 g, 20 mmol). The
mixture was refluxed for 48 h and allowed to cool to room temperature at which
time two layers separated. The aqueous layer was extracted four times with 100 ml
of CH2Cl2 and the combined organic layers were washed free of base. Removal of
the solvent and the recrystallization of the brown solid from 95% EtOH yielded
35.5 g (57%) of a light tan solid, mp 68–70.5ЊC (lit. (29) 62ЊC and lit. (30) 77ЊC).

SYNTHESIS AND SEPARATION OF ANTITUMOR AGENTS
87
1H NMR (CDCl3; 60 MHz): ͳ 3.68 (t, J ϭ 6.3Hz, 2H, CH2Br), 4.39 (t, J ϭ 6.3Hz,
2H, CH2O), 6.98 (d, 2H, ArH), 7.39–7.89 (m, 7H, ArH).
4-(2-Bromoethoxy)-4Ј-methoxybenzophenone (21). 4-Methoxybenzoic acid (18;
13.0 g, 85.5 mmol) and ͱ-bromophenetole (19; 13 g, 65 mmol) were added to a
flask equipped with a mechanical stirrer containing pyrophosphoric acid (PPA; 140
g). The reaction mixture was warmed to 100ЊC and stirred vigorously for 4 h. It
was then cooled, ice/water was added, and the mixture was filtered. The filtrate
was extracted with ether. The solvent was removed under vacuum, and residue was
dissolved in CHCl3 and extracted with 10% aqueous NaOH solution. Removal of
solvent and recrystallization from MeOH afforded 15.7 g (72%) solid, mp 110–111ЊC
(lit. (31) 111.5–113ЊC). 1H NMR (CDCl3; 60 MHz): ͳ 3.67 (t, 2H, J ϭ 6.3Hz, CH2Br),
3.91 (s, 3H, OCH3), 4.39 (t, 2H, J ϭ 6.3Hz, CH2O), 6.96 (b, 4H, ArH), 7.85 (b,
4H, ArH).
(Z/E)-1-[4-(2-Bromoethoxy)phenyl]-1,2-diphenyl ethene (22). To a solution of
ketone (20; 17.5 g, 57 mmol) in dry THF (50 ml) was added benzyl magnesium
chloride in THF (2
M
solution, 30 ml, 60 mmol), which was diluted with additional
THF (20 ml). The resulting reaction mixture was refluxed overnight. The reaction
mixture was cooled and a saturated solution of NH4Cl (25 ml) was added. The
product was extracted four times with 100 ml of ether which was washed with water
until neutral (4 ϫ 50 ml). Removal of the solvent afforded the intermediate carbinol,
which was dehydrated using p-TSA (1.15 g) by heating to reflux for 4 h in benzene
(90 ml). The reaction mixture was cooled and the organic layer was extracted with
water until neutral (3 ϫ 50 ml). Removal of solvent yielded an oil which was purified
over silica gel (pet ether/CH2Cl2, 4 : 1) to afford a white solid 16.6 g (77%) of a
1
mixture of Z/E-ethene, mp 92–94ЊC. H NMR (CDCl3; 300 MHz): ͳ 3.67 (t, 2H,
J ϭ 6.3 Hz, CH2Br), 4.32 (t, 2H, J ϭ 6.3Hz, CH2O), 6.87 (s, 1H, vinyl H), 6.90–7.36
(m, 14 H, ArH).
(Z/E)-1-[4-(2-Bromoethoxy)phenyl]-1-(4-methoxyphenyl)-2-phenyl ethene (23).
Ethene 23 was prepared as described above using the ketone (21; 5.50 g, 16.4 mmol)
and benzyl chloride (2
M
solution, 10.7 ml, 21.4 mmol) in THF. The reaction mixture
was heated to reflux which continued overnight. The reaction mixture was cooled
and ether (35 ml) and water (15 ml) were added. The two layers were separated
and the aqueous layer was extracted with ether (3 ϫ 35 ml). The combined ether
layers were dried (anhydrous MgSO4), filtered, and concentrated to obtain 7.78 g
crude carbinol, which was dehydrated with p-TSA (0.6 g) in benzene (70 ml) while
refluxing overnight. The benzene layer was washed with water (3 ϫ 30 ml) until
free of acid and stripped off under vacuum. Purification of the product by column
chromatography over silica gel (pet ether/CH2Cl2, 4 : 1) yielded 5.20 g (78%) oil.
1H NMR (CDCl3; 300 MHz): ͳ 3.67, 3.69 (t, J ϭ 6.6 Hz, 2H, CH3Br), 3.86, 3.87 (s,
3H, OCH3), 4.33, 4.34 (t, J ϭ 6.6Hz, 2H, CH2O), 6.89, 6.90 (s, 1H, vinyl H), 6.85–7.52
(m, 13H, ArH).
(Z/E)-1,1-Dichloro-2,3-diphenyl-2-[4-(2-bromoethoxy)phenyl]cycloproane (24).
Ethene (22; 5.0 g, 13.2 mmol), TEBA (0.5 g,2.2 mmol), 50% aqueous NaOH solution
(30 ml), and chloroform (55 ml) were stirred at room temperature for 95 h. The
two layers were separated and the aqueous layer was extracted with CH2Cl2 (4 ϫ
25 ml). The combined organic layers were dried and concentrated. After purification

