Preparation, resolution, and biological evaluation of 5-aryl-1,2- dihydro-5H-chromeno[3,4-f]quinolines: Potent, orally active, nonsteroidal progesterone receptor agonists

Article abstract of DOI:10.1021/jm980190c

Two potent nonsteroidal progestins from the 5-aryl-1,2-dihydro-5H- chromeno[3,4-f]quinoline class (LG120746 and LG120747) were selected for scale-up, resolution, and biological evaluation of the purified enantiomers. For each quinoline, the levorotatory enantiomer was determined to be the more potent agonist of the human progesterone receptor isoform B (hPR-B) (EC₅₀ < 3 nM), but the dextrorotatory enantiomers retained significant PR modulatory activity (EC₅₀ < 200 nM). In two in vivo rodent models of progestational activity, a pregnancy maintenance assay and a uterine wet weight assay, the two eutomers displayed potent progesterone-like effects. In a third model for progestational activity, the mammary end bud assay, these compounds were significantly less active. These studies demonstrate that certain members of this class of selective progesterone receptor modulators display encouraging and potentially useful tissue-selective progestational effects.

Full text of DOI:10.1021/jm980190c

J . Med. Chem. 1998, 41, 2779-2785
2779
P r ep a r a tion , Resolu tion , a n d Biologica l Eva lu a tion of
5-Ar yl-1,2-d ih yd r o-5H-ch r om en o[3,4-f]qu in olin es: P oten t, Or a lly Active,
Non ster oid a l P r ogester on e Recep tor Agon ists
J ames P. Edwards,*,† Lin Zhi,† Charlotte L. F. Pooley,† Christopher M. Tegley,† Sarah J . West,†
Ming-Wei Wang,‡,§ Marco M. Gottardis,‡ Charles Pathirana,† William T. Schrader,‡ and Todd K. J ones†
Departments of Medicinal Chemistry and Endocrine Research, Ligand Pharmaceuticals Inc., San Diego, California 92121
Received March 27, 1998
Two potent nonsteroidal progestins from the 5-aryl-1,2-dihydro-5H-chromeno[3,4-f]quinoline
class (LG120746 and LG120747) were selected for scale-up, resolution, and biological evaluation
of the purified enantiomers. For each quinoline, the levorotatory enantiomer was determined
to be the more potent agonist of the human progesterone receptor isoform B (hPR-B) (EC50
<
3 nM), but the dextrorotatory enantiomers retained significant PR modulatory activity (EC50
< 200 nM). In two in vivo rodent models of progestational activity, a pregnancy maintenance
assay and a uterine wet weight assay, the two eutomers displayed potent progesterone-like
effects. In a third model for progestational activity, the mammary end bud assay, these
compounds were significantly less active. These studies demonstrate that certain members of
this class of selective progesterone receptor modulators display encouraging and potentially
useful tissue-selective progestational effects.
In tr od u ction
The regulation of gene expression by small molecule
modulators of intracellular receptors (IRs)1 is an attrac-
tive opportunity for drug discovery.2 For example,
progesterone plays a central role in regulating female
reproductive function via the human progesterone re-
ceptor (hPR).3 Although a number of steroidal progestins
are currently marketed (Figure 1), the search for novel
agonists of hPR promises to yield important new drugs
to address women’s health issues, including hormone
replacement therapy4 and cancers of the female repro-
ductive system.5 In contrast to the human androgen
(hAR) and estrogen (hER) receptors, nonsteroidal modu-
lators of hPR have only recently been discovered.
Nonsteroidal hPR modulators6 are particularly interest-
ing due to the possibility of developing drugs with less
cross-reactivity than steroids to related IRs such as the
human glucocorticoid (hGR), mineralocorticoid (hMR),
and androgen (hAR) receptors, as well as molecules
which exhibit novel pharmacology.7
F igu r e 1. Selected currently marketed steroidal hPR ago-
nists.
although preliminary work indicated that one enanti-
omer is significantly more active than the other.8a
To further study the progestational effects of this
series of compounds, two C(9) fluoro derivatives (2 and
3, Figure 2) were selected for further biological profiling.
Although both C(9) fluoro and C(9) chloro derivatives
have potent in vitro and in vivo progestational activity,8b
certain C(9) chloro compounds also display significant
binding affinity for hGR (Table 1), and they were not
pursued as hPR modulators. This report describes the
scale-up, preparative HPLC separation of enantiomers,
and in vitro studies on compounds 2 and 3. In addition,
the in vivo progestational effects of the eutomers (-)-2
and (-)-3 were characterized in three rodent models.
We have recently described the discovery and pre-
liminary structure-activity relationship studies of a
class of potent nonsteroidal hPR modulators based on
the 5-aryl-1,2-dihydro-5H-chromeno[3,4-f]quinoline phar-
macophore (1, Figure 2).8 Several members of this class
of PR modulators have subnanomolar binding affinities
to baculovirus-expressed hPR-A9 and are as potent and
more efficacious in a functional assay using recombinant
hPR-B10 than progesterone, the cognate hormone. These
initial studies dealt primarily with racemic compounds,
* Address correspondence to this author at: Ligand Pharmaceuti-
cals, 10275 Science Center Drive, San Diego, CA 92121. Phone: (619)
550-7723. Fax: (619) 550-7249. E-mail: jedwards@ligand.com.
† Department of Medicinal Chemistry.
Ch em istr y
‡ Department of Endocrine Research.
The preparation of multigram quantities of 2 and 3
required optimization of several steps of the previously
§ Current address: GC BioTechnologies, LLC, 11099 N. Torrey Pines
Rd, La J olla, CA 92037.
