Quinol-4-ones as steroid A-ring mimetics in nonsteroidal dissociated glucocorticoid agonists

Article abstract of DOI:10.1021/jm061273t

We report on the nuclear receptor binding affinities, cellular activities of transrepression and transactivation, and anti-inflammatory properties of a quinol-4-one and other A-ring mimetic containing nonsteroidal class of glucocorticoid agonists.

Full text of DOI:10.1021/jm061273t

J. Med. Chem. 2006, 49, 7887-7896
7887
Quinol-4-ones as Steroid A-Ring Mimetics in Nonsteroidal Dissociated Glucocorticoid Agonists
John Regan,*,† Thomas W. Lee,† Rene´e M. Zindell,† Younes Bekkali,† Jo¨rg Bentzien,† Thomas Gilmore,†
Abdelhakim Hammach,† Thomas M. Kirrane,† Alison J. Kukulka,† Daniel Kuzmich,† Richard M. Nelson,† John R. Proudfoot,†
Mark Ralph,† Josephine Pelletier,‡ Donald Souza,‡ Lijiljana Zuvela-Jelaska,‡ Gerald Nabozny,‡ and David S. Thomson†
Departments of Medicinal Chemistry and Immunology & Inflammation, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road,
Ridgefield, Connecticut 06877
ReceiVed October 31, 2006
We report on the nuclear receptor binding affinities, cellular activities of transrepression and transactivation,
and anti-inflammatory properties of a quinol-4-one and other A-ring mimetic containing nonsteroidal class
of glucocorticoid agonists.
Introduction
Therefore, the search for GC agonists with a dissociated profile
(greater transrepression than transactivation activity) has ac-
celerated in recent years10,11 with an appreciation of the complex
molecular pathways and the anticipation of an improved safety
margin.12,13
During the past 50 years the successful treatment of inflam-
matory diseases has relied on the use of glucocorticoid (GC)a
agonists such as dexamethasone (1a) and prednisolone (1b)
(Chart 1). While effective in controlling asthma,1 rheumatoid
arthritis,2 and other disorders, GC therapy is fraught with a
number of severe side effects. The seriousness of the complica-
tions often hampers high dose and chronic administration. In
addition, cross reactivity with other steroid hormone receptors,
such as progesterone receptor (PR) and mineral corticoid
receptor (MR), of the commonly prescribed GCs can provoke
off-target pharmacology. In direct response to the current state
of GC therapy, finding drugs with good anti-inflammatory
properties and reduced side effects remains an ongoing quest.
A primary objective in our GC agonist anti-inflammatory
program is identifying nonsteroidal anti-inflammatory agents
with the added benefit of exhibiting dissociation. Toward this
end, we sought to discover pharmacophores capable of playing
the hydrogen bond acceptor’s role of the C-3 carbonyl of
dexamethasone that could be combined with a nonsteroidal
scaffold. Dissociated GC agonists 2a and 2b14 represent a good
starting point for such a venture as they contain a benzoxazinone
group as a readily replaceable A-ring mimetic.15,16 Other
important features include the alcohol as a steroidal C-11
hydroxy surrogate, a trifluoromethyl substituent to maintain
agonist activity,17 and a phenyl ring which is believed to reside
adjacent to the area of steroid D-ring binding in the glucocor-
ticoid receptor-ligand binding domain (GR-LBD). Expanding
the scope of this effort to include modifications to the phenyl
ring provides an opportunity to gauge whether changes to this
region of the scaffold are a viable option to attenuate nuclear
receptor potency and selectivity, as well as transrepression or
transactivation pathways. We envision preparing the target
molecules, represented by 5, by the opening of epoxide 3 (Chart
1) with a nucleophilic amine embedded in a lipophilic ring
containing a hydrogen bond acceptor functionality18 (i.e., atom
A in 4). The syntheses and biological profiles of compounds
such as 5 are the subject of this paper.
The probability of identifying a GC agonist with a better
safety profile compared to existing therapies has substantially
increased with newer understandings of the molecular mecha-
nism of action.3-8 After an agonist enters a target cell and binds
to the glucocorticoid hormone receptor (GR), the ligand-
activated complex (GRC) translocates into the nucleus where
direct and indirect functional pathways can be accessed. Acting
directly, the GRC serves as an endogenous transcription factor
by binding to specific DNA sequences and coactivator proteins,
thereby initiating transcription of metabolic and endocrine genes.
GRC-mediated transactivation of these genes is believed to
contribute to the side effect profile of GC therapy.9 Acting
indirectly, the GRC adopts a conformation with an affinity for
transcription factors (e.g., NF-kB and AP-1). Subsequent binding
to these transcription factors results in the inhibition of
expression of pro-inflammatory cytokines such as TNF-R and
IL-6. This process, known as transrepression, is thought to
contribute, in part, to the anti-inflammatory component of GCs.
Chemistry
The preparations of a quinol-4-one-containing analogue (11)
and a piperid-4-one (14) are shown in Scheme 1 as representa-
tive examples of 5. The key step in this sequence is the opening
of an epoxide (i.e., 9) with a nucleophilic amine. Briefly, the
preparation of epoxide 9 begins with the copper-mediated 1,4-
addition of Grignard reagent 6 to 1,1,1-trifluorobut-3-en-2-one
(7)19 to provide ketone 8. Trimethylsulfoxonium iodide and NaH
transform 8 to epoxide 9. Quinolone 11 is obtained by reacting
9 with 4-hydroxyquinoline (10) and sodium ethoxide in etha-
nol.20 Conversion of the methoxy group in 11 to a hydroxy in
12 is accomplished with BBr3. Piperidin-4-one analogue 14 is
prepared from the reaction of 9, cis-3,5-dimethyl-4-piperidone
hydrochloride (13)21,22 and K2CO3 in DMF.
* To whom correspondence should be addressed. Phone: (203) 798-
4768. Fax: (203) 791-6072. E-mail: jregan@rdg.boehringer-ingelheim.com.
† Department of Medicinal Chemistry.
‡ Department of Immunology & Inflammation.
a Abbreviations: AP-1, activator protein-1; DBF, dihydrobenzofuran;
DELFIA, dissociation-enhanced lanthanide fluorescent immunoassay; DMEM,
Dulbecco’s modified Eagle’s medium; D-gal, D-galactose; CHAPS, 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate; EDTA, ethylene-
diamimetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FP,
fluorescence polarization; GC, glucocorticoid; GRC, glucocorticoid recep-
tor-ligand complex; GR, glucocorticoid receptor; GR-LBD, glucocorticoid
receptor-ligand binding domain; HFF, human foreskin fibroblast; HSP,
heat-shock protein; IL-6, interleukin-6; LPS, lipopolysaccharide; MMTV,
mouse mammary tumor virus; MR, mineral corticoid receptor; NF, nuclear
factor; PR, progesterone receptor; TES, N-[tris(hydroxymethyl)methyl]-2-
aminoethanesulfonic acid; TNF-R, tumor necrosis factor-R.
10.1021/jm061273t CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2006

7888 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Regan et al.
Chart 1
Scheme 1a
a Reagents and conditions: (a) CuI (1.1 equiv)/THF, -20 °C, 30 min, then 7 (1.1 equiv), room temperature overnight; (b) trimethylsulfoxonium iodide/
NaH/DMSO; (c) 10/NaOEt/ethanol, heat; (d) BBr3 (10 equiv)/CH2Cl2, room temperature overnight; (e) 13 (2 equiv)/K2CO3 (5 equiv)/DMF, 100 °C, 1.5 h.
Results and Discussion
perhaps provide a water shield to the hydrogen bond network
consisting of the A-ring mimetic acceptor and the GR-LBD
donor. The outcome is a significant enhancement in GR binding
affinity. For example, cis-dimethylpiperidin-4-one 14 exhibits
a 140-fold gain in GR binding (IC50 ) 10 nM). Other methylated
analogues include cis-dimethylmorpholine 20, dimethylpyridin-
4-one 21, and dimethylimidazole 22 with IC50 values of 38,
120, and 650 nM, respectively. Introducing methyl groups onto
the A-ring replacements has no effect on PR binding affinity,
whereas modest activity against MR is seen. In the transrepres-
sion anti-inflammatory cellular assay, compounds 14 and 21
inhibit IL-6 release from HFF with IC50 values of 75 and 53
nM, respectively, and 87-89% efficacy compared to dexa-
methasone.
The nuclear receptor binding potency of the target compounds
is determined by their ability to compete for receptor binding
with tetramethylrhodamine-labeled dexamethasone (GR, MR)
or mifepristone (PR).23 The transrepression potential is measured
in human foreskin fibroblast (HFF) cells. Inhibition of IL-1
stimulated IL-6 production, and the percent efficacy is compared
with that of dexamethasone.24,25 In an effort to establish
transactivation activity, compounds are counterscreened for the
induction of aromatase in HFF cells26 and the ability to activate
the MMTV promoter in HeLa cells transfected with an MMTV
luciferase contruct.27 Comparisons with dexamethasone establish
a basis for identifying compounds with a dissociated profile.
Table 1 shows the effects of A-ring replacements in scaffold
5. Morpholine 15, pyridin-4-one 17, and imidazole 18 are devoid
of activity, while piperidin-4-one 16 demonstrates weak GR
binding affinity (IC50 ) 1400 nM). Introducing methyl groups
on the ring is an opportunity to increase the lipophilicity and
Further increasing the lipophilicity of the A-ring mimetic from
methyl substitutions to fusing an aromatic nucleus to the
hydrogen bond acceptor ring provides analogues shown in Table
2. Changes in GR binding avidity with this modification vary

Quinol-4-ones as Steroid A-Ring Mimetics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7889
Table 1. Methyl-Substituted A-Ring Mimetics
a IC50 values were determined from duplicate 11-point concentration-response curves. See ref 23. b IC50 values represent the mean of at least two independent
determinations. See ref 25. c Not tested.
