A tissue-selective nonsteroidal progesterone receptor modulator: 7,9-difluoro-5-(3-methylcyclohex-2-enyl)-2,2,4-trimethyl-1,2-dihydrochromeno[3, 4-f]quinoline

Article abstract of DOI:10.1021/jm8004256

The progesterone receptor plays an important role in the female reproductive system. Here we describe the discovery of a new selective progesterone receptor modulator (SPRM). In rats, the lead compound, 7,9-difluoro-5-(3-methylcyclohex-2-enyl)-2,2,4-trimethyl-1,2-dihydrochromeno[3, 4-f]quinoline (5c), inhibited ovulation and showed full efficacy in uterine and vaginal tissue but was a mixed partial agonist/antagonist in breast tissue. The compound also suppressed ovulation in monkeys, but in contrast to currently approved steroidal PR agonists, it did not suppress estradiol levels.

Full text of DOI:10.1021/jm8004256

3696
J. Med. Chem. 2008, 51, 3696–3699
Scheme 1a
A Tissue-Selective Nonsteroidal Progesterone
Receptor Modulator: 7,9-Difluoro-5-(3-
methylcyclohex-2-enyl)-2,2,4-trimethyl-1,2-
dihydrochromeno[3,4-f]quinoline
Bijan Pedram,† Arjan van Oeveren,*,† Dale E. Mais,†
Keith B. Marschke,† Pieter M. Verbost,‡ Marinus B. Groen,‡
and Lin Zhi†
DiscoVery Research, Ligand Pharmaceuticals Inc., 10275 Science
Center DriVe, San Diego, California 92121, Lead Optimization Unit, N.
V. Organon, Molenstraat 110, 5340 BH Oss, The Netherlands
a Reagents: (a) (i) DIBAL-H, THF, -78°C, (ii) MeOH, p-TsOH, 71%;
(b) BF3 ·OEt2, CH2Cl2 55-90%; (c) m-CPBA, CH2Cl2, 4%.
ReceiVed April 14, 2008
Scheme 2a
Abstract: The progesterone receptor plays an important role in the
female reproductive system. Here we describe the discovery of a new
selective progesterone receptor modulator (SPRM). In rats, the lead
compound, 7,9-difluoro-5-(3-methylcyclohex-2-enyl)-2,2,4-trimethyl-
1,2-dihydrochromeno[3,4-f]quinoline (5c), inhibited ovulation and
showed full efficacy in uterine and vaginal tissue but was a mixed partial
agonist/antagonist in breast tissue. The compound also suppressed
ovulation in monkeys, but in contrast to currently approved steroidal
PR agonists, it did not suppress estradiol levels.
a Reagents: (a) (i) Me2PhSiLi, CuCN, MeLi, THF, (ii) PhN(Tf)2,
45-78%; (b) Pd(OAc)2, PPh3, Et3N, HCO2H, DMF, 65-75%.
medroxyprogesterone acetate (MPA). Selective progesterone
receptor modulators (SPRMs) can potentially alleviate some of
the risks associated with currently available progestins. In an
effort to further optimize a series of selective PR modulators,4
we investigated 5-cycloalkenyl substituted chromenoquinolines
that have been reported as very efficacious and potent gluco-
corticoid receptor (GR) modulators developed from the PR
template.5 Here we report results on the series of SPRMs that
demonstrated tissue selectivity in various animal models. The
lead compound 5c is a highly potent and efficacious selective
PR modulator and demonstrated full efficacy in a rat ovulation
inhibition assay. The compound was a partial agonist/antagonist
in breast tissue.
Scheme 1 describes the synthesis of the target compounds 4,
5, and 6. Reduction of the lactone 15 with diisobutylaluminium
hydride followed by treatment with methanol in the presence
of a catalytic amount of para-toluenesulfonic acid afforded the
stable methoxyacetal 2 as a racemic mixture. Nucleophilic
addition of cycloalkenylsilanes 3 to the oxycarbenium ion
generated from this acetal was used to prepare 5-cycloalkenyl
compounds 4 and 5. Racemic substituted cyclohexenylsilanes
3 were prepared in a two-step procedure as described in Scheme
2. Copper mediated conjugate addition of PhMe2SiLi to a
cyclohexenone (8) and trapping the enolate with phenyltriflimide
afforded the enol triflate (9).6 The triflate was reduced to the
cyclohexenyl silane (3) with palladium acetate and formic acid/
triethylamine.7 Addition of the substituted allylic silanes (3)
furnishes products with two new chiral centers, and generally
a mixture of two racemic diastereomers was obtained. The anti
(4) and syn (5) diastereomers were separated by chromatographic
methods initially. When tested in various biological assays (vide
infra), it was found that the syn diastereomer of 5-(3-methyl-
cycohex-2-enyl)-chromenoquinolines was much more active
than the anti diastereomer. Therefore we sought to improve the
diastereoselectivity in the addition reaction in favor of the syn
diastereomers (5). When we initially prepared these compounds
via a Lewis acid catalyzed reaction of methoxy acetal 2 with
dimethyl(1-methylcyclohex-2-enyl)phenylsilane, the syn/anti
The progesterone receptor (PRa) is a member of the nuclear
receptor superfamily of ligand-dependent transcription factors.