S0022-2623(98)00190-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/23/1998

2780 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Edwards et al.
Sch em e 1a
F igu r e 2. 5-Aryl-1,2-dihydro-5H-chromeno[3,4-f]quinoline
general structure (1), LG120746 (2), and LG120747 (3).
Ta ble 1. In Vitro hPR-B Activity in Cotransfected CV-1 Cells
and Binding Affinities to Baculovirus-Expressed hPR-A and
hGRa
a
(i) Methyl 2-bromo-5-nitrobenzoate (7), (Ph3P)4Pd, CsF, DME,
80 °C; (ii) 20% KOH, EtOH, THF, room temperature; (iii) SOCl2,
DCE, 80 °C, then AlCl3, (iv) Zn, CaCl2-2H2O, EtOH, reflux; (v)
acetone, I2, 125-135 °C; (vi) ArLi, THF, -65 °C; (vii) BF3-OEt2,
Et3SiH, CH2Cl2, room temperature.
hPR activity
hGR
b
ligand
X
EC50
efficacyc
Kib
Kib
(()-2
(()-4
(()-3
(()-5
F
Cl
F
2.8 ( 0.7
3.6 ( 0.7
2.7 ( 0.2
9.4 ( 2.6
117 ( 7
95 ( 12
97 ( 6
0.32 ( 0.11
0.48 ( 0.18
0.49 ( 0.16
0.50 ( 0.15
133
6.3
210
14
the lithium-halogen exchange process and during the
addition of quinoline 11.13 Allowing the reaction tem-
perature to rise above approximately -60 °C resulted
in significant decomposition of both the aryllithium
reagent and the substrate, presumably due to ortho-
lithiation processes. The reduction of the intermediate
lactols occurred uneventfully; however, the acidic reac-
tion medium had to be neutralized very carefully to
avoid isomerization of the C(3)-C(4) olefin to the exo
isomers (12, 13). Using these minor precautions, quino-
lines 2 and 3 could be prepared reproducibly in 70-
80% overall yield from 11 on a 5 g scale.
With a preparative method in hand for multigram
quantities of racemic 2 and 3, the HPLC resolution of
the enantiomers was then explored. After a survey of
a number of chiral stationary phases, it was found that
the “Whelk-O” column developed by Pirkle and co-
workers14 cleanly separated the enantiomers of both 2
and 3. Using a 10 cm (R,R)-Whelk-O column, optimiza-
tion of the mobile phase (heptane/CH2Cl2/Et3N) afforded
conditions suitable for separation of up to 400 mg of
racemate per 40-min injection, and several grams of
each enantiomer could be separated to a purity of
greater than 97% ee in a day.
Cl
73 ( 10
a
Cotransfection experiment values represent at least triplicate
determinations. Values are in nM, mean ( SEM, N g 2. EC50
values represent the concentration required to give half-maximal
activation for that ligand. c Efficacy expressed as percent relative
to progesterone ) 100%.
b
described synthetic route (Scheme 1). Specifically, the
palladium-catalyzed coupling reaction of boronic acid 6
with aryl bromide 7 was not scalable (10-15 g scale)
due to hydrolysis of the methyl esters during the
reaction. This was avoided by utilizing a fluoride-
mediated Suzuki cross-coupling reaction,11 affording the
biaryl ester 8 in 75-85% yields on a 20 g scale.
Conversion of 8 to the nitrobenzocoumarin 9 proceeded
uneventfully using the previously described conditions.8b
Palladium-catalyzed hydrogenation of the nitro group
of 9 also proved problematic upon scale-up, and recourse
was made to a zinc-mediated reduction.12 Subjection
of aniline 10 to typical Skraup reaction conditions,
which requires high temperatures (120-130 °C) and a
high proportion of iodine (30-40 wt %), afforded moder-
ate yields (20-40%) of 1,2-dihydro-2,2,4-trimethylquino-
line 11 and gave variable results on a multigram scale.
One solution to this problem was simply to run the
reaction at high dilution (<0.01 M), affording quinoline
11 in a reproducible 50-60% yield. The next step,
generation of the aryllithium reagents, required strict
control of reaction temperature (<-65 °C), both during
Whereas the free bases of (-)- and (+)-2 could be
recrystallized from hexane (ee >99.5%, mp 133-4 °C),
(-)- and (+)-3 were foams that resisted recrystallization.
The low basicity of the quinoline nitrogen of 3 limited
the number of potential salts that could be formed, but

Progesterone Receptor Agonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2781
F igu r e 3. Chem3D representation of (-)-(S)-2 based on X-ray crystallographic coordinates.
Ta ble 2. In Vitro hPR-B Activity in Cotransfected CV-1 Cells
and Binding Affinities to Baculovirus-Expressed hPR-Aa
hPR activity
b
ligand
EC50
efficacyc
Kib
progesterone
(-)-2
1.5 ( 0.5
1.3 ( 0.3
100 ( 0
125 ( 8
83 ( 9
99 ( 8
43 ( 5
3.8 ( 0.2
0.1 ( 0.1
(+)-2
135 ( 28
30
(-)-3-sulfate
(+)-3-sulfate
2.4 ( 0.6
147 ( 65
0.4 ( 0.3
60
a
Cotransfection experiment values represent at least triplicate
b
determinations. Values are in nM, mean ( SEM, N g 2. If no
SEM is noted, value is from a single determination. EC50 values
represent the concentration required to give half-maximal activa-
tion for that ligand. c Efficacy expressed as percent relative to
progesterone ) 100%.
suitable crystals of the sulfate hydrate (“3-sulfate”,
>99.5% ee, mp ∼230 °C dec) were eventually obtained.