Table 2. Phenyl-Fused A-Ring Mimetics
a IC50 values were determined from duplicate 11-point concentration-response curves. See ref 23. b IC50 values represent the mean of at least two independent
determinations. See ref 25. c Not tested.
between 2- and 10-fold. For example, compare 14 with 24, 22
with 26, and 21 with 11. However, of these compounds, only
transrepression activity.10,28,29 A likely explanation is that the
flexibility of the GR-LBD can accommodate these ligands but,
in doing so, is rendered incapable of effectively interacting with
anti-inflammatory transcription factors.30,31 Support for the
critical role of the hydrogen bond accepting atom in the A-ring
quinolone 11 potently inhibits cellular IL-6 production (IC50
)
21 nM and 82% efficacy). These results reinforce the often
observed lack of correlation between GR binding affinity and

7890 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Table 3. Phenyl Ring Modifications
Regan et al.
and 90% efficacy). In addition, a good GR/PR binding ratio is
re-established. Switching the hydroxy group at C-2 to a methyl
(i.e., R2 ) Me, 32 and 33) maintains the GR binding affinity
but lessens the GR/MR ratio. Reductions in IL-6 inhibition (IC50
values of 30-60 nM) and lower efficacies are also observed
(63-66%) for this change. Substituting an electronegative atom
at C-5 for an electropositive atom (34) retains the GR binding
and cellular activity with an increase in PR and MR binding
affinities compared to those of 12. Further changing the
electronic character at C-5 by introducing a methoxy improves
the agonist activity compared to that of 33. However, placing
a hydroxy group at C-5 (36) causes a significant loss of GR
binding, implying a negative interaction of a hydrogen bond
donor at this site. Phenyl ring analogues involving substitution
at C-4 reveal a very narrow range of acceptable pharmacophores.
Only fluorine at this site provides good GR binding affinity
and cellular activity (37). Groups larger than fluorine (methyl,
methoxy, hydroxy) generally lead to a significant loss of binding
affinity (data not shown). Incorporating the oxygen atom at C-2
into a dihydrobenzofuran (DBF) ring (38) significantly dimin-
ishes the nuclear receptor selectivity. The transrepression activity
is comparable to that of 11. The above survey of substituents
on the phenyl ring of the quinolone-containing steroid mimetic
generally demonstrates a range of nuclear receptor binding
affinity smaller than a factor of 10. However, pharmacophores
such as methoxy or hydroxy at C-2 (11, 12, 31, 34, and 37)
and DHB (38) are required for achieving the best agonist profile.
Another general trend (data not shown) for this series is a
hydroxy group at C-2 displays better IL-6 inhibition and efficacy
compared to a methoxy despite similar GR binding affinities
(e.g., 11 vs 12). Also important are the substitution patterns at
C-2 and C-5 (34 vs 35) and steric constraints at C-4. Taken
together, these results highlight the important relationships of
size and polarity required for favorable interactions adjacent to
the steroid D-ring domain within the GR-LBD. In addition,
similar observations are noted with phenyl ring substitutions
for molecules containing A-ring mimetics other than quinol-4-
one. See Table 5 for examples.
IL-6 inhib in
HFF IC50b (nM)
a
a
a
compd
no.
GR IC50 PR IC50 MR IC50 (efficacy, % vs
R2 R4 R5
(nM)
(nM)
(nM)
dexamethasone)
11
12
30
31
32
33
34
35
36
37
38d
OMe
OH
H
OH
Me
Me
OH
Me
Me
OH
H
H
H
H
H
H
H
H
H
F
F
F
H
H
H
F
Me
OMe
OH
H
10
5
28
6
22
10
8
11
780
3
475
960
106
555
240
240
160
525
120
580
350
450
150
120
160
91
27 (82)
6 (92)
78 (48)
6 (90)
61 (63)
34 (66)
5.6 (93)
17 (86)
ntc
>2000
340
>2000
880
98
3 (88)
11 (86)
4
48
a IC50 values were determined from duplicate 11-point concentration-
response curves. See ref 23. b IC50 values represent the mean of at least
two independent determinations. See ref 25. c Not tested. d See Table 5 for
the structure.
replacements is highlighted with tetrahydroquinoline 25. A factor
of 200 decrease in GR binding affinity, compared to that of
dihydroquinolone 24, underscores the importance of achieving
a hydrogen bond network in the GR-LBD with this series. The
3-fold difference in GR binding affinity between benzo[1,4]-
oxazine 23 and dihydroquinolone 24 may reflect a stronger
hydrogen bond acceptor pharmacophore and an advantageous
distance for 24 between the hydrogen bond donor of the protein
and the acceptor group on the ligand. One consequence of fusing
a phenyl ring to this scaffold is an erosion of the GR/PR ratio
from greater than 75-fold for compounds without a fused
aromatic nucleus (14 and 20) to less than 50-fold for those with
a phenyl ring attached (11, 23, 24). The GR/MR binding ratio
was nearly unchanged. Interestingly, benzimidazole 26 does not
show a loss of nuclear receptor selectivity. Additional changes
to the A-ring mimetic involve embedding a nitrogen atom into
the aromatic nucleus as a goal to define polarity limits. Table
2 describes the consequences of a quinazolinone (27) and
naphthyridin-4-ones (28, 29) acting as A-ring replacements.
Compared to 11, a significant diminution in GR binding is
observed for 27 and less so with 28 and 29. Nevertheless, both
28 and 29 retain satisfactory transrepression activity. These data
suggest the lipophilic environment of the A-ring binding domain
in GR can tolerate ligands with somewhat increased polarity
whereas the PR-LBD does not readily accommodate these
changes. Of the phenyl-fused A-ring replacements shown in
Table 2, compounds 11, 23, 24, 26, 28, and 29 achieve the
highest GR binding affinity, but only 11, 28, and 29 reach IC50
values <100 nM in the transrepression assay.
A primary goal of this project is identifying GC agonists that
can distinguish between transrepression and transactivation
pathways. Two assays are used as a counterscreen to determine
the extent of transactivation. Dexamethasone activates the
MMTV promoter in HeLa cells transfected with an MMTV
luciferase construct. In addition, dexamethasone induces expres-
sion of aromatase in HFF.32 Several compounds were evaluated
for their transactivation potential to activate MMTV and induce
aromatase expression (EC50 and percent efficacy). A compound
can be characterized as dissociated with either weak activity or
low efficacy, or both, in the transactivation assays compared to
the transrepression assay. The role that phenyl ring substitutions
play in defining dissociation is seen in Table 4. In the MMTV
assay, 2-hydroxyphenyl compounds 12 and 37 are partial
agonists (EC50 ) 15-37 nM and 20-35% efficacy) while the
other compounds are devoid of activity compared to dexa-
methasone. In the aromatase induction assay, 12 and 37 exhibit
pronounced activity with EC50 ) 15-17 nM and 65-74%
efficacies. Alternatively, 2-methoxyphenyl, 2-methylphenyl, and
DBF analogues (11, 32, and 38) display low potency or efficacy
in the aromatase assay. Of the modifications at C-5 of the phenyl
ring, methoxy 35 displays moderate activity against aromatase
(EC50 ) 95 nM, 65% efficacy) while hydrogen or fluorine (32
and 33) are weakly potent and efficacious derivatives. These
data suggest groups on the phenyl ring have an important
influence on the degree of dissociation and parallel the observa-
Molecular modeling suggests that, upon binding to the GR-
LBD, the phenyl ring of 2b resides adjacent to the steroid D-ring
domain.15,16 The contribution to GR binding and transrepression
activity of simple groups attached to the phenyl ring in the
quinolone-containing scaffold is summarized in Table 3.
Conversion of the methoxy group at C-2 (11, R2 ) OMe) to a
hydroxy (12, R2 ) OH) improves the transrepression activity.
Removal of the hydroxy group and fluorine atom from 12
(compound 30) weakens the GR binding affinity (IC50 ) 28
nM) and decreases the agonist activity in the cellular assay (IC50
) 78 nM and 48% efficacy). Also seen is a decline in the GR/
PR ratio. Maintaining only a hydroxy group (31) restores the
binding (GR IC50 ) 6 nM) and cellular activity (IC50 ) 6 nM

Quinol-4-ones as Steroid A-Ring Mimetics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7891
Table 5. In Vivo Activities of Dissociated Agonists
Table 4. Dissociation Profiles
IL-6 inhib in
MMTV activation in aromatase inhib in
HFF IC50b (nM)
HeLa EC50c (nM)
(efficacy, % vs
dexamethasone)
HFF EC50d (nM)
(efficacy, % vs
dexamethasone)
a
compd GR IC50 (efficacy, % vs
no.