88
SINGH, MEYER, AND MAGARIAN
of the crude product over silica gel (pet ether/CHCl3, 10 : 1 to 1 : 1) an oil was
obtained. Fractional crystallization from MeCN afforded the E-isomer of 24 (1.5
g, 25%). The filtrate was concentrated and the oil was crystallized from pet ether/
benzene to afford the Z isomer (24; 1.34 g, 20%) as a white solid (Table 1 for
1H NMR).
(Z/E)-1,1-Dichloro-2-[4-(2-bromoethoxy)phenyl]-2-(4-methoxyphenyl)-3-phenyl
cyclopropane (25). Ethene (23; 5.21 g, 12.7 mmol), TEBA (0.5 g, 2.2 mmol), chloro-
form (60 ml), and 50% aqueous NaOH solution (30 ml) were combined and stirred
at room temperature for 72 h. The two layers were separated and the aqueous
layer was extracted with CH2Cl2 (4 ϫ 25 ml). The solvent was removed under
vacuum, which yielded 5.5 g of a brown solid, which was purified over silica gel (pet
ether/CH2Cl2, 1 : 1) to afford 4.0 g of a light yellow solid. The complete separation of
the Z/E isomers could not be achieved; however, fractions enriched in either Z-
or E-isomer were obtained. The Z-enriched isomer (25) was obtained after two
recrystallizations from MeCN (0.43 g) (Table 1). The mother liquor was reduced
and the solid was crystallized from abs. EtOH giving the E-enriched-isomer (1.84
g) (Table 1).
(Z) - 1,1 - Dichloro - 2,3 - diphenyl - 2 - [4 - (2 - dimethylaminoethoxy)phenyl]cyclo-
propane (6). Z-bromide 24 (0.6 g, 1.3 mmol) and dimethyl amine (7.8
M
aqueous
solution, 6 ml, 46.8 mmol) were dissolved in CHCl3 : MeCN : 2-PrOH (3 : 5 : 5, 13
ml) and heated at 48 Ϯ 2ЊC for 20 h. The solution was cooled, and the solvent
mixture was removed under vacuum, leaving a residue that was dissolved in ether.
The ether solution was extracted with water (10 ml), 10% aq NaOH, dried over
anhydrous MgSO4, filtered, and the solvent stipped off. The residue was crystallized
from pet ether to afford 0.28 g (51%) of a white solid (Table 1). The E-isomer of
6 was similarly obtained from E-1,1-dichloro-2,3-diphenyl-2-[4-(2-bromoethoxy)-
phenyl]cyclopropane and crystallized from EtOH in 39% yield (Table 1).
(Z)-1,1-Dichloro-2-[4-(2-dimethylaminoethoxy)phenyl]-2-(4-methoxyphenyl)-3-
phenyl cyclopropane (7). Z-bromide 25 (0.52 g, 1.06 mmol) and dimethyl amine
(7.8
M
aq solution 7 ml, 54.6 mmol) in CHCl3 : MeCN : 2-PrOH (3 : 5 : 5, 15 ml) were
heated at 48 Ϯ 2ЊC for 20 h. The solution was cooled to room temperature and
the solvent mixture was removed under vacuum leaving a solid residue, which was
dissolved in ether. The ether solution was extracted with water (10 ml), 10% aqueous
NaOH, dried over anhydrous MgSO4, filtered, and the solvent stripped off. The
resulting solid was crystallized from pet ether to afford 0.14 g (29%) white solid.
The E-isomer of 7 was similarly prepared from E-1,1-dichloro-2-[4-(2-bromoethoxy)
phenyl]-2-(4-methoxyphenyl)-3-phenyl cyclopropane and crystallized from pet
1
ether in 35% yield (see Table 1 for H NMR data of Z- and E-isomers).
RESULTS AND DISCUSSION
The Z-isomers of four 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs) (Chart
2) were synthesized as shown in Schemes I–III. In addition, their E-diastereomers
were also obtained. The stereoselective synthesis of 8 has been previously reported
by Meyer and Magarian (32). In the di- and triaryl structures, the phenyl rings