The absolute configuration of (-)-2 was determined to
be S by X-ray crystallography,15 and the absolute
configuration of (-)-3 was assigned by analogy. A stereo
3D depiction of (-)-(S)-2 is shown in Figure 3.
F igu r e 4. Concentration-response curves for progesterone,
(-)-(S)-2, and (-)-(S)-3-sulfate. See Experimental Section for
details.
pregnancy during simultaneous treatment with the
potent steroidal antiprogestin mifepristone (RU486) was
also tested.
In Vitr o Biologica l Activities
In h ibition of Uter in e Wet Weigh t. Inhibition of
estrogen-induced uterine wet weight in the immature
rat was evaluated as a classical endpoint for progestin
response. Both enantiomers of 2 were tested in this
model (Figure 5, upper panel) in addition to the eutomer
of 3 (Figure 5, lower panel). In agreement with the in
vitro cotransfection and binding data described above,19
(+)-(R)-2 was inactive in inhibiting uterine wet weight
gains in estradiol-treated animals (Figure 5, upper
panel). Conversely, (-)-(S)-2 was as effective as medrox-
yprogesterone acetate (MPA), a standard marketed
progestin, in this assay. Both (-)-(S)-2 and MPA
significantly decreased (p < 0.01) mean uterine wet
weights at doses greater than or equal to 1 mg/kg with
a maximum mean efficacy of approximately 35% com-
pared to estrogen-treated animals. (-)-(S)-3-sulfate was
also tested in this assay (Figure 5, lower panel) and was
found to be equipotent to MPA throughout the entire
dose range evaluated. Both MPA and (-)-(S)-3-sulfate
significantly (p < 0.01) decreased estrogen-induced
uterine wet weight gain at doses greater than or equal
to 0.3 mg/kg with a maximum mean efficacy similar to
(-)-(S)-2 and MPA in the previous experiment.
As expected from earlier work, the levorotatory enan-
tiomers (-)-(S)-2 and (-)-(S)-3 were the more active
enantiomers (Table 2); however, the distomers retained
significant (130-150 nM) hPR agonist activity. Indeed,
the hPR-A binding affinities of 30 and 60 nM for the
“distomers” (+)-(R)-2 and (+)-(R)-3, respectively, ranked
among the most potent reported for nonsteroidal hPR
modulators from other compound classes.6 The binding
affinities of 0.11 and 0.36 nM for (-)-(S)-2 and (-)-(S)-3
were among the most potent for any hPR modulator
reported to date, exceeding by 1 order of magnitude the
Ki of progesterone (3.8 nM) and comparable to that of
medroxyprogesterone acetate (0.3 nM) in this assay. In
the cotransfection assay, (-)-(S)-2 and (-)-(S)-3 were
virtually indistinguishable from progesterone (Figure
4).
In Vivo Biologica l Activities
To characterize the progestational effects of (-)-(S)-2
and (-)-(S)-3 in vivo, three assays targeting different
endpoints were examined. In the uterine wet weight
assay, progestins blunt the effects of estrogens on the
female uterus.16 The breast bud assay has been used
to characterize the effects of antiprogestins on the
differentiation of the mammary gland in estrogen-
primed ovariectomized animals17 and was modified to
test for progestational effects in this study.18 Finally,
the ability of these nonsteroidal progestins to maintain
Ma m m a r y Lob u la r -Aveola r Bu d P r olifer a t ion
Assa y. Induction of differentiation and proliferation of
lobular-aveolar buds in the rat mammary gland has
been demonstrated morphologically and proliferatively
as a hallmark of progestational action. In ovariecto-

2782 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Edwards et al.
increase in mitotic proliferation (as measured by BrdU
incorporation) of lobular-aveolar buds.16 To compare the
progestational activity of (-)-(S)-2 and (-)-(S)-3-sulfate
to MPA, these compounds were evaluated in this assay.
As described above, (-)-(S)-2, (-)-(S)-3-sulfate, and
MPA are fully efficacious at a dose of 3 mg/kg in the
uterine wet weight assay. As expected, at this dose
MPA caused a maximum increase of over 20-fold induc-
tion of BrdU labeling in lobular-aveolar buds of the
mammary gland over estrone-alone treated animals
(Table 3). However, (-)-(S)-2 and (-)-(S)-3-sulfate
induced BrdU labeling only 3.6- and 2.5-fold, respec-
tively, over estrone-alone treated animals. These data
suggest a tissue-selective action for the nonsteroidal
progestins, which display at least 6-fold less efficacy
than MPA at an equivalent high dose.
An tifer tility Assa y. Implantation in mice occurs
around day 4 of pregnancy and is accompanied by a
steep increase in circulating progesterone.20 This ex-
tremely sensitive window of progesterone dependency
can be manipulated with antiprogestin treatment.21
When given before the expected time of implantation,
a progesterone receptor antagonist, such as mifepristone
(RU486),22 will specifically block implantation and hence
the establishment of pregnancy.23 These well-known
antifertility properties of mifepristone can be reversed
by high-dose progestin treatment, and the effects of (-)-
(S)-2 and (-)-(S)-3-sulfate were examined using 10-
12-week-old pregnant, mature ICR mice. These data
are shown in Table 4. Oral administration of RU486
(0.5 mg or ∼15 mg/kg) between days 2 and 4 post coitum
reduced the pregnancy rate from a control level of 88.2%
to 18.8% (∼80% inhibition; P < 0.001), and this dose
was chosen for further studies. The observed antifertil-
ity effect of RU486 was partially reversed by coadmin-
istration of MPA but only with a much higher daily dose
(10 mg or ∼300 mg/kg). Of the two test compounds, (-)-
(S)-2 completely restored pregnancy in RU486-treated
females at doses between 1.0 and 3.0 mg (∼30-100 mg/
kg; P < 0.001) per day, whereas (-)-(S)-3-sulfate
produced a partial reversal in the same dose range
(Table 4), similar to the effects observed with MPA but
at lower doses.