(nM)
dexamethasone)
1a
1b
2a
11
12
14
21
28
29
32
33
35
37
38g
39g
40g
41g
3.4
15
3
10
5
10
120
77
34
22
10
11
3
0.5 (100)
6.6 (96)
59 (80)
27 (82)
6 (92)
75 (89)
53 (87)
56 (78)
35 (72)
61 (63)
34 (66)
17 (86)
3 (88)
17 (100)
16 (91)
>2000 (14)
>2000 (10)
37 (20)
nte
1.9 (100)
19 (92)
91 (56)
220 (67)
14 (74)
240 (93)
170 (77)
ipf (41)
ip (39)
>2000 (29)
ip (22)
95 (65)
17 (82)
ip (60)
ip (26)
>2000 (3)
nt
>2000 (1)
>2000 (1)
>2000 (2)
>2000 (6)
15 (35)
nt
nt
nt
nt
4
11 (86)
39 (77)
47 (59)
70 (71)
34
48
125
>2000 (14)
>2000 (20)
IL-6 inhib in
inhib of
a IC50 values were determined from duplicate 11-point concentration-
response curves. See ref 23. b IC50 values represent the mean of at least
two independent determinations. See ref 25. c EC50 values represent the
mean of at least two independent determinations. See ref 27. d EC50 values
represent the mean of at least two independent determinations. See ref 26.
e Not tested. f Due to the flat shape of the dose-response curve, an EC50
value beyond 2000 nM could not be calculated. g See Table 5 for the
structures.
HFF IC50b (nM) TNF-R in micec
a
compd
no.
phenyl GR IC50
(efficacy, % vs
dexamethasone)
(%) (n ) 8)
(10 mg/kg)
A
ring
(nM)
1bd
12
32
33
38
39
40
41
see Chart 1
see Table 3
15
5
7 (96)
6 (92)
84 ( 8
55 ( 6
II
II
II
I
III
I
II
I
III
III
I
22
10
4
34
48
125
61 (63)
34 (66)
11 (86)
39 (77)
47 (59)
70 (71)
36 ( 10
50 ( 17
49 ( 10
49 ( 6
58 ( 12
48 ( 11
tion that some steroids containing modified D-rings show
dissociated activities.33 The contribution of the A-ring replace-
ment to the dissociation profile is seen in Table 4. Comparing
compounds with the 2-methoxy-5-fluorophenyl ring, dimeth-
ylpiperidin-4-one 14, dimethylpyridin-4-one 21, and naphthy-
ridin-4-one 28 demonstrate a somewhat weaker transactivation
response compared to quinol-4-one 12. However, the 2-methyl-
5-fluorophenyl and DBF rings impart a much more dissociated
profile for dimethylpyridin-4-one (39 and 41), naphthyridin-4-
one (40), and quinol-4-one (33 and 38) A-ring mimetics. Thus,
a distinction between potent transrepression activity and dimin-
ished transactivation is achieved with the proper choice of A-ring
replacements and phenyl substitution with this class of com-
pounds.
Selected dissociated compounds were evaluated for their anti-
inflammatory properties in an LPS-stimulated mouse model of
TNF-R production. Test compounds were administered orally
in Cremophor 60 min prior to LPS challenge. TNF-R levels
were measured 60 min later. Table 5 summarizes the GR binding
and cellular and in vivo transrepression profiles of various
combinations of A-ring (dimethylpyridin-4-one, quinol-4-one,
and naphthyridin-4-one) and phenyl ring analogues. When dosed
at 10 mg/kg, the nondissociated analogue 12 and dissociated
compounds 33 and 38-41 inhibit TNF-R production (48-58%).
I
a IC50 values were determined from duplicate 11-point concentration-
response curves. See ref 23. b IC50 values represent the mean of at least
two independent determinations. c See the Experimental Section. d 3 mg/
kg, po.
at C-2 and C-5 are well tolerated. The dissociated profile of
this series in either the MMTV or aromatase assays was
dependent upon the choice of the A-ring mimetic and the
substitution on the phenyl ring. As A-ring replacements, fused
rings 11, 28, and 29 exhibit a superior dissociated profile due
to differences in either potency or efficacy in the transactivation
assays compared to monocyclic rings 14 and 21. In general,
methyl and methoxy groups at C-2 of the phenyl ring demon-
strate weaker transactivation activities than hydroxy at this site.
In an animal model of inflammation, compounds 12, 33, and
38-41 potently inhibit TNF-R production. Thus, the nuclear
receptor binding affinities, the cellular levels of transrepression
and transactivation, and the anti-inflammatory properties have
been demonstrated with the quinol-4-one-containing class of
GR agonists. Further work in this series will be reported in due
course.
Experimental Section
All solvents and reagents were obtained from commercial sources
and used without further purification. Flash chromatography was
performed according to the procedure of Still et al. (EM Science
Kieselgel 60, 70-230 mesh). Melting points were obtained from a
Mel-temp 3.0 or Fisher-Johns melting point apparatus and are
uncorrected. The 1H NMR spectra were recorded on a Bruker DPX
400 spectrometer. Chemical shifts are reported in parts per million
(δ) from the tetramethylsilane resonance in the indicated solvent.
Infrared spectra were recorded without solvent on a Nicolet Impact
410. Mass spectra were obtained from a Finnigan-SSQ7000
spectrometer. Samples were generally introduced by a particle beam
and ionized with NH4Cl. The analytical HPLC purity was estab-
lished with the following protocols. Method A: Gilson chromato-
graphic system, Waters SureFire reversed-phase C18 analytical
column (4.6 × 50 mm, 5 µm, part number 186002557), flow rate
5 mL/min, detection with a Gilson UV/VIS-155 at 254 nm, sample
Conclusion
Several new types of pharmacophores can serve as A-ring
mimetics with good binding affinity for GR for this scaffold.
These include dimethylpiperidin-4-one (14), quinolone (11), cis-
dimethylmorpholine (20), benzo[1,4]oxazine (23), dihydroqui-
nolone (24), benzimidazole (26), and naphthyridin-4-one (29).
In addition, good nuclear receptor selectivity is achieved. Of
these, 11, 29, and dimethylpyridin-4-one (21) exhibit good
transrepression activity in a cellular assay of IL-6 production.
The substitution patterns on the phenyl ring play an important
role in defining GR binding affinity and the degree of trans-
repression. Compounds with a hydroxy group at C-2 obtain the
highest efficacy levels of transrepression, while a C-5 hydroxy
is deleterious toward activity. Methyl and methoxy substituents

7892 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Regan et al.
size 25 µL, gradient 5% acetonitrile in water with 0.1% TFA to
95% acetonitrile in water with 0.1% TFA over 6 min and held.
Method B: Rainin chromatography system, model SD-200, Tosoh
Bioscience TSK Super ODS column (4.6 mm × 10 cm, 2 µM,
part number G0030-90F), flow rate 1.2 mL/min, detection with
Dynamax absorbance detector at 254 nM, gradient 5% acetonitrile
in water with 0.05% TFA to 95% acetonitrile in water with 0.05%
TFA over 10 min and held. Method C: Rainin chromatography
system, model SD-200, Whatman PartiSphere C-8 column (4.6 ×
125 mm, 5 µm, part number 4621-0503), detection with Dynamax
absorbance detector at 254 nM, gradient 5% acetonitrile in water
with 0.1% TFA to 95% acetonitrile in water with 0.1% TFA.
purity: method A 99%, retention time (tR) 3.53 min; method B
98%, tR 7.98 min.
1-[4-(5-Fluoro-2-hydroxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (12). To a solution of 11
(0.082 g, 0.19 mmol) in CH2Cl2 (3 mL) at -20 °C was added
dropwise BBr3 (1.9 mL of a 1 M solution in CH2Cl2, 1.9 mmol).
The mixture was stirred for 1 h and quenched with saturated
NaHCO3 and ethyl acetate. The organic layer was washed with
saturated NaHCO3 and brine and dried (MgSO4). Removal of the
volatiles in vacuo left a residue which was purified by trituration
in ether and ethyl acetate and provided 0.041 g of product. Mp:
1
222-223 °C. H NMR (400 MHz, DMSO-d6): δ 9.98 (s, 1H),
1,1,1-Trifluoro-4-methylpent-3-en-2-one (7). To a 0-5 °C
mixture of N,O-dimethylhydroxylamine hydrochloride (15.8 g) in
CH2Cl2 (400 mL) was added dropwise trifluoroacetic anhydride
(21.7 mL). Pyridine (37 mL) was added to the mixture dropwise.
The resulting mixture was stirred at 0-5 °C for 0.5 h and diluted
with water. The organic layer was washed with water, 1 N aqueous
HCl, water, and brine and dried (MgSO4). Removal of the volatiles
in vacuo for 5 min provided 2,2,2-trifluoro-N-methoxy-N-methyl-
acetamide as a colorless oil.
To a 0-5 °C mixture of 2,2,2-trifluoro-N-methoxy-N-methyl-
acetamide (3 g) in 30 mL of anhydrous ether was added (2-
methylpropenyl)magnesium bromide in THF (42 mL of a 0.5 M
solution). The reaction was stirred at 0-5 °C for 0.5 h, warmed to
room temperature, and stirred overnight. The reaction was quenched
with saturated aqueous NH4Cl and extracted with ether three times.
The organic layers were combined, washed with water and brine,
and dried (MgSO4). Most of the volatiles were removed at
atmospheric pressure, providing 7 as an approximately 75% pure
solution containing ether and THF that was used without further
purification.