Two major isoforms of the progesterone receptor, PR-A and
PR-B, have been identified.1 The progesterone receptor is mainly
expressed in female reproductive tissues, and the actions in these
tissues have been the target of drugs acting on PR. Synthetic
steroidal PR agonists, in combination with estrogens, have been
widely used in oral contraception (OC), female hormone
replacement therapy (HRT), and the treatment of a number of
female reproductive disorders for decades. PR antagonists, like
mifepristone, effectively terminate pregnancy and offer thera-
peutic promise for endometriosis, uterine fibroids, and breast
cancer.2 Recently, clinical data on asoprisnil, a selective PR
modulator, has been reported, targeting endometriosis.3 Like
the natural ligand progesterone, all of the currently marketed
progestins possess some degree of cross-reactivity with other
steroid hormone receptors, especially androgen and glucocor-
ticoid receptor, which can cause potential side-effects. Addition
of a progestin in estrogen replacement therapy has proven to
significantly reduce endometrial cancer risk due to its anties-
trogenic activity in the uterus. However, in breasts, progestins
demonstrate stimulative activity in addition to the proliferative
effect of estrogens. This has raised some concern about a
potential breast cancer risk for women with long-term use of
progestins in OC and HRT in combination with estrogens even
though the clinical results have been controversial. The latest
results from the Women’s Health Initiative confirmed the
increased breast cancer risk for women who used combined
hormone preparation of conjugated equine estrogens and
* To whom correspondence should be addressed. Phone: 858-550-7870.
Fax: 858-550-7249. E-mail: avanoeveren@ligand.com.
†
Discovery Research, Ligand Pharmaceuticals Inc.
Lead Optimization Unit, N. V. Organon.
‡
a Abbreviations: PR, progesterone receptor; SPRM, selective progesterone
receptor modulator; MPA, medroxyprogesterone acetate; OC, oral contra-
ception; HRT, hormone replacement therapy; MCPBA, meta-chloroper-
benzoic acid; OVX, ovariectomized.
10.1021/jm8004256 CCC: $40.75
2008 American Chemical Society
Published on Web 06/14/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3697
Table 1. Cotransfection, T47D Alkaline Phosphatase Assay, and Competitive Binding Data for SPRMs, Progesterone, and MPA
hPR T47D
assay agonist
EC50 (nM)
hPR T47D
assay agonist
Eff (%)
hPR agonist
EC50 (nM)
hPR binding
Ki (nM)
compound
R1
R2
hPR agonista Eff (%)
5a (rac, syn)
5b (rac, syn)
4b (rac, anti)
(-)-5c (syn)
(+)-5d (syn)
5e (rac,syn)
(-)-6
H
H
H
H
H
H
CH3
3.4 ( 0.4
0.6 ( 0.2
5.4 ( 0.7
0.5 ( 0.1
50.8 ( 3.1
10.7 ( 1.6
0.4 ( 0.1
0.5 ( 0.0
0.5 ( 0.3
171 ( 32
162 ( 12
100 ( 16
145 ( 12
11 ( 5
97 ( 17
177 ( 15
100
27 ( 3
66 ( 4
54 ( 3
63 ( 3
55 ( 2
4.8 ( 0.6
3.7 ( 0.5
14
CH3
CH3
CH3
CH3
CH3
3.5 ( 0.9
33 ( 4
4.3 ( 0.9
4.4 ( 1.8
>1000
96 ( 9
66 ( 5
59 ( 6
100
n.d.
2.8 ( 0.3
2.2 ( 0.1
0.4 ( 0.1
1.2 ( 0.2
3.4 ( 0.1
1.4 ( 0.3
progesterone
MPA
98 ( 11
98 ( 6
a Agonist efficacies were determined relative to progesterone (100%), a hyphen indicates efiicacy <10%.
Scheme 3a
of the alkene (-)5c with MCPBA, but oxidation mainly took
place at the dihydroquinoline double bond and the epoxide 6
was only a minor product and isolated in 4% yield.