F igu r e 5. Comparison of inhibition of estradiol-induced
uterine wet weight increases by MPA, (+)-(R)-2, (-)-(S)-2, and
(-)-(S)-3-sulfate. Bars are designated as follows. Upper
panel: control/vehicle (striped bar); estradiol (black bar); MPA
+ estradiol (gray bar); (-)-(S)-2 + estradiol (dotted bar); (+)-
(R)-2 + estradiol (hatched bar). Lower panel: control/vehicle
(striped bar); estradiol (black bar); MPA + estradiol (gray bar);
(-)-(S)-3-sulfate + estradiol (dotted bar). Statistically signifi-
cant (ANOVA) Points are designated: *** p < 0.001; ** P <
0.01; * p < 0.05 vs estradiol-alone treated animals (black bar).
See Experimental Section for details.
Ta ble 3. Induction of BrdU Labeling in Lobular-Aveolar Rat
Mammary Buds by Progestin Treatmenta
ligand
fold induction
estrone
1
MPA (3.0 mg/kg)
24
(-)-2 (3.0 mg/kg)
(-)-3-sulfate (3.0 mg/kg)
3.6
2.5
a
Fold induction by progestin treatment is calculated as the fold
increase of the BrdU labeling index over estrone treatment alone.
Index is measured from the average of 12 microscopic fields of
each inguinal mammary fat pad of treated animals (4 rats/group).
Discu ssion
The relative in vitro activity of the enantiomers of 2
and 3 demonstrated that the levorotatory isomers were
100 times more potent progestins than the dextrorota-
mized rats primed with estrone, treatment with
progestins such as MPA can induce a dose dependent
Ta ble 4. Effects of Daily Doses of MPA, (-)-(S)-2, and (-)-(S)-3-sulfate on the Outcome of Pregnancy in Mice Simultaneously
Treated with RU486 (0.5 mg)a
dose
(mg/mouse)
no. pregnant/
no. mated
%
treatment
control
RU486
MPA + RU486
mice
CL
implants
pregnant
0.1 mL
0.5 mg
30
10
3
1
3
1
3
17
16
10
10
12
10
9
11
10
10
14.2 ( 1.0
13.4 ( 0.9
10.7 ( 1.7
12.3 ( 1.2
13.6 ( 0.8
11.7 ( 1.2
14.1 ( 0.4
14.1 ( 0.6
13.8 ( 0.4
13.0 ( 0.8
13.9 ( 1.0*
3.3 ( 1.6
15/17
3/16
5/10
1/10
2/12
1/10
9/9
10/11
5/10
3/10
88.2
18.8
50.0
10.0
16.7
10.0
100
91
6.7 ( 2.1**
1.4 ( 1.3***
2.6 ( 1.7***
1.2 ( 1.1***
13.7 ( 0.4*
12.0 ( 1.6*
6.6 ( 2.2**
2.2 ( 1.3***
(-)-2 + RU486
(-)-3 + RU486
50
30
1
a
Pregnant ICR mice were treated orally with RU486 (0.5 mg/animal) or RU486 (0.5 mg/animal) plus different doses of the test compounds
between day 2 and day 4 after mating. Control animals received similar doses of MPA (positive control) or an equivalent volume of corn
oil (negative control). Necropsies were performed on day 8 post coitum to determine the outcome of the pregnancy. *P < 0.001 compared
to RU486-treated group; **P < 0.005 and ***P < 0.001 compared to oil-treated group (Student t-test). CL ) corpora lutea.

Progesterone Receptor Agonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2783
9-F lu or o-1,2-d ih yd r o-2,2,4-tr im eth yl-5-cou m a r in o[3,4-
f]qu in olin e (11). In a 600-mL resealable pressure tube, a
suspension of 10 (0.92 g) in acetone (400 mL) was treated with
iodine (0.40 g) and heated to 120-130 °C for 36 h. The
reaction mixture was cooled to room temperature, concentrated
to remove the bulk of the acetone, and dissolved in CH2Cl2
(200 mL). The organic layer was washed with 0.5 N Na2S2O3
(2 × 200 mL) and saturated NaHCO3 (1 × 100 mL). The
aqueous layers were extracted with CH2Cl2 (2 × 100 mL). The
combined organic layers were dried (K2CO3), filtered, and
concentrated to afford an orange solid. Purification by silica
gel chromatography (hexane/EtOAc, 8:1 to 2:1 gradient) af-
forded 0.70 g (56%) of 11 as a bright yellow solid, identical to
samples prepared previously.8b
(()-5-(3-Ch lor op h en yl)-9-flu or o-1,2-d ih yd r o-2,2,4-t r i-
m eth yl-5H-ch r om en o[3,4-f]qu in olin e (2). In a 500-mL
three-neck flask, a solution of 3-bromochlorobenzene (7.0 mL,
60 mmol, 7.0 equiv) in THF (75 mL) was cooled to -69 °C
(internal temp). A 2.5 M solution of n-BuLi in hexanes (24
mL, 60 mmol, 7.0 equiv) was added via syringe pump over a
45-min period (internal temperature e-68 °C) and the reac-
tion mixture maintained at e-68 °C for 30 min. A solution
of 11 (2.64 g, 8.53 mmol) in THF (30 mL) was added via syringe
pump over a 45-min period (internal temperature <-64 °C)
and the reaction mixture maintained at e-65 °C for 30 min.