8.30 (d, 1H), 7.87 (d, 1H), 7.75 (t, 1H), 7.52 (t, 1H), 7.33 (m, 2H),
7.13 (m, 1H), 7.05 (m, 1H), 7.48 (s, 1H), 6.20 (d, 1H), 4.21 (m,
2H), 3.24 (d, 1H), 2.15 (d, 1H), 1.75 (s, 3H), 1.55 (s, 3H). MS
(ESI): m/e 424 (MH+). HPLC purity: method A 98%, tR 3.11 min;
method B 95%, tR 7.16 min.
cis-1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-
(trifluoromethyl)pentyl]-3,5-dimethylpiperidin-4-one Hydro-
chloride (14). A mixture of 9 (0.092 g, 0.32 mmol), cis-3,5-
dimethylpiperidin-4-one hydrochloride (13; 0.103 g, 0.64 mmol),
and powdered K2CO3 (0.218 g, 1.58 mmol) in DMF (3 mL) was
heated at 100 °C for 1.5 h, cooled to room temperature, diluted
with ether, washed thoroughly with water and brine, and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was treated with ethereal HCl. The mixture was filtered and
the solid washed with ether and dried in vacuo to provide 0.015 g
of product. Mp: 77-80 °C. 1H NMR (400 MHz, CDCl3): δ 7.02
(dd, 1H), 6.78 (m, 1H), 6.68 (m, 1H), 3.75 (s, 3H), 1.52 (s, 3H),
1.28 (s, 3H), 0.78 (d, 6H). MS (ESI): m/e 420 (MH+). HPLC
purity: method C 92%, tR 10.25 min.
1,1,1-Trifluoro-4-(5-fluoro-2-methoxyphenyl)-4-methyl-2-(mor-
1,1,1-Trifluoro-4-(5-fluoro-2-methoxyphenyl)-4-methylpentan-
2-one (8). To a 0-5 °C solution of 7 (24 g, approximately 75%
pure solution containing ether and THF) and CuI (30.1 g) in ether
(300 mL) was added (5-fluoro-2-methylphenyl)magnesium bromide
(315 mL, 157 mmol, 0.5 M solution in THF). The mixture was
warmed to room temperature, stirred overnight, and diluted with
saturated aqueous NH4Cl (15 mL) and ethyl acetate. The organic
layer was washed with water and brine and dried (MgSO4).
Removal of the volatiles in vacuo provided a residue which was
purified by flash silica gel chromatography using 0-5% ethyl
acetate in hexanes as the eluent. The product-rich fractions were
collected and the volatiles removed in vacuo to yield 20.3 g (46%)
of 8 as an oil.
2-[2-(5-Fluoro-2-methoxyphenyl)-2-methylpropyl]-2-(trifluo-
romethyl)oxirane (9). To a suspension of trimethylsulfoxonium
iodide (4.76 g, 21.5 mmol) in DMSO (25 mL) was added NaH
(0.863 g of a 60% dispersion in oil, 21.5 mmol) in four portions.
After being stirred for 0.5 h, the solution was added to 8 (5.00 g,
17.9 mmol) in DMSO (20 mL). After 2 h the mixture was diluted
with water (150 mL) and extracted with ether (2 × 125 mL). The
combined organic layers were washed with water and brine and
dried (MgSO4). Removal of the volatiles in vacuo provided 9 as
an oil which was used without further purification.
pholin-4-ylmethyl)pentan-2-ol Hydrochloride (15). Mp: 195-
1
200 °C. H NMR (400 MHz, CDCl3): δ 6.97 (dd, 1H), 6.78 (m,
1H), 6.68 (m, 1H), 4.55 (s, 1H), 3.75 (s, 3H), 3.46 (br s, 4H), 2.33
(m, 4H), 2.14 (d, 2H), 1.97 (d, 2H), 1.52 (s, 3H), 1.32 (s, 3H). MS
(ESI): m/e 380 (MH+). HPLC purity: method A 99%, tR 2.41 min;
method B 99%, tR 6.35 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]piperidin-4-one Hydrochloride (16). Mp:
134-136 °C. 1H NMR (400 MHz, DMSO-d6): δ 6.8 (m, 3H), 4.55
(br s, 1H), 3.62 (s, 3H), 2.7-2.0 (m, 12H), 1.36 (s, 3H), 1.18 (s,
3H). MS (ESI): m/e 392 (MH+). HPLC purity: method C 94%, tR
7.42 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-pyridin-4-one (17). Mp: 205-206 °C.
1H NMR (400 MHz, DMSO-d6): δ 7.48 (d, 2H), 7.12 (m, 3H),
6.47 (s, 1H), 6.07 (d, 2H), 3.92 (s, 3H), 3.81 (d, 1H), 3.60 (d, 1H),
2.92 (d, 1H), 2.18 (d, 1H), 1.65 (s, 3H), 1.45 (s, 3H). IR (cm-1):
1639. MS (ESI): m/e 388 (MH+). HPLC purity: method A 99%,
tR 2.72 min; method B 99%, tR 6.84 min.
1,1,1-Trifluoro-4-(5-fluoro-2-methoxyphenyl)-2-(imidazol-1-
ylmethyl)-4-methylpentan-2-ol (18). Mp: 166-167 °C. 1H NMR
(400 MHz, CDCl3): δ 8.1 (br s, 1H), 7.2-6.7 (m, 6H), 3.94 (dd,
2H), 3.78 (s, 3H), 2.45 (s, 2H), 1.44 (s, 3H), 1.33 (s, 3H). MS
(ESI): m/e 361 (MH+). HPLC purity: method A 99%, tR 2.56 min;
method B 98%, tR 6.59 min; method C 96%, tR 8.67 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (11). A mixture of 9
(0.226, 0.773 mmol), 4-hydroxyquinoline (10; 0.112 g, 0.773
mmol), sodium ethoxide (0.28 mL of a 21 wt % solution in ethanol,
0.77 mmol), and anhydrous ethanol (4 mL) was heated at 85 °C
for 6 h, cooled to room temperature, diluted with ethyl acetate and
acetic acid, washed with water and brine, and dried (MgSO4).
Removal of the volatiles in vacuo provided a residue which was
purified with flash silica gel chromatography using ethyl acetate
as the eluent. The product-rich fractions were collected and the
volatiles removed in vacuo to yield 0.13 g (38%) of 11. Mp: 198-
2-[(trans-2,6-Dimethylmorpholin-4-yl)methyl]-1,1,1-trifluoro-
4-(5-fluoro-2-methoxyphenyl)-4-methylpentan-2-ol Hydrochlo-
ride (19). Mp: 50-55 °C. Free base 1H NMR (400 MHz,
CDCl3): δ 7.02 (m, 1H), 6.81 (m, 1H), 6.69 (m, 1H), 4.68 (br d,
1H), 3.76 (m, 5H), 2.26 (m, 4H), 2.1-1.85 (m, 4H), 1.51 (s, 3H),
1.30 (s, 3H), 1.03 (br s, 6H). MS (ESI): m/e 408 (MH+). HPLC
purity: method A 99%, tR 3.16 and 3.61 min; method B 99%, tR
8.19 and 9.23 min.
1
2-[(cis-2,6-Dimethylmorpholin-4-yl)methyl]-1,1,1-trifluoro-4-
200 °C. H NMR (400 MHz, CDCl3): δ 8.21 (d, 1H), 7.42 (m,
2H), 7.25 (s, 1H), 7.1 (m, 1H), 6.92 (m, 2H), 6.80 (m, 1H), 4.15
(dd, 2H), 3.80 (s, 3H), 2.55 (dd, 2H), 1.5 (s, 3H), 1.33 (s, 3H).
FTIR neat (cm-1): 1624, 1610. MS (ESI): m/e 438 (MH+). HPLC
(5-fluoro-2-methoxyphenyl)-4-methylpentan-2-ol Hydrochloride
1
(20). Mp: 133-135 °C. H NMR (400 MHz, DMSO-d6): δ 6.8
(m, 3H), 4.13 (br s, 1H), 3.74 (br s, 2H), 3.58 (s, 3H), 3.0-1.7 (m,

Quinol-4-ones as Steroid A-Ring Mimetics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7893
8H), 1.30 (s, 3H), 1.14 (s, 3H), 0.8 (br s, 6H). MS (ESI): m/e 408
(MH+). HPLC purity: method A 99%, tR 2.76 min; method B 99%,
tR 6.96 min.
layer was washed with water and dried (MgSO4). Removal of the
volatiles provided a residue which was purified with silica gel
chromatography using 80% ethyl acetate in petroleum ether as the
eluent. The product-rich fractions were collected and the volatiles
removed in vacuo. The residue was recrystallized from petroleum
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-3,5-dimethyl-1H-pyridin-4-one (21). Mp:
1
1
ether to provide 0.051 g of product. Mp: 180-182 °C. H NMR
246-247 °C. H NMR (400 MHz, DMSO-d6): δ 7.29 (s, 2H),
(400 MHz, CDCl3): δ 7.65 (m, 2H), 7.12 (m, 5H), 6.79 (m, 1H),
6.82 (m, 1H), 4.11 (s, 2H), 3.71 (s, 3H), 2.62 (d, 1H), 2.39 (d,
1H), 1.58 (s, 3H), 1.36 (s, 3H). MS (ESI): m/e 411 (MH+). HPLC
purity: method A 94%, tR 2.90 min; method B 96%, tR 7.67 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinazolin-4-one (27). (a) A solution of
2-[2-(5-fluoro-2-methoxyphenyl)-2-methylpropyl]-2-(trifluoromethyl)-
oxirane (500 mg, 1.71 mmol) and 2-aminobenzamide (1.17 g, 8.56
mmol) in DMF (2.5 mL) was heated at 140 °C for 20 h. The
reaction mixture was diluted with ether and washed with water and
brine. The organic layer was dried (Na2SO4), filtered, and concen-
trated. Purification of the residue by column chromatography (15-
30% ethyl acetate in hexanes) afforded 447 mg (61%) of 2-[[4-
(5-fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(trifluoro-
methyl)pentyl]amino]benzamide as a white foam: 1H NMR (400
MHz, DMSO-d6): δ 8.19 (m, 1H, NH), 7.72 (s, 1H, NH), 7.51 (d,
1H, J ) 7.9 Hz), 7.08 (m, 1H), 7.06 (m, 1H, NH), 6.98 (dd, 1H,
J ) 10.8, 3.0 Hz), 6.85 (m, 2H), 6.46 (m, 1H), 5.91 (d, 1H, J )
8.2 Hz), 5.87 (s, 1H, OH), 3.72 (s, 3H), 2.88 (m, 2H), 2.68 (m,
1H), 1.94 (d, 1H, J ) 15.0 Hz), 1.49 (s, 3H), 1.33 (s, 3H). MS
(ES): m/e 429 ([M + H]+). (b) To a solution of 2-[[4-(5-fluoro-
2-methoxyphenyl)-2-hydroxy-4-methyl-2-(trifluoromethyl)pentyl]-
amino]benzamide (106 mg, 0.247 mmol) in 6.0 mL of trimethyl
orthoformate was added 0.1 mL of trifluoroacetic acid. After 1.5 h
at room temperature, the reaction mixture was concentrated.