SYNTHESIS AND SEPARATION OF ANTITUMOR AGENTS
89
S
CHEME I
S
CHEME II

90
SINGH, MEYER, AND MAGARIAN
S
CHEME III
are denoted Ͱ, ͰЈ, and ͱ to conform with current notations used in nonsteroidal
antiestrogens.
In general, the preparation of these compounds required the synthesis of the
appropriately substituted ethenes via a Grignand reaction followed by the dehydra-
tion of the intermediate carbinols. Dichlorocyclopropanation of the ethene followed,
which was accomplished by the stereospecific addition of the dichlorocarbene gener-
ated from chloroform and 50% aq NaOH in the presence of TEBA as the anion
transfer agent (33). Fractional crystallization was employed to separate the diaste-
reomers. Displacement of the side chain bromide with dimethyl amine followed by
fractional crystallization furnished the basic side chain compounds (34).

SYNTHESIS AND SEPARATION OF ANTITUMOR AGENTS
91
TABLE 2
Retention Time and Characterization of Enantiomers
Compound
No.
Retention time
Enantiomeric
Solvent systema
(min)
Optical rotationb
purity (%)c
4
5
6
7
A
B
C
C
5.3
6.5
4.2
4.6
4.9
6.3
6.7
9.5
Ϫ30.76
ϩ26.02
Ϫ31.94
ϩ34.41
Ϫ38.69
ϩ38.97
Ϫ26.89
ϩ26.75
100
99.1
99.5
100
100
100
100
99.8
a A, hexane : ethanol (9 : 1); B, hexane : 2-propanol (9 : 1); C, hexane : 2-propanol : diethylamine
(9 : 1 : 0.01).
b Optical rotations were determined in CH2Cl2 at ambient temperature, ␭ ϭ 589 nm.
c The overall purity of enantiomers was Ͼ98% as determined from HPLC chromatograms.
Synthesis of 4 involved the protection of the 4-hydroxyl group of ketone 9 before
subjecting it to a Grignand reaction to obtain the ethene 12 as shown in Scheme
I. Fractional crystallization at the ethene stage afforded enriched Z- and E-isomers.
The individual isomers were converted into their dichlorocyclopropanes to obtain
both the Z- and E-isomers of 4.
The synthesis of 5 required two steps. Two different Grignard reagents were
used for the synthesis of ethene 16 as shown in Scheme II. The yields with these
reagents (11 and 15) were 79 and 56%, respectively. Although, the yield with reagent
11 was higher, the availability of 15 commercially made the synthesis simple and
less tedious. After the cyclopropanation reaction fractional crystallization was per-
formed in pet ether/acetone to afford the Z- and E-isomers of 5.
The synthesis of 6 and 7 was performed as depicted in Scheme III. The first step
involved the synthesis of the bromoethoxy ketones (20, 21). The bromoethyl group
was used for two purposes: first it served as the protective group in 17 for the
Grignard reaction, and second, the bromo group was easily displaced by the dimethyl
amino group. Ketone 20 was prepared by the O-alkylation of 17 using 1,2-dibromo-
ethane in a phase transfer reaction (29). However, 21 was obtained by the electro-
philic aromatic substitution involving 18 and 19 in the presence of PPA, which
served as the dehydrating agent. The ethenes (22, 23) were then obtained from the
Grignard reaction. The fractional crystallization of the cyclopropanes (24, 25) and
the subsequent dimethylamination of the individual diastereoisomers, followed by
fractional crystallization, afforded the pure diastereoisomers 6 and 7.
The 1H NMR resonances for the O-methylene protons in: (1) the benzyloxy side
chain (OCH2C6H5) in Z-4, (2) the O-methyl protons in Z-5, (3) the O-methylene
protons of the bromoethoxy side chain (OCH2CH2Br) in 24 and 25, and (4) the
N,N-dimethylethoxy side chain (OCH2CH2N(CH3)2) in 6 and 7 appear at a relatively
higher field in comparison to their E-isomers, owing to the shielding effect of the
1
adjacent phenyl ring (35, 36). The relevant H NMR data are shown in Table 1.