A 1:1 solution of THF/H2O (40 mL) was added slowly via
syringe, and the reaction mixture was allowed to warm to room
temperature and poured into saturated NH4Cl (100 mL). The
product was extracted with EtOAc (3 × 100 mL); the extracts
were washed with brine (1 × 100 mL), combined, dried
(MgSO4), filtered, and concentrated to afford an orange oil.
Purification by silica gel chromatography (hexanes/EtOAc, 2:1)
afforded 3.42 g (95%) of the hemiacetal as an orange oil, which
was not characterized.8
tory isomers. These in vitro progestational effects were
comfirmed in two animal models, while in a third, the
mammary bud assay, the nonsteroidal compounds were
much less active. Both (-)-(S)-2 and (-)-(S)-3-sulfate
had progestational activity in the uterine wet weight
assay comparable to MPA, the most widely prescribed
marketed progestin. In the breast, however, both of the
nonsteroidal compounds were at least 6-fold less effica-
cious compared to MPA at high doses, indicating a
potentially useful tissue-selective effect.
In the pregnancy maintenance assay, (-)-(S)-2 was
able to completely reverse the antifertility effect of the
potent steroidal antiprogestin mifepristone (RU486) at
a daily oral dose equal to or above 1.0 mg (30 mg/kg);
(-)-(S)-3-sulfate was less efficacious and only produced
a partial reversal in this study. Although MPA showed
an effect similar to (-)-(S)-3-sulfate, this steroid re-
quired a dose 10 times higher than the nonsteroidal
compound. While both test compounds are more potent
than MPA, (-)-(S)-2 is both more potent and more
efficacious. These results decisively demonstrate that
the in vivo activities of the two nonsteroidal progester-
one receptor agonists {(-)-(S)-2 and (-)-(S)-3-sulfate}
are progestational in nature and can specifically mimic
the action of the natural hormone in regulating preg-
nancy. These are the first nonsteroidal compounds
reported to demonstrate this activity.
The levorotatory S enantiomers of the nonsteroidal
progestins LG120746 (2) and LG120747 (3) have potent
hPR activity in vitro that translates well to the in vivo
situation. As would be anticipated from their nonste-
roidal structure, these compounds display novel phar-
macology in that the potent progestational effects
observed in the rodent uterus are not observed in the
breast. Moreover, the biological activity of these non-
steroidal selective progesterone receptor modulators was
demonstrated in a mouse pregnancy maintenance assay,
a definitive assay for in vivo progestational effects.
In
a 200-mL round bottom flask, the hemiacetal was
dissolved in CH2Cl2 (50 mL) and treated with Et3SiH (8.1 mL,
51 mmol, 6.0 equiv) and BF3-OEt2 (6.45 mL, 51 mmol, 6.0
equiv). The reaction mixture turned deep green and was
stirred at room temperature for 4 h. The reaction mixture was
quenched by addition of saturated NaHCO3 (50 mL) added all
at once; the resulting biphasic mixture was stirred vigorously
for 15 min, diluted with H2O (50 mL), and extracted with
EtOAc (2 × 100 mL). The extracts were washed with brine
(1 × 100 mL), combined, dried (Na2SO4), filtered, and concen-
trated. Purification by silica gel chromatography (hexane/
EtOAc, 10:1 to 6:1 gradient) afforded 2.78 g (84%, 80% for two
steps) of (()-2 as a white solid. The analytical data matched
that of samples prepared previously.8b
Exp er im en ta l Section 24
Meth yl (5′-F lu or o-2′-m eth oxy-4-n itr o-2-bip h en yl)ca r -
boxyla te (8). In a 500-mL flask, a solution of methyl 2-bromo-
5-nitrobenzoate (11.6 g, 44.6 mmol) in DME (60 mL) was
treated with tetrakis(triphenylphosphine)palladium (1.54 g,
1.33 mmol, 3.0 mol %). The reaction mixture was stirred at
room temperature for 10 min. Cesium fluoride (21.4 g, 141
mmol, 3.10 equiv) was added, followed by a solution of 5-fluoro-
2-methoxyphenylboronic acid8b (12 g, 71 mmol, 1.5 equiv) in
DME (40 mL). The reaction mixture was heated to 80 °C for
6 h, cooled to room temperature, poured into 2.0 M Na2CO3
(200 mL), and extracted with EtOAc (3 × 200 mL). The
extracts were washed with brine (1 × 100 mL), combined, dried
(MgSO4), filtered, and concentrated to an orange oil. Purifica-
tion by silica gel chromatography (hexane/EtOAc, 9:1) afforded
10.4 g (76%) of 8 as a yellow-orange solid, identical to samples
prepared previously.8b
8-Am in o-2-flu or o-6H-d iben zo[b,d ]p yr a n -6-on e (10). In
a 500-mL round bottom flask, a slurry of 2-fluoro-8-nitro-6H-
dibenzo[b,d]pyran-6-one8b (4.5 g, 17.4 mmol) in 95% EtOH/
water (250 mL) was treated with zinc dust (5.1 g, 78.3 mmol,
4.5 equiv) and CaCl2‚2H2O (5.1 g, 34.8 mmol, 2 equiv). The
reaction mixture was heated to reflux for 15 h, filtered hot
through a bed of Celite, and concentrated to half volume. The
solution was diluted with EtOAc (400 mL) and washed with
2% HCl (2 × 100 mL) and brine (1 × 100 mL). The aqueous
layers were extracted with EtOAc (2 × 250 mL). The combined
organic layers were dried (MgSO4), filtered, and concentrated
to afford 3.70 g (93%) of the aniline 10 as a yellow solid,
identical to samples prepared previously.8b
(()-5-(4-Ch lor o-3-m eth ylp h en yl)-9-flu or o-1,2-d ih yd r o-
2,2,4-tr im eth yl-5H-ch r om en o[3,4-f]qu in olin e (3). In
a
500-mL three-neck flask, a solution of 5-bromo-2-chlorotoluene
(12 g, 58 mmol, 6.0 equiv) in THF (100 mL) was cooled to -69
°C (internal temperature). A 2.5 M solution of n-BuLi in
hexanes (23 mL, 58 mmol, 6.0 equiv) was added via syringe
pump over a 45-min period (internal temperature e-65 °C)
and the reaction mixture maintained at e-65 °C for 30 min.