Purification by column chromatography (70% EtOAc/hexanes)
afforded 82.1 mg (76%) of the title compound as a white solid.
7.05 (m, 3H), 6.27 (s, 1H, OH), 3.80 (s, 3H), 3.61 (d, 1H, J )
15.0 Hz), 3.37 (d, 1H, J ) 15.0 Hz), 2.80 (d, 1H, J ) 15.6 Hz),
2.00 (d, 1H, J ) 15.6 Hz), 1.75 (s, 6H), 1.53 (s, 3H), 1.31 (s, 3H).
FTIR (neat): 1649 cm-1. MS (ES): m/e 416 ([M + H]+). HPLC
purity: method A 99%, tR 2.95 min; method B 94%, tR 7.69 min.
2-[(2,4-Dimethylimidazol-1-yl)methyl]-1,1,1-trifluoro-4-(5-
fluoro-2-methoxyphenyl)-4-methylpentan-2-ol (22). Mp: 185-
1
186 °C. H NMR (400 MHz, CDCl3): δ 7.04 (m, 1H), 6.87 (m,
1H), 6.78 (m, 1H), 6.40 (s, 1H), 3.80 (s, 3H), 3.70 (m, 2H), 2.45
(m, 2H), 2.22 (s, 3H), 2.15 (s, 3H), 1.47 (s, 3H), 1.36 (s, 3H). MS
(ESI): m/e 389 (MH+). HPLC purity: method A 99%, tR 2.75 min;
method C 96%, tR 7.04 min.
2-[(2,3-Dihydrobenzo[1,4]oxazin-4-yl)methyl]-1,1,1-trifluoro-
4-(5-fluoro-2-methoxyphenyl)-4-methylpentan-2-ol (23). Mp: 98-
1
99 °C. H NMR (400 MHz, CDCl3): δ 7.13 (dd, 1H), 6.88 (m,
1H), 6.72 (m, 1H), 6.65 (m, 1H), 6.56 (m, 2H), 5.85 (d, 1H), 4.03
(m, 2H), 3.73 (s, 3H), 3.20 (d, 1H), 3.05 (m, 2H), 2.98 (d, 1H)
2.52 (d, 1H), 2.10 (d, 1H) 1.58 (s, 3H), 1.35 (s, 3H). MS (ESI):
m/e 428 (MH+). HPLC purity: method A 98%, tR 5.25 min; method
B 99%, tR 11.83 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-2,3-dihydro-1H-quinolin-4-one (24). To a
mixture of LAH (0.003 g) in anhydrous THF (0.8 mL) at 0-5 °C
was added 11 (0.031 g). The mixture was stirred for 1 h and an
additional 3-4 mg of LAH added. After 2 h the reaction was
quenched with acetic acid, water, and ether. The organic layer was
washed with water, saturated NaHCO3, and brine and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was purified with flash silica gel chromatography using 33%
ethyl acetate in hexanes as the eluent. The product-rich fractions
were collected and the volatiles removed in vacuo to provide 0.015
g of 1-[4-(5-fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1,2,3,4-tetrahydroquinolin-4-ol. A mixture of
this alcohol (0.015 g) and MnO2 (0.2 g) in 2 mL of CH2Cl2 was
stirred overnight, applied to a column of flash silica gel, and eluted
with 25% ethyl acetate in hexanes. The product-rich fractions were
collected and the volatiles removed in vacuo. Mass: 0.010 g. Mp:
1
Mp: 118-121 °C. H NMR (400 MHz, MeOH-d4): δ 8.20 (s,
1H), 8.16 (d, 1H, J ) 8.0 Hz), 7.69 (m, 1H), 7.49 (m, 1H), 7.27
(d, 1H, J ) 9.8 Hz), 7.08 (d, 1H, J ) 8.6 Hz), 7.03 (m, 2H), 4.14
(m, 2H), 3.93 (s, 3H), 3.01 (d, 1H, J ) 15.2 Hz), 2.16 (d, 1H, J )
15.2 Hz), 1.69 (s, 3H), 1.47 (s, 3H). MS (ES): m/e 439 ([M +
H]+). HPLC purity: method A 99%, tR 3.21 min; method B 98%,
tR 7.97 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-[1,5]naphthyridin-4-one (28). Mp: 193-
195 °C. 1H NMR (400 MHz, MeOH-d4): δ 8.64 (m, 1H), 7.78 (d,
1H, J ) 7.9 Hz), 7.54 (m, 2H), 7.28 (d, 1H, J ) 9.8 Hz), 7.02 (m,
2H), 6.37 (d, 1H, J ) 7.9 Hz), 4.23 (d, 1H, J ) 16.0 Hz), 4.11 (d,
1H, J ) 16.0 Hz), 3.91 (s, 3H), 2.95 (d, 1H, J ) 15.2 Hz), 2.15 (d,
1H, J ) 15.2 Hz), 1.68 (s, 3H), 1.47 (s, 3H). FTIR (neat): 1625,
1584 cm-1. MS (ES): m/e 440 ([M + H]+).
1
132-134 °C. H NMR (400 MHz, CDCl3): δ 7.80 (d, 1H), 7.13
(m, 3H), 6.88 (m, 1H), 6.77 (m, 1H), 6.67 (m, 1H), 5.91 (d, 1H),
3.78 (s, 3H), 3.52 (d, 1H), 3.28 (m, 2H), 3.02 (d, 1H), 2.55 (m,
4H) 2.18 (d, 1H), 1.56 (s, 3H), 1.38 (s, 3H). IR (cm-1): 1672,
1603. MS (ESI): m/e 440 (MH+). HPLC purity: method A 96%,
tR 4.74 min; method B 98%, tR 10.82 min.
1-[4-(5-Fluoro-2-methoxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-[1,6]naphthyridin-4-one (29). Mp: 229-
2-[(3,4-Dihydro-2H-quinolin-1-yl)methyl]-1,1,1-trifluoro-4-(5-
fluoro-2-methoxyphenyl)-4-methylpentan-2-ol (25). To a mixture
of 9 (0.21 g, 0.72 mmol) and 1,2,3,4-tetrahydoquinoline (0.096 g;
0.72 mmol) in methylene chloride (5 mL) was added flash silica
gel (0.5 g). The volatiles were removed in vacuo. The residue was
heated in a microwave at 150 °C for 4 min, applied to a column of
flash silica gel, and eluted with 5% ethyl acetate in hexanes. The
product-rich fractions were concentrated in vacuo. The residue was
treated with ethereal HCl and the volatiles removed in vacuo.
Trituration with hexanes and neutralization provided 0.06 g of the
1
230 °C. H NMR (400 MHz, MeOH-d4): δ 9.28 (s, 1H), 8.43 (d,
1H, J ) 6.4 Hz), 7.77 (d, 1H, J ) 8.0 Hz), 7.29 (m, 1H), 7.03 (m,
2H), 6.94 (d, 1H, J ) 6.4 Hz), 6.27 (d, 1H, J ) 8.0 Hz), 4.13 (d,
1H, J ) 15.9 Hz), 4.05 (d, 1H, J ) 15.9 Hz), 3.92 (s, 3H), 2.98 (d,
1H, J ) 15.2 Hz), 2.14 (d, 1H, J ) 15.2 Hz), 1.69 (s, 3H), 1.47 (s,
3H). FTIR (neat): 1642, 1587 cm-1. MS (ES): m/e 439 ([M +
H]+). HPLC purity: method A 99%, tR 2.59 min; method B 99%,
tR 6.91 min.
1-(2-Hydroxy-4-methyl-4-phenyl-2-(trifluoromethyl)pentyl)-
1
1
oily product. H NMR (400 MHz, CDCl3): δ 7.11 (dd, 1H), 6.87
1H-quinolin-4-one (30). Mp: 80-82 °C. H NMR (400 MHz,
(m, 3H), 6.73 (m, 2H), 6.60 (m, 1H), 5.97 (d, 1H), 3.73 (s, 3H),
3.27 (d, 1H), 3.15-2.90 (m, 3H), 2.67 (m, 2H), 2.55 (d, 1H) 2.12
(d, 1H), 1.57 (s, 3H), 1.40 (s, 3H). MS (ESI): m/e 426 (MH+).
HPLC purity: method A 99%, tR 4.21 min; method B 98%, tR 13.07
min.