92
SINGH, MEYER, AND MAGARIAN
F
IG. 1. HPLC chromatograms of (A) (Ϯ)-Z-4, (B) (Ϯ)-Z-5, (C) (Ϯ)-Z-6, and (D) (Ϯ)-Z-7. (For
separation conditions see Experimental.)
Single crystal X-ray analysis was used to determine the configuration of both the
Z- and E-isomers (37, 38).
Enantiomeric resolution is one of the most important subjects in the field of fine
chemistry. A variety of racemates have been resolved (39) using two different
techniques: the direct and indirect method (40). The direct technique involves the
chromatographic separation of enantiomers using chiral stationary phase or chiral
mobile phase, and the indirect technique is the method of derivatization of diastere-
oisomers prior to chromatographic separation. The direct method was chosen to
separate the enantiomers in this work.
The semipreparative resolution of the enantiomers from the (Z) diastereoisomers
(4–8) into their respective enantiomers was accomplished using chiral chromatogra-
phy. Table 2 shows the results of the chiral separation. Solvent systems containing
hexane and an alcohol are recommended for separation on a chiral stationary phase,
amylose tris-3,5-dimethylphenyl carbamate, to prevent deterioration of the column
material. Three solvent systems consisting of hexane/EtOH (A; 9 : 1), hexane/2-
PrOH (B; 9 : 1), and hexane/2-PrOH/diethylamine (C; 9 : 1 : 0.01) were evaluated
in this study. In order to find the best conditions for the chiral separation of each

SYNTHESIS AND SEPARATION OF ANTITUMOR AGENTS
93
compound, their capacity factor (K), column plate number (N), separation factor
(Ͱ), and resolution factor (Rs) were determined using solvent systems A, B, and
C. These parameters were calculated using the chiral analytical column. When
selecting the appropriate solvent system for each compound, emphasis was placed
upon the resolution factor since this value incorporates all other parameters. The
addition of diethylamine to solvent system B resulted in significant improvement
in the resolution factor of 6 (6.18 vs 4.15) and 7 (8.14 vs 6.51); therefore, solvent
C was chosen for the separation of compounds 6 and 7 using a semipreparative
column. The addition of diethylamine led to shorter retention times. The values of
resolution factors for 4 and 5 using solvent systems A and B were 5.00 vs 5.21 and
1.36 vs 2.23, respectively. The chromatograms of compounds 4–7 are shown in Fig.
1 (A–D). Since these compounds were only partially soluble in the HPLC eluent,
they were dissolved in hexane/CHCl3 (4 : 1; 4), hexane/2-PrOH (2 : 1; 5), or hexane/
2-PrOH (3 : 2; 6, 7). Following the separation of the individual enantiomers, samples
were reinjected on the analytical column to establish enantiomeric purity. It is
important to note that the enantiomers of the E-diastereoisomers could not be
separated successfully on this column under similar separation conditions. Other
chiral columns were also evaluated for the separation of the Z-isomers, but were
unsuccessful in separating the optical isomers.
In summary, the Z-isomers of 1,1-dichloro-2,2,3-triarylcyclopropanes as potent
antitumor agents were synthesized and characterized by high-resolution NMR.
Enantiomers of each isomer were separated using semipreparative chiral HPLC.
Appropriate solvent systems were evaluated and optical rotations were determined.
ACKNOWLEDGMENTS
This work was supported by NCI (NIH) Grant CA40458 (R.A.M.). The authors thank Scott Perrin
from the Regis Chemical Company for his assistance in testing chiral columns and in the selection of
the one used in this work.
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