A solution of 11 (3.0 g, 9.7 mmol) in THF (50 mL) was added
via syringe pump over a 45-min period (internal temperature
<-68 °C) and the reaction mixture maintained at e-65 °C
for 30 min. A 1:1 solution of THF/H2O (40 mL) was added
slowly via syringe, and the reaction mixture was allowed to
warm to room temperature and poured into saturated NH4Cl
(100 mL). The product was extracted with EtOAc (3 × 100
mL); the extracts were washed with brine (1 × 100 mL),
combined, dried (MgSO4), filtered, and concentrated to afford
an orange solid. The solid was dissolved in EtOAc (50 mL)
and triturated with hexanes (300 mL) to afford 3.7 g (88%) of
the hemiacetal as an orange solid, which was not character-
ized.8
In
a 200-mL round bottom flask, the hemiacetal was
dissolved in CH2Cl2 (50 mL) and treated with Et3SiH (8.1 mL,
51 mmol, 6.0 equiv) and BF3-OEt2 (6.45 mL, 51 mmol, 6.0
equiv). The reaction mixture turned deep green and was
stirred at room temperature for 12 h. The reaction mixture

2784 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Edwards et al.
was quenched by addition of saturated NaHCO3 (50 mL) added
all at once; the resulting biphasic mixture was stirred vigor-
ously for 15 min, diluted with H2O (50 mL), and extracted with
EtOAc (2 × 100 mL). The extracts were washed with brine
(1 × 100 mL), combined, dried (Na2SO4), filtered, and concen-
trated. Purification by silica gel chromatography (hexane/
EtOAc, 10:1 to 6:1 gradient) afforded 3.1 g (87%, 76% for two
steps) of (()-3 as a white foam. The analytical data matched
that of samples prepared previously.8b
HP LC Sep a r a tion of En a n tiom er s of 2 a n d 3. An (R,R)-
Whelk-O1 preparative column (10 µm particle size, 21.1 × 250
mm; Regis Technologies, Inc.) on a Waters Delta Prep 4000
was equilibrated with an eluent of 3% Et3N in heptane/CH2-
Cl2 (90:10) at a flow rate of 15 mL/min. A 1 g/10 mL solution
of 2 in heptane/CH2Cl2 (70:30) was prepared, and 4.0 mL of
this solution was injected per run. Compound elution was
monitored by absorbance detection at 275 nm. The retention
time of the first peak was approximately 18 min. The first-
eluting enantiomer (levorotatory) was collected until the
absorbance began to decline on the LCD at which point the
mixed fractions were collected separately. Once the absor-
bance had returned to a fixed height, the second (dextrorota-
tory) enantiomer was collected and the flow rate was increased
to 40 mL/min until all of the second enantiomer had been
collected. The flow rate was lowered to 15 mL/min, and
another injection was made. The purity of the combined
fractions was 98% ee with 85-90% recovery. Mixed fractions
were combined and resubjected to the HPLC conditions. The
enantiomers of 3 were resolved in an identical manner to that
described above for 2; 98% ee, 80-85% recovery.
The NMR data matched that of (-)-3-sulfate described above.
(R,R)-Whelk-O1; 1.2 mL/min (heptane/CH2Cl2, 80:20) tR ) 6.3
min, 99% ee; [R]22D ) +190°; mp >230 °C dec. Anal. (C26H25
ClFNO5S-H2O) C, H, N.
-
In Vivo Assa ys. Uter in e Wet Weigh t Assa ys. Immature
21-day-old female Harlan Sprague-Dawley rats (Harlan Spra-
gue Dawley Inc., Indianapolis, IN) were housed and com-
pounds were administered under similar conditions as de-
scribed below for the mammary end bud assay. Briefly, on
day 1 of the assay 17â-estradiol (Sigma; St. Louis, MO)
dissolved in 100% ethanol and diluted in corn oil was admin-
istered sc in an injection volume of 0.1 mL. On the second
day of the assay test compounds were dissolved in corn oil
(Mazola) in a similar manner and administered by oral gavage
in 0.5 mL of vehicle. 24 h after administration of test
compounds animals were euthanized, and uteri were excised
and blotted dry on filter paper before weighing.
Ma m m a r y Lobu la r -Aveola r Bu d P r olifer a tion Assa y.
Sprague-Dawley (4-5-week-old, ∼100 g) female rats (HSD,
Indianapolis, IN.) were ovariectomized by the vendor (bilateral-
dorsal surgery) and allowed to acclimate for at least one week.
All animals were given food (Harlan Teklab LM485-7012,
Indianapolis, IN) and acidified water ad libitum throughout
the experiment. Test compounds or progestin standard (MPA,
medroxy progesterone acetate; Sigma, St. Louis, MO) were
dissolved in 10% EtOH and corn oil (Mazola) and kept at room
temperature. Estrone (Sigma, St. Louis, MO) was dissolved
in 100% EtOH and corn oil with the EtOH blown off under
nitrogen. Animals were randomized into groups of four or five
animals and treated for 3 consecutive days with estrone sc (10
µg/animal) and test compounds po in a 0.5 mL volume of
vehicle. Estrone-only treated animals were given oral vehicle
alone.