2-(Benzoimidazol-1-ylmethyl)-1,1,1-trifluoro-4-(5-fluoro-2-
methoxyphenyl)-4-methylpentan-2-ol (26). A mixture of 9 (0.12
g, 0.41 mmol), benzimidazole (0.048 g; 0.41 mmol), and potassium
tert-butoxide (0.1 mL of a 1 M solution in THF, 0.1 mmol) in
DMSO (3 mL) was heated at 130 °C for 2 h, cooled to room
temperature, and diluted with ethyl acetate and water. The organic
CDCl3): δ 8.30 (d, 1H, J ) 7.3 Hz), 7.53 (m, 1H), 7.39 (m, 5H),
7.26 (m, 2H), 7.18 (d, 1H, J ) 8.7 Hz), 6.09 (d, 1H, J ) 7.9 Hz),
4.08 (d, 1H, J ) 15.8 Hz), 3.91 (d, 1H, J ) 15.8 Hz), 3.71 (s, 1H,
OH), 2.39 (d, 1H, J ) 15.4 Hz), 2.25 (d, 1H, J ) 15.4 Hz), 1.61
(s, 3H), 1.37 (s, 3H). MS (ES): m/e 390 ([M + H]+). HPLC
purity: method A 99%, tR 3.25 min; method B 99%, tR 7.60 min.
1-[2-Hydroxy-4-(2-hydroxyphenyl)-4-methyl-2-(trifluorometh-
1
yl)pentyl]-1H-quinolin-4-one (31). Mp: 157 °C. H NMR (400
MHz, MeOH-d4): δ 8.6 (d, 1H), 8.45 (d, 1H), 7.9 (t, 1H), 7.75 (t,
1H), 7.55 (d, 1H), 7.5 (d, 1H), 7.18 (t, 1H), 7.05, (d, 1H), 6.98 (t,
1H), 6.85(d, 1H), 3.25 (m, 2H), 4.6 (s, 2H), 2.2 (d, 1H), 1.73 (s,

7894 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Regan et al.
3H), 1.55 (s, 3H). MS (ESI): m/e 406 (M+), 404 (M-). HPLC
purity: method A 92%, tR 3.02 min; method B 85%, tR 7.08 min.
1-[2-Hydroxy-4-methyl-4-o-tolyl-2-(trifluoromethyl)pentyl]-
1H-quinolin-4-one (32). Mp: 174 °C. 1H NMR (400 MHz,
CDCl3): δ 8.26 (m, 1H), 7.52 (m, 2H), 7.4 (d,1H), 7.3-7.15 (m,
4H), 7.08 (d, 1H), 6.1 (d, 1H), 4.05 (q, 2H), 2.5 (s, 3H), 2.4 (q,
2H), 1.7 (s, 3H), 1.48 (s, 3H). MS (ESI): m/e 404 (M+). HPLC
purity: method A 96%, tR 3.44 min; method B 99%, tR 8.03 min.
1-[4-(5-Fluoro-2-methylphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (33). Mp: 103-105 °C.
1H NMR (400 MHz, CDCl3): δ 8.16 (dd, 1H, J ) 8.1, 1.5 Hz),
7.50 (m, 1H), 7.37 (d, 1H, J ) 7.8 Hz), 7.28 (m, 1H), 7.23 (m,
1H), 7.07 (m, 2H), 6.87 (m, 1H), 6.01 (d, 1H, J ) 7.8 Hz), 4.52 (s,
1H, OH), 4.10 (d, 1H, J ) 15.7 Hz), 3.95 (d, 1H, J ) 15.7 Hz),
2.50 (s, 3H), 2.47 (d, 1H, J ) 15.6 Hz), 2.33 (d, 1H, J ) 15.6 Hz),
1.72 (s, 3H), 1.48 (s, 3H). MS (ES): m/e 422 ([M + H]+). HPLC
purity: method A 98%, tR 3.47 min; method B 99%, tR 8.08 min.
1-[2-Hydroxy-4-(2-hydroxy-5-methylphenyl)-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (34). Mp: 160-161 °C.
1H NMR (400 MHz, MeOH-d4): δ 8.21 (d, 1H), 7.73 (d, 1H),
7.52 (t, 1H), 7.30 (m, 2H), 7.13 (d, 1H), 6.94 (d, 1H), 6.72 (d,
1H), 6.19 (d, 1H) 4.27 (d, 1H), 4.13 (d, 1H), 3.15 (d, 1H), 2.29 (s,
3H), 2.13 (d, 1H), 1.69 (s, 3H), 1.50 (s, 3H). MS (ESI): m/e 419
(M+).
1-[2-Hydroxy-4-(5-methoxy-2-methylphenyl)-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (35). Mp: 196-198 °C.
1H NMR (400 MHz, CDCl3): δ 8.37 (d, 1H), 7.55 (t, 1H), 7.4 (d,
1H), 7.3 (t, 1H), 7.27-7.08 (m, 3H), 6.72 (d, 1H), 6.15 (d, 1H),
4.1 (q, 2H), 3.8 (s, 3H), 2.98 (s, 1H), 2.4 (s, 3H), 2.38 (q, 2H),
1.65 (s, 3H), 1.43 (s, 3H). MS (ESI): m/e 434 (M+). HPLC
purity: method A 98%, tR 3.43 min; method B 98%, tR 7.96 min.
1-[2-Hydroxy-4-(5-hydroxy-2-methylphenyl)-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (36). Mp: 125-127 °C.
1H NMR (400 MHz, MeOH-d4): δ 8.28 (d, 1H), 7.83 (d, 1H),
7.68 (t, 1H), 7.4 (t, 1H), 7.29 (d, 1H), 7.16 (d, 1H), 7.06 (d, 1H),
6.62 (m, 1H), 6.39 (d, 1H), 4.16 (q, 2H), 2.72 (d, 1H), 2.58 (s,
3H), 2.18 (d, 1H), 1.78 (s, 3H), 1.5 (s, 3H). MS (ESI): m/e 421
(M+). HPLC purity: method A 97%, tR 2.89 min; method B 94%,
tR 6.85 min.
1-[4-(4-Fluoro-2-hydroxyphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (37). Mp: 293-295 °C.
1H NMR (400 MHz, DMSO-d6): δ 10.3 (s, 1H, OH), 8.07 (d, 1H,
J ) 7.5 Hz), 7.61 (d, 1H, J ) 7.8 Hz), 7.53 (m, 1H), 7.34 (m, 1H),
7.27 (m, 1H), 7.13 (d, 1H, J ) 8.6 Hz), 6.63 (m, 2H), 6.22 (s, 1H,
OH), 5.97 (d, 1H, J ) 7.8 Hz), 4.05 (d, 1H, J ) 16.2 Hz), 3.94 (d,
1H, J ) 16.2 Hz), 2.99 (d, 1H, J ) 15.0 Hz), 2.03 (d, 1H, J )
15.0 Hz), 1.61 (s, 3H), 1.38 (s, 3H). MS (ES): m/e 424 ([M +
H]+). HPLC purity: method A 95%, tR 3.14 min; method B 96%,
tR 7.26 min.
1-[4-(2,3-Dihydrobenzofuran-7-yl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-quinolin-4-one (38). Mp: 187-188 °C.
1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, 1H), 7.70 (m, 2H),
7.38 (7, 1H), 7.29 (m, 3H), 6.94 (t, 1H), 6.32 (s, 1H), 6.08 (d, 1H),
4.68 (t, 2H), 4.05 (dd, 2H), 3.22 (t, 1H), 2.88 (d, 1H), 2.12 (d,
1H), 1.70 (s, 3H), 1.43 (s, 3H). FTIR neat (cm-1): 1624, 1610.
MS (ES): m/e 432 ([M + H]+). HPLC purity: method A 97%, tR
3.38 min; method B 97%, tR 8.07 min; method C 98%, tR 10.50
min.
12.1 Hz), 7.21 (dd, 1H, J ) 6.6, 8.3 Hz), 7.00 (d, 1H, J ) 6.3 Hz),
6.93 (m, 1H), 6.27 (d, 1H, J ) 8.0 Hz), 4.08 (m, 2H), 2.67 (d, 1H,
J ) 15.6 Hz), 2.62 (s, 3H), 2.23 (d, 1H, J ) 15.6 Hz), 1.77 (s,
3H), 1.54 (s, 3H). MS (ES): m/e 423 ([M + H]+). HPLC purity:
method A 99%, tR 2.44 min; method B 99%, tR 6.42 min.
1-[4-(5-Fluoro-2-methylphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-3,5-dimethyl-1H-pyridin-4-one (41). Mp:
205-207 °C. 1H NMR (400 MHz, MeOH-d4): δ 7.38 (s, 2H), 7.25
(m, 1H), 7.16 (m, 1H), 6.87 (m, 1H), 3.71 (d, 1H, J ) 15.0 Hz),
3.59 (d, 1H, J ) 15.0 Hz), 2.64 (d, 1H, J ) 15.6 Hz), 2.55 (s, 3H),
2.11 (d, 1H, J ) 15.6 Hz), 1.95 (s, 6H), 1.71 (s, 3H), 1.48 (s, 3H).
MS (ES): m/e 401 ([M + H]+). HPLC purity: method A 99%, tR
2.78 min; method B 99%, tR 7.21 min.