Table 1 summarizes the SAR results of several cycloalkenyl
substituted dihydrochromeno-quinolines in cotransfection and
competitive binding assays. In line with previous findings on
D-ring SAR,10 it was found that 7,9-difluoro substituents were
superior to other D-ring substitution patterns tested,5b and the
results shown here are for compounds with a 7,9-difluoro
substituted D-ring. As can be seen from the data, the substituents
on the cycloalkene ring have a significant influence on the
activity of the compounds. The unsubstituted cyclohexenyl
compound 5a showed quite good efficacy and potency, whereas
the 2,3 dimethylcyclohexenyl analog, 5e, was less efficacious
and potent. The 3-methylcyclohexenyl (5b) is most optimal and
showed the best activity with more than 5-fold EC50 (0.6 nM)
improvement over 5a (3.4 nM). As we have pointed out, the
stereochemistry of the compounds affects their biological
activity, the anti-isomer 4b is much less active (EC50 of 5.4
nM) than the syn-isomer 5b, although there is no difference
between their binding affinities. For the compounds that were
tested in enantiomerically pure form, it was clear that almost
all of the activity resides in one of the enantiomers, which is
consistent with the results of all other related series. As can be
seen, eutomer (-)5c has activity almost identical to racemate
compound 5b and distomer (+)5d is inactive. Epoxide 6 showed
very good in vitro biological activity, indicating the epoxide
can be used as a bioisostere of olefin that is an essential piece
of the activity. However, we did not pursue this compound
further because of its complexity of synthesis.
a Reagents: (a) Phenylisocyanate, Et3N, DMAP, Et2O, 0°C, 70%; (b)
n-Butyllithium, Et2O -85°C, 50%; (c) (t-Bu)2PhSiLi, CuI, PPh3, THF, 80%.
products were obtained in a 2:1 ratio. Changing the reaction
conditions, by varying temperature, Lewis acid, stoichiometry,
solvent, concentration, order of addition, and rate of addition,
led only to minor improvements in the syn/anti ratio of the
product. It was found later that the syn/anti ratio of the product
was highly dependent on the size of the silane substituent of
the 1-methylcyclohex-2-enylsilanes and using the bulky diphen-
yl-t-butylsilane improved the diastereoselectivity of the reaction
significantly to furnish the addition product with an 8:1 syn/
anti ratio. As can be seen in Table 1, the (5S)-enantiomer (-)5c
had much greater hPR activity than the (5R)-enantiomer (+)5d.
As a result, we focused our attention on developing an
enantioselective synthesis of product (-)5c. We were pleased
to find that the Lewis acid promoted reaction of the chiral silane
3a with the racemate methoxyacetal 2 is highly stereospecific.
In fact, when enantiomerically enriched silane was used, the
products 4 and 5 that were obtained had the same enantiomeric
ratio as the starting silane, and we were not able to detect any
other enantiomer by analytical chiral HPLC when enantiomeri-
cally pure allylsilane was used. The required enantiomerically
pure cyclohexenylsilane 3a was obtained from the enantiomeri-
cally pure 2-iodo-3-methylcyclohex-2-enol8 9 following litera-
ture procedures for similar systems (Scheme 3). Conversion of
the alcohol to the carbamate with phenylisocyanate, followed
by an SN2 displacement with the silylcuprate was regio- and
enantiospecific9 and afforded the enantiomerically pure cy-
cloalkenylsilane 3a. The epoxide 6 was obtained from treatment
Compound (-)5c was one of the most efficacious (145% of
progesterone) and potent compounds tested in the cotransfection
assay, and it also had excellent binding affinity for the human
progesterone receptor. When tested for PR regulated alkaline
phosphatase activity in the T47D breast cancer cell line, which

3698 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Letters
Figure 3. Effect of (-)5c (open diamonds) and 11 (open circles) in
combination with MPA (1 mg/kg) and estrone on uterus (A), vagina
(B), and breast (C) epithelium in ovariectomized rats. Effect of MPA
and estrone is shown as black circles. Open bar represents effect of
vehicle and hatched bar represents effect of estrone treatment.
Figure 1. Ovulation inhibition by MPA (black bar) or (-)5c (striped
bar) in rats. Rats (n ) 6) were dosed with vehicle or progestin for 5
days. The number of ova per rat and the number rats ovulating were
determined.
Figure 2. Progestational effects of MPA (black dots) or (-)5c (open
diamonds) in combination with estrone on uterus (A), vagina (B), and
breast (C) epithelium in ovariectomized rats. Open bar represents effect
of vehicle and hatched bar represents effect of estrone treatment.
was used as a surrogate marker for breast activity, the compound
showed only partial efficacy (55%), whereas the steroidal
reference compounds (progesterone, MPA) showed full efficacy
in this assay. Cross-reactivity of (-)5c with other steroidal
hormone receptors was also tested in a cotransfection assay,
and the compound was found to exhibit more than 100-fold
selectivity toward the hPR versus human androgen receptor
(hAR), human mineralocorticoid receptor (hMR), and human
estrogen receptor alpha (hERR), with weak antagonist activity
on the human glucocorticoid receptor (hGR), but no in vivo
antiglucocorticoid activity was observed at doses up to 30 mg/
kg.11 (cotransfection assay data in Supporting Information).