(-)-2: To a 250-mL round bottom flask was added 5.95 g of
(-)-2 prepared above and diethyl ether (30 mL). Mild warming
resulted in complete dissolution. To this solution was added
hexane (150 mL), and the flask was placed on a rotary
evaporator and the ether was removed. During the evapora-
tion crystallization began to occur and the flask was removed
and cooled to -4 °C for approximately 3 h. The mother liquors
were removed, and the pale yellow crystals were rinsed with
cold hexane (3 × 75 mL). The crystals were then collected
and dried in vacuo (1 Torr) for 20 h to afford 5.0 g of (-)-2.
Data for (-)-2: mp 134-136 °C; HPLC (R,R)-Whelk-O1 (5 µm
particle size, 4 × 250 mm, EM Separations); 1.0 mL/min
After 3 days of consecutive dosing (on the morning of the
fourth day) animals were given BrdU (Sigma, St. Louis, MO)
200 mg/kg ip in a volume of 2 mL of phosphate-buffered saline
(PBS). Three hours after BrdU administration animals were
euthanized with carbon dioxide asphyxiation and necropsied
for mammary glands. Right inguinal mammary glands were
dissected, taking care to remove the majority of adjacent fat.
Mammary gland was embedded in OCT freezing media and
frozen sections were cut at 5 µM thickness. Samples were
stored at -20 °C until final processing.
(heptane/CH2Cl2, 80:20) tR ) 6.3, 99.5% ee; [R]22 ) -372.9°.
D
Anal. (C25H21ClFNO) C, H, N.
Slides were fixed for 10 min at 4 °C in a cold solution of
acetone/methanol/formaldehyde (19:9:2), washed with PBS for
10 min at room temperature, and denatured in 0.1 N HCl for
1 h at 37 °C in a water bath under gentle shaking. Slides
were neutralized in 0.1 M borate buffer, pH 8.5, at room
temperature for 2 × 15 min under gentle agitation and washed
in PBS for 10 min at room temperature. Samples were blocked
by incubating with 3% bovine serum albumin (BSA) and 3%
normal goat serum (NGS) in PBS for 30-60 min at room
temperature. Incubation of samples with a primary antibody
[anti BrdU mouse monoclonal IgG1; (Becton Dickinson) at a
dilution of 1:50 with blocking solution] was completed at 1 h
at room temperature. Samples were then washed with PBS
for 2 × 5 min at room temperature and incubated with
secondary antibody [goat anti-mouse (GAM) IgG conjugated
with FITC molecules (Southern Biotechnology Associates,
Inc.)], diluted to 1:100 with blocking solution for 1 h at room
temperature. Slides were evaluated for FITC staining by
capturing digitized images of mammary gland using a Lieca
flouresence microscrope (model DMRB) and a Optronix CCD
camera system (model VI-470, Goleta, CA). Digitized images
were downloaded to a HP P166 computer with a Oculus TCX
frame grabber (Coreco, Quebec, Canada). Images were quan-
tified using image analysis software Optimas 6.0 Seattle, WA).
Raw data was generated in a blinded fashion and restricted
to areas containing lobular buds from each field (3 fields/slide).
Labeling index for each field was generated as labeled area
divided by lobular areas selected within the field and expressed
as a fold increase over estrone alone treated animals.
(+)-2: (+)-2 was recrystallized in a manner identical to that
described above for (-)-2: 5.91 g of HPLC-purified (+)-2
afforded 4.75 g of recrystallized (+)-2. Data for (+)-2: mp
134-135 °C; HPLC (R,R)-Whelk-O1; 1.0 mL/min (heptane/
CH2Cl2, 80:20) tR ) 7.7 min, 99.8% ee; [R]22D ) +373.9°. Anal.
(C25H21ClFNO) C, H, N.
(-)-3-Su lfa te: The free base (-)-3 (5.4 g, 13 mmol) was
dissolved in 150 mL of anhydrous ether and was titrated with
concentrated H2SO4 ether solution (1/10 volume) until no
further precipitation was observed (8 mL of the solution was
used, ∼14 mmol). The salt was filtered and washed with ether
(2 × 30 mL). The solid salt was dissolved in methanol (30
mL) and was concentrated and dried to give 6.8 g of white
foam (> 98% mass recovery, 96% ee). The white foam was
dissolved in CH2Cl2 (260 mL) and diluted slowly with pentane
(230 mL). The solution was left standing at room temperature
overnight to afford a white crystalline solid. The solid was
filtered, washed with 2:1 mixture of pentane and CH2Cl2 (3 ×
15 mL), and dried (1 Torr) to afford 6.2 g of (-)-3-hydrogen
sulfate monohydrate (93% recovery): (R,R)-Whelk-O1; 1.2 mL/
min (heptane/CH2Cl2, 80:20) tR ) 4.9 min, 99% ee; [R]22
)
D
-197°; mp >230 °C dec; 1H NMR (400 MHz, CD3OD) 7.98 (bs,
1H), 7.62 (bs, 1H), 7.55 (bs, 1H), 7.18 (d, J ) 8.3, 1H), 7.07 (s,
1H), 6.97 (bs, 1H), 6.95-6.82 (m, 3H), 5.99 (bs, 1H), 2.21 (s,
3H), 2.08 (s, 3H), 1.51 (s, 3H), 1.45 (s, 3H). 13C NMR (100
MHz, CD3OD) 138.4, 137.4, 136.0, 132.0, 130.1, 128.4, 126.0,
124.5, 120.8, 76.0, 51.7, 24.3, 23.1, 20.1. Anal. (C26H25
-
ClFNO5S‚H2O) C, H, N.