Nuclear Receptor Assays. GR, PR, and MR binding assays were
performed in a fluorescence polarization microplate format that
measures competition for binding to the nuclear receptor between
a test compound and a fluorescently labeled receptor ligand, or
probe. The probes used for the assays were as follows: GR and
MR assays, 5 nM tetramethylrhodamine-labeled dexamethasone;
PR assay, 5 nM tetramethylrhodamine-labeled RU-486. For GR
and MR assays, receptors present in lysates of baculovirus-infected
insect cells co-infected with receptor, HSP-70, HSP-90, and p23
were used. For PR binding assays, lysates were generated from
insect cells infected with receptor-containing baculovirus alone.
Binding reactions consisted of lysate, the test compound, and the
probe combined in an assay buffer containing 10 mM TES, 50 mM
KCl, 20 mM Na2MoO4‚2H2O, 1.5 mM EDTA, 0.04% (w/v)
CHAPS, 10% (v/v) glycerol, and 1 mM dithiothreitol, pH 7.4.
Lysates containing GR, PR, and MR were diluted into the assay
300-fold, 13-fold, and 17-fold in the respective binding reactions.
The DMSO concentration in all binding reactions was 1% (v/v).
Following 1 h of incubation in the dark at room temperature, the
fluorescence polarization signal was measured using a Molecular
Devices Analyst plate reader with excitation and emission filters
of 550 and 580 nm selected and with a 561 nm dichroic mirror
installed. The positive control FP signal was measured in assay
wells containing binding reactions without inhibitor. The fluores-
cence polarization signal produced by maximal inhibition (back-
ground control) was determined using the signals from wells
containing 2 µM concentrations of a standard inhibitor for each
receptor: dexamethasone for GR and MR and RU-486 for PR.
Fluorescence polarization signals from duplicate 11-point concen-
tration-response curves were fitted to a 4-parameter logistic
equation to determine IC50 values.
Cellular Assays. The fibroblast IL-6 assay measures the ability
of test compounds to inhibit the elaboration of IL-6 by human
foreskin fibroblasts following stimulation by IL-1 in vitro. Human
foreskin fibroblasts (ATCC no. CRL-2429) were grown in Iscove’s
medium supplemented with 10% (v/v) charcoal/dexamethasone-
treated fetal bovine serum and plated at 20000 cells/well 2 days
prior to the assay. On the day of the assay, the medium was
replaced, followed by the addition of the test compound (0.4%
DMSO final assay concentration) and recombinant human IL-1
(final concentration 1 ng/mL). After incubation at 37 °C for 18-
24 h, IL-6 in the tissue culture medium was quantitated using
standard electrochemiluminescence, DELFIA, or ELISA methods.
Test compound IC50 values were determined by fitting the data
from duplicate 11-point concentration-response curves to a
4-parameter logistic equation. In this assay, IL-6 inhibition is
considered a measure of the agonist response of the glucocorticoid
receptor, and dexamethasone is considered to possess 100%
efficacy.
The fibroblast aromatase assay measures the ability of test
compounds to induce aromatase activity in human foreskin
fibroblasts, as indicated by the production of â-estradiol in the
presence of exogenously added testosterone. Human foreskin
fibroblasts (ATCC no. CRL-2429) were grown in Iscove’s medium
supplemented with 10% (v/v) charcoal/dexamethasone-treated fetal
bovine serum and plated at 10000 cells/well 5 days prior to the
assay. On the day of the assay, the medium was replaced, followed
by the addition of the test compound (0.4% DMSO final assay
1-[4-(2,3-Dihydrobenzofuran-7-yl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-3,5-dimethyl-1H-pyridin-4-one (39). Mp:
237-238 °C. 1H NMR (400 MHz, MeOH-d4): δ 7.35 (s, 2H), 7.17
(d, 1H, J ) 7.8 Hz), 7.13 (d, 1H, J ) 7.3 Hz), 6.85 (m, 1H), 4.59
(m, 2H), 3.74 (d, 1H, J ) 15.1 Hz), 3.50 (d, 1H, J ) 15.1 Hz),
3.18 (m, 2H), 2.92 (d, 1H, J ) 15.3 Hz), 1.99 (d, 1H, J ) 15.3
Hz), 1.95 (s, 6H), 1.64 (s, 3H), 1.38 (s, 3H). MS (ES): m/e 410
([M + H]+). HPLC purity: method A 99%, tR 2.81 min; method
B 98%, tR 7.30 min.
1-[4-(5-Fluoro-2-methylphenyl)-2-hydroxy-4-methyl-2-(tri-
fluoromethyl)pentyl]-1H-[1,6]naphthyridin-4-one (40). Mp: 253-
1
255 °C. H NMR (400 MHz, MeOH-d4): δ 9.27 (s, 1H), 8.45 (d,
1H, J ) 6.3 Hz), 7.76 (d, 1H, J ) 8.0 Hz), 7.38 (dd, 1H, J ) 2.7,

Quinol-4-ones as Steroid A-Ring Mimetics
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7895
(8) Katzenellenbogen, J. A.; Katzenellenbogen, B. S. Nuclear hormone
receptors: ligand-activated regulators of transcription and diverse
cell responses. Chem. Biol. 1996, 3, 529-536.
(9) Burnstein, K. L.; Cidlowski, J. A. The down side of glucocorticoid
receptor regulation. Mol. Cell. Endocrinol. 1992, 83, C1-C8.
(10) Coghlan, M. J.; Elmore, S. W.; Kym, P. R.; Kort, M. E. Selective
Glucocorticoid Receptor Modulators. In Annual Reports in Medicinal
Chemistry; Doherty, A. M., Ed.; Academic Press: New York, 2002;
pp 167-176.
(11) Schacke, H.; Rehwinkel, H.; Asadullah, K.; Cato, A. C. Insight into
the molecular mechanisms of glucocorticoid receptor action promotes
identification of novel ligands with an improved therapeutic index-
amethasone. Exp. Dermatol. 2006, 15, 565-573.
concentration) and testosterone (final concentration 1 µM). After
incubation at 37 °C for 18-24 h, estradiol was quantitated in the
tissue culture medium using a commercial ELISA (ALPCO no.
020-DR-2693). Test compound EC50 values were determined by
fitting the data from duplicate 11-point concentration-response
curves to a 4-parameter logistic equation. For test compound
comparison, dexamethasone was considered to possess 100%
efficacy in this assay.
HeLa MMTV transactivation assay measures the ability of test
compounds to activate MMTV promoter in HeLa cells stably
transfected with MMTV luciferase construct. HeLa MMTV cells
were grown in DMEM medium supplemented with 10% (v/v) fetal
bovine serum (Hyclone), 100 U/mL penicillin, 100 µg/mL strep-
tomycin, 2 mM L-glutamine, and 300 µg/mL genetecin. The day
prior to assay the cells were plated at 25000 cells/well in 96-well
white clear bottom cell culture plates in DMEM medium supple-
mented with 3% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 2 mM L-glutamine. On the day of the assay, the
medium was replaced, followed by addition of the test compound
(0.2% DMSO final assay concentration). Positive control wells
received 1 µM dexamethasone and represent 100% induction, while
negative control wells received vehicle only and represent back-
ground. After final addition of the compounds the plates were
incubated for 6 h at 37 °C and 5% CO2. After the incubation period
luminescence was detected upon cell lysis with Steady Glow
luciferase reagent (Promega no. E2520). Activation of MMTV
promoter by test compounds is expressed in percentage relative to
the positive control. Test compound EC50 values were determined
by nonlinear curve fitting of the data from duplicate eight-point
concentration-response curves.
In Vivo LPS Challenge Assay. Female Balb/c mice, weighing
approximately 20 g, were used. Mice were administered 1a, the
test compound, and vehicle in cremophor (po) approximately 60
min prior to LPS/D-gal administration. The volume of oral gavage
was 0.15 mL. Then the mice were administered LPS (Escherichia
coli LPS 055:85, 1.0 µg/mouse) plus D-gal (50 mg/kg) intravenously
in 0.2 mL of pyrogen-free saline. One hour after LPS/D-gal
administration, each mouse was anesthetized and bled by cardiac
puncture and the blood collected for serum TNF-R and compound
levels. Blood samples were centrifuged at 2500 rpm for 10-15
min, the serum was decanted, and the samples were stored frozen
at -70 °C until transfer either for TNF-R determination or to Drug
Metabolism and Pharmacokinetics for plasma concentration analysis
by HPLC. The concentration of TNF-R in the serum was measured
by a commercially available ELISA kit (R&D Systems, Min-
neapolis, MN). ELISA was performed according to the manufac-
turer’s assay procedure. All samples were assayed in duplicate.
(12) Belvisi, M. G.; Brown, T. J.; Wicks, S.; Foster, M. L. New
Glucocorticosteroids with an improved therapeutic ratio? Pulm.
Pharmacol. Ther. 2001, 14, 221-227.
(13) Miner, J. N. Designer glucocorticoids. Biochem. Pharmacol. 2002,
64, 355-361.
(14) Schacke, H.; Schottelius, A.; Docke, W. D.; Strehlke, P.; Jaroch, S.;
Schmees, N.; Rehwinkel, H.; Hennekes, H.; Asadullah, K. Dissocia-
tion of transactivation from transrepression by a selective glucocor-
ticoid receptor agonist leads to separation of therapeutic effects from
side effects. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 227-232.
(15) Barker, M.; Clackers, M.; Demaine, D. A.; Humphreys, D.; Johnston,
M. J.; Jones, H. T.; Pacquet, F.; Pritchard, J. M.; Salter, M.; Shanahan,
S. E.; Skone, P. A.; Vinader, V. M.; Uings, I.; McLay, I. M.;
Macdonald, S. J. Design and synthesis of new nonsteroidal gluco-
corticoid modulators through application of an “agreement docking”
method. J. Med. Chem. 2005, 48, 4507-4510.