In the classic Clauberg-McPhail assay to read in vivo
progestational activity in rabbit uterine epithelium, compound
(-)5c was active at an oral dose of 0.10 mg/kg for 3 days.
(data not shown).
(-)5c was tested to determine its ability to inhibit ovulation
in the female rat (Figure 1). (-)5c was able to dramatically
reduce the number of ova at doses of 0.25, 1.0, or 2.0 mg/kg.
To determine tissue selectivity of (-)5c, the effects on uterine
epithelium cell height, vaginal epithelial thickness, and mam-
mary alveolar bud proliferation in estrogen treated ovariecto-
mized (OVX) rats were measured. While (-)5c shows similar
efficacy as MPA in the uterus and vagina, it demonstrated
significantly reduced efficacy and potency on breast alveolar
bud proliferation compared to MPA (Figure 2).
Figure 4. Ovulation inhibition by (-)5c in cynomolgus monkeys.
Estradiol (open squares) and progesterone (closed circles) were
measured in a cynomolgus monkey before and after 3 weeks of daily
treatment with 0.06 mg/kg (A) or 0.3 mg/kg (B) of (-)5c (stippled
bar).
in a dose response from 1 to 30 mg/kg. The results are shown
in Figure 3. As can be seen in this figure, (-)5c was able to
partially (∼60% efficacy) antagonize the effect of MPA in breast
tissue similar to a typical steroidal PR antagonist 11.11 (-)5c
had no impact on the effect of MPA on the uterine epithelium
cell height or vaginal epithelial thickness, whereas 11 totally
reversed the effect of MPA, thereby demonstrating the unique
tissue dependent partial agonist/antagonist activity of this
compound.
To demonstrate the ability of (-)5c to inhibit ovulation in
nonhuman primates, the compound was dosed orally in cyno-
molgus monkeys for 3 weeks. Four monkeys were given either
0.06 mg/kg or 0.3 mg/kg. Estradiol and progesterone levels were
measured as indicators of the ovulatory cycle. As can be seen
in Figure 4, daily dosing of (-)5c at 0.06 mg/kg (Figure 4A)
or 0.3 mg/kg (Figure 4B) resulted in suppression of progesterone
The reduced efficacy seen in breast tissue prompted us to
run the same experiment in antagonist mode. In this experiment
MPA was dosed at its ED50 of 1 mg/kg, and (-)5c was given

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3699
2003, 68, 1019–1032. (b) Chwalisz, K.; Perez, M. C.; Demanno, D.;
Winkel, C.; Schubert, G.; Elger, W. Selective progesterone receptor
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levels, indicating inhibition of ovulation. Similar results to those
shown in Figure 4 were seen in all monkeys (n ) 4).
Interestingly the estradiol levels in these animals were not fully
suppressed and followed a cyclic pattern during treatment. This
is in contrast to known steroidal progesterone agonists, where
a suppression of estradiol levels is observed. These results are
another indication of the unique activity of (-)5c in vivo.
In summary, (-)5c is a potent nonsteroidal selective hPR
modulator. The compound exhibits tissue selectivity, with full
suppression of estrogen induced uterine wet weight in rats but
partial agonistic/antagonistic effects on alveolar bud proliferation
in the same animals. Compound (-)5c is a potent inhibitor of
ovulation in cynomolgus monkeys but does not fully suppress
estrogen levels in contrast with steroidal progesterone agonists,
which provides an opportunity to develop progestin alone
contraceptives.
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M. J.; Carter, G.; Lane, B. C. trans-Activation and Repression
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2,5-Dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclo-
hexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and Its
Four Stereoisomers. Mol. Pharmacol. 2002, 62, 297–303. (b) Zhi, L.;
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Acknowledgment. We thank the departments of New Leads
and Molecular and Cell Biology at Ligand Pharmaceuticals for
performing the in vitro assays and the multiendpoint (uterus,
vagina, breast) in vivo assay, and the Pharmacology department
at Organon for all other in vivo assays.
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Supporting Information Available: Synthetic procedures,
chemical characterization data for compounds 4-6 and a description
of the biological assays, including ovulation inhibition results in
cynomolgus monkeys. This material is available free of charge via
the Internet at http://pubs.acs.org.
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