(+)-3-Su lfa te. The procedure as described above afforded
1.2 g of (+)-3-hydrogen sulfate monohydrate (81% recovery).
Sta tistica l Meth od s. Statistical significance was evalu-
ated in all bioassays using ANOVA followed by one-tailed

Progesterone Receptor Agonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2785
D.; Kiddoe, M.; Kraft, P.; Lai, M. T.; Campen, C.; Palmer, S.;
Phillips, A. Synthesis and Progesterone Receptor Binding Af-
finity of Substituted 1-Phenyl-7-benzyl-4,5,6,7-Tetrahydro-1H-
Indazoles. Bioorg. Med. Chem. Lett. 1997, 7, 2551-2556.
(7) (a) Clemm, D. L.; Macy, B. L.; Santiso-Mere, D.; McDonnell, D.
P. Definition of the Critical Cellular Components Which Dis-
tinguish Between Hormone and Antihormone Activated Proges-
terone Receptor. J . Steroid Biochem. Mol. Biol. 1995, 53, 487-
495. (b) Edwards, D. P.; Altmann, M.; DeMarzo, A.; Zhang, Y.;
Weigel, N. L.; Beck, C. A. Progesterone Receptor and the
Mechanism of Action of Progesterone Antagonists. J . Steroid
Biochem. Mol. Biol. 1995, 53, 449-458.
(8) (a) Zhi, L.; Tegley, C. M.; Kallel, E. A.; Marschke, K. B.; Mais,
D. E.; Gottardis, M. M.; J ones, T. K. 5-Aryl 1,2-Dihydro-
chromeno[3,4-f]quinolines, A Novel Class of Nonsteroidal Human
Progesterone Receptor Agonists. J . Med. Chem. 1998, 41, 291-
302. (b) Edwards, J . P.; West, S. J .; Marschke, K. B.; Mais, D.
E.; Gottardis, M. M.; Jones, T. K. 5-Aryl-1,2-dihydro-5H-chromeno-
[3,4-f]quinolines as Potent, Orally Active, Nonsteroidal Proges-
terone Receptor Agonists: The Effects of D-Ring Substituents.
J . Med. Chem. 1998, 41, 303-310.
(9) Christensen, K.; Estes, P. A.; On˜ate, S. A.; Beck, C. A.; DeMarzo,
A.; Altmann, M.; Lieberman, B. A.; St. J ohn, J .; Nordeen, S. K.;
Edwards, D. P. Characterization and Functional Properties of
the A and B Forms of Human Progesterone Receptors Synthe-
sized in a Baculovirus System. Mol. Endocrinol. 1991, 5, 1755-
1770.
(10) The human progesterone receptor exists as two forms, differing
only at the N-terminus, the B isoform (116 kDa) and the A
isoform (94 kDa). Emerging evidence suggests differential
biological roles for these two receptors, see: (a) Wen, D. X.; Xu,
Y.-F.; Mais, D. E.; Goldman, M. E.; McDonnel, D. P. The A and
Dunnet’s t test using SuperANOVA (Abacus Concepts Inc,
Berkely,CA).
P r egn a n cy Ma in ten a n ce Assa y. Mature virgin ICR mice
(10-12-weeks-old; Harlan Sprague Dawley Inc., Indianapolis,
IN) were housed in a light (14 h light:10 h darkness; lights off
at 20.00 h) and temperature (22 °C) controlled room, and fed
and watered ad libitum (Diet LM-485, Teklad, Madison, WI).
Females were caged with fertile males of the same strain
overnight, and examined the following morning for vaginal
plugs (day 1 of pregnancy). Mating was assumed to have
taken place at 02.00 h (time 0).25 Pregnant mice were treated
orally with 0.5 mg of RU486 (∼15 mg/kg) alone, or in
combination with different doses of MPA, (-)-2 or (-)-3-sulfate,
between days 2 and 4 of pregnancy. Compounds were dis-
solved in 100% ethanol and diluted with corn oil to appropriate
concentrations to give an injection volume of 0.1 mL. Control
animals received an equivalent volume of corn oil. Autopsies
were carried out at day 8 of pregnancy and the number of
implantation sites recorded.
Ack n ow led gm en t. We thank Dr. Keith Marschke
for hPR-B cotransfection experimental data, Dr. Dale
Mais for hPR-A and hGR binding data, and Murriel
Wagoner for assistance in animal experiments. The
authors also thank Professor William Pirkle (University
of Illinois at Urbana-Champaign) for his invaluable
assistance during the optimization phase of the chiral
HPLC separation of rac-2 and rac-3.
B
Isoforms of the Human Progesterone Receptor Operate
through Distinct Signaling Pathways within Target Cells. Mol.
Cell Biol. 1994, 14, 8356-8364. (b) Vegeto, E.; Shahbaz, M. M.;
Wen, D. X.; Goldman, M. E.; O’Malley, B. W.; McDonnell, D. P.
Human Progesterone Receptor A Form is a Cell- and Promoter-
Specific Repressor of Human Progesterone Receptor B Function.
Mol. Endocrinol. 1993, 7, 1244-1255. (c) Chalbos, D.; Galtier,
F. Differential Effect of Forms A and B of Human Progesterone
Receptor on Estradiol-dependent Transcription. J . Biol. Chem.
1994, 269, 23007-23012.
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