(16) Bledsoe, R. K; Lambert, M. H.; Montana, V. G.; Stewart, E. L.; Xu,
E. H. Structure of a Glucocorticoid Ligand Binding Domain
Comprising an Expanded Binding Pocket, and Methods Using
Nuclear Receptors Structure for Drug Design. Eur. Pat. Appl.
1375517, 2004.
(17) Betageri, R.; Zhang, Y.; Zindell, R. M.; Kuzmich, D.; Kirrane, T.
M.; Bentzien, J.; Cardozo, M.; Capolino, A. J.; Fadra, T. N.; Nelson,
R. M.; Paw, Z.; Shih, D. T.; Shih, C. K.; Zuvela-Jelaska, L.; Nabozny,
G.; Thomson, D. S. Trifluoromethyl group as a pharmacophore:
effect of replacing a CF3 group on binding and agonist activity of a
glucocorticoid receptor ligand. Bioorg. Med. Chem. Lett. 2005, 15,
4761-4769.
(18) Abraham, M. H.; Duce, P. P.; Prior, D. V.; Barratt, D. G.; Morris, J.
J.; Taylor, P. J. Hydrogen bonding. Part 9. Solute proton donor and
proton acceptor scales for use in drug design. J. Chem. Soc., Perkin
Trans. 2 1989, 1355-1375.
(19) Bekkali, Y.; Cardozo, M.; Kirrane, T. M.; Kuzmich, D.; Proudfoot,
J. R.; Takahashi, H.; Thomson, D.; Wang, J.; Zindell, R.; Harcken,
C. H. J. J.; Razavi, H. Glucocorticoid Mimetics, Methods of Making
Them, Pharmaceutical Compositions, and Uses Thereof. WO 03/
082787, 2003.
(20) Proudfoot, J. R.; Regan, J. R.; Thomson, D. S.; Kuzmich, D.; Lee,
T. W-H.; Hammach, A.; Ralph, M. S.; Zindell, R.; Bekkali, Y.
1-Propanol and 1-Propylamine Derivatives and Their Use as Glu-
cocorticoid Ligands. WO 04/063163, 2004.
(21) Harper, N.; Chignell, C.; Kirk, G. F. Synthesis of some N-substituted
3,5-dimethyl-4-piperdinols and their derivatives as potential analge-
sics. J. Med. Chem. 1964, 7, 726-728.
Acknowledgment. We thank Gayle DeBaun for help in the
(22) McCabe, P. H.; Milne, N. J.; Sim, G. A. Conformational study of
bridgehead lactams. Preparation and x-ray structural analysis of
1-azabicyclo[3.3.1]nonane-2,6-dione. J. Chem. Soc., Perkin Trans.
2 1989, 10, 1459-1462.
preparation of this manuscript.
References
(23) Briefly, GR, PR, and MR binding assays were performed in a
fluorescence polarization format that measures competition for
binding to the nuclear receptor, present in lysates of baculovirus-
infected insect cells, between a test compound and a fluorescently
labeled receptor ligand, or probe. IC50 values were determined by
fitting the fluorescence polarization signal data to a four-parameter
logistic equation. All IC50 values shown represent the mean of at
least two independent determinations. Repeated testing of reference
compounds in these assays demonstrates typical IC50 standard
deviations of 20-40% about the mean. See the Experimental Section
for complete details.
(1) Gupta, R.; Jindal, D. P.; Kumar, G. Corticosteroids: the mainstay in
asthma therapy. Bioorg. Med. Chem. 2004, 12, 6331-6342.
(2) Buttgereit, F.; Saag, K. G.; Cutolo, M.; da Silva, J. A.; Bijlsma, J.
W. The molecular basis for the effectiveness, toxicity, and resistance
to glucocorticoids: focus on the treatment of rheumatoid arthritis.
Scand. J. Rheumatol. 2005, 34, 14-21.
(3) Zhou, J.; Cidlowski, J. A. The human glucocorticoid receptor: one
gene, multiple proteins and diverse responses. Steroids 2005, 70,
407-417.
(4) Resche-Rigon, M.; Gronemeyer, H. Therapeutic potential of selective
modulators of nuclear receptor action. Curr. Opin. Chem. Biol. 1998,
2, 501-507.
(5) Adcock, I. M. Molecular mechanisms of glucocorticosteroid actions.
Pulm. Pharmacol. Ther. 2000, 13, 115-126.
(24) Waage, A.; Slupphaug, G.; Shalaby, R. Glucocorticoids inhibit the
production of IL6 from monocytes, endothelial cells and fibroblasts.
Eur. J. Immunol. 1990, 20, 2439-2443.
(25) IL-6 levels were quantitated in tissue culture supernatants 18-24 h
following compound addition and IL-1 stimulation, and compound
IC50 values were determined by fitting the data from duplicate 11-
point concentration-response curves to a 4-parameter logistic
equation. In this assay, IL-6 inhibition is considered a measure of
the agonist response of the glucocorticoid receptor, and dexametha-
sone is considered to possess 100% efficacy. All IC50 values shown
(6) Heck, S.; Kullmann, M.; Gast, A.; Ponta, H.; Rahmsdorf, H. J.;
Herrlich, P.; Cato, A. C. A distinct modulating domain in glucocor-
ticoid receptor monomers in the repression of activity of the
transcription factor AP-1. EMBO J. 1994, 13, 4087-4095.
(7) Schacke, H.; Docke, W. D.; Asadullah, K. Mechanisms involved in
the side effects of glucocorticoids. Pharmacol. Ther. 2002, 96, 23-
43.

7896 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Regan et al.
represent the mean of at least two independent determinations.
Repeated testing of reference compounds in these assays demonstrates
typical IC50 standard deviations of 20-30% about the mean. See
the Experimental Section for complete details.
J.; Miller, D. K.; Yamin, T. T.; Thompson, C. M.; O’Neill, E. A.;
Zaller, D.; Forrest, M. J.; Carballo-Jane, E.; Luell, S. Novel
heterocyclic glucocorticoids: in vitro profile and in vivo efficacy.
Bioorg. Med. Chem. Lett. 2005, 15, 2163-2167.
(29) Smith, C. J.; Ali, A.; Balkovec, J. M.; Graham, D. W.; Hammond,
M. L.; Patel, G. F.; Rouen, G. P.; Smith, S. K.; Tata, J. R.; Einstein,
M.; Ge, L.; Harris, G. S.; Kelly, T. M.; Mazur, P.; Thompson, C.
M.; Wang, C. F.; Williamson, J. M.; Miller, D. K.; Pandit, S.; Santoro,
J. C.; Sitlani, A.; Yamin, T. T.; O’Neill, E. A.; Zaller, D. M.;
Carballo-Jane, E.; Forrest, M. J.; Luell, S. Novel ketal ligands for
the glucocorticoid receptor: in vitro and in vivo activity. Bioorg.
Med. Chem. Lett. 2005, 15, 2926-2931.
(30) Bourguet, W.; Germain, P.; Gronemeyer, H. Nuclear receptor ligand-
binding domains: three-dimensional structures, molecular interactions
and pharmacological implications. Trends Pharmacol. Sci. 2000, 21,
381-388.
(31) Bledsoe, R. K.; Stewart, E. L.; Pearce, K. H. Structure and function
of the glucocorticoid receptor ligand binding domain. Vitam. Horm.
2004, 68, 49-91.
(32) Berkovitz, G. D.; Bisat, T.; Carter, K. M. Aromatase activity in
microsomal preparations of human genital skin fibroblasts: influence
of glucocorticoids. J. Steroid Biochem. 1989, 33, 341-347.
(33) Vayssiere, B. M.; Dupont, S.; Choquart, A.; Petit, F.; Garcia, T.;
Marchandeau, C.; Gronemeyer, H.; Resche-Rigon, M. Synthetic
glucocorticoids that dissociate transactivation and AP-1 transrepres-
sion exhibit antiinflammatory activity in vivo. Mol. Endocrinol. 1997,
11, 1245-1255.
(26) The assay measures the ability of test compounds to induce aromatase
activity in human foreskin fibroblasts, as indicated by the production
of â-estradiol in the presence of exogenously added testosterone.
â-Estradiol levels were quantitated in the tissue culture media 18-
24 h following test compound and testosterone addition, and test
compound EC50 values were determined by fitting data from duplicate
11-point concentration-response curves to a 4-parameter logistic
equation. For test compound comparison, dexamethasone was
considered to possess 100% efficacy in this assay. All EC50 values
shown represent the mean of at least two independent determinations.
Repeated testing of reference compounds in these assays demonstrates
typical EC50 standard deviations of 30-50% about the mean. See
the Experimental Section for complete details.
(27) HeLa cells stably transfected with MMTV luciferase construct are
incubated in the presence of the test compounds for 6 h. After the
incubation period, luminescence is detected in cell lysates. Test
compound EC50 values are determined by nonlinear curve fitting of
the data from duplicate eight-point concentration-response curves.
Repeated testing of reference compounds in these assays demonstrates
a 30% coefficient of variation. See the Experimental Section for
complete details.
(28) Thompson, C. F.; Quraishi, N.; Ali, A.; Tata, J. R.; Hammond, M.
L.; Balkovec, J. M.; Einstein, M.; Ge, L.; Harris, G.; Kelly, T. M.;
Mazur, P.; Pandit, S.; Santoro, J.; Sitlani, A.; Wang, C.; Williamson,
JM061273T