Discovery and SAR study of novel dihydroquinoline containing glucocorticoid receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2005.12.043

We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.

Full text of DOI:10.1016/j.bmcl.2005.12.043

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1549–1552
Discovery and SAR study of novel dihydroquinoline
containing glucocorticoid receptor ligands
Hidenori Takahashi,* Younes Bekkali, Alison J. Capolino, Thomas Gilmore,
Susan E. Goldrick, Richard M. Nelson, Donna Terenzio, Ji Wang, Ljiljana Zuvela-Jelaska,
John Proudfoot, Gerald Nabozny and David Thomson
Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA
Received 12 October 2005; revised 7 December 2005; accepted 9 December 2005
Available online 28 December 2005
Abstract—We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular
scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.
Ó 2005 Elsevier Ltd. All rights reserved.
Synthetic glucocorticoids are widely used to treat many
serious inflammatory and autoimmune disorders.1 How-
ever, a major drawback in the clinical use of synthetic
glucocorticoids is their association with a number of
severe and life-threatening adverse events, such as
enhanced bone resorption and muscle weakening.
Discovery of glucocorticoid receptor (GR) agonists that
are dissociated, that exhibit a reduced incidence or a re-
duced severity of side effects while maintaining potent
anti-inflammatory activity, is currently an intensely
sought goal.2 There are also additional efforts underway
to identify selective GR antagonists with the expectation
that these may be useful in treating diabetes.3
tive ligands exemplified by 3.6 Literature data have indi-
cated that both series of nuclear receptor ligands, 2 and
3, may bind to the receptors in a similar manner, and that
the choice of substituents at the C5-position determines
the degree of dissociation, while an optimally sized func-
tional group on C10-position in AL-438 is essential for the
desired GR/PR selectivity profile (Fig. 1).
This article discloses our initial attempts to identify a
novel series of dissociated GR ligands that is also
N
Mifepristone (RU-486) 1 has been reported as a GR
antagonist and shown to be effective for blocking gene
transcription mediated by endogenous glucocorticoids.
The utility of Mifepristone has been demonstrated for
treatment of Cushing’s syndrome, diabetes, glaucoma,
and depression.4 Although structural similarity between
Mifepristone and endogenous glucocorticoids is ob-
served, it also shares structural features with a series
from Abbott/Ligand, exemplified by 2,5 that is reported
to be dissociated in both cellular and in vivo systems.
OH
11
H
H
O
1
Mifepristone (RU-486)
5
O
C
D
Based upon literature reports it is likely that this series
evolved from a series of progesterone receptor (PR) selec-
10
A
B
O
N
H
N
H
Keywords: Glucocorticoid receptor; Ligand; SAR.
*
3 LG001447
2
AL-438
Corresponding author. Tel.: +1 203 798 5049; fax: +1 203 798
5297; e-mail: htakahas@rdg.boehringer-ingelheim.com
Figure 1. Steroidal and non-steroidal nuclear receptor ligands.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.043

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H. Takahashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1549–1552
structurally related to RU-486 wherein we append the
C11-position substituents of RU-486, believed to be nec-
essary for dissociation and GR/PR selectivity, to the
A-ring rather than the C-ring, while also opening up
the C-ring.
chose the bromide 8 as an advanced intermediate to
explore the effect of C4-position substitution. The syn-
thesis of 8 is depicted in Scheme 1. A palladium cata-
lyzed cross-coupling7 of 1-bromo-4-nitrobenzene and
2-methoxy-phenylboronic acid, followed by palladium
catalyzed hydrogen transfer reaction, afforded a precur-
sor for the Skraup reaction. Treatment of aniline 6 with
catalytic amount of iodine and acetone afforded
dihydroquinoline scaffold in modest yield. Bromide
intermediate 8 was reacted with a variety of nucleophiles
to furnish the desired analogs in acceptable overall yield
(Scheme 2).
We had initially focused on identifying short and versa-
tile synthetic routes that would allow us to explore a
variety of substituents and substitution patterns on the
dihydroquinoline core efficiently (Schemes 1–3). We
HO
OH
Br
B
For SAR studies of substituents on the phenyl ring at
the C6-position of the 1,2-dihydroquinoline, which we
envisioned as mimicking the D-ring of AL-438, we chose
known compound 106 as the key branching point inter-
mediate. For the cross-coupling reaction, we applied
modified Suzuki coupling conditions reported by Buch-
wald et al.8 due to a low yield of the products obtained
by applying standard Suzuki cross-coupling conditions.
Subsequently, compound 11 was brominated under the
same conditions as described in Scheme 1, followed by
displacement of the bromide atom by allyl mercaptan
in the presence of potassium carbonate, as base, to give
the desired analogs 13 in acceptable yields (Scheme 3).
O
a, b
+
O
H2N
NO2
4
5
6
Br
d
c
O
O
N
H
N
H
7
8
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2 (5 mol%),
DMF, 160 °C, 15 min, microwave, 70%; (b) Pd/C, NH4COOH,
MeOH, rt, 3 h, 99%; (c) acetone, iodine (30 mol%), MgSO4, 160 °C,
25 min, microwave, 43%; (d) N-bromosuccinimide, CH3CN, À20 °C,
30 min, 65%.
All the compounds were tested in binding assays against
a panel of human nuclear hormone receptors (GR, PR,
estrogen receptor (ER), and mineralocorticoid receptor
(MR)) using a fluorescence polarization competitive
binding assay.9
R
Introduction of an allyl group containing a sulfur linker,
in the C4-position of 1,2-dihydroquinoline scaffold, gave
the novel, non-selective GR ligand 16 (IC50 = 360 nM).
Those compounds with nitrogen 14 or oxygen 15
replacements for the sulfur linker did not exhibit either
GR or PR binding activity at concentrations up to
2000 nM (Table 1).
Br
X
O
O
N
N
H
H
9
X=N, O, S
8
Scheme 2. Reagents: X = N; allylamine, K2CO3, DMSO, rt, 50%;
X = O; allylalcohol, NaHMDS, THF, rt, 80%; X = S; RSH, K2CO3,
DMSO, rt 30–75%.
Replacing the allyl substituent on compound 16 had a
remarkable effect on GR binding affinity, and we
learned that the SAR at this position was not straight-
forward. For example, (i) reduction of the allyl group
to a n-propyl group resulted in a loss of binding affinity,
(ii) phenethyl and phenyl groups were tolerated but a
benzyl group was not, (iii) branched butyl and cyclic
Br
Ar
b
a
N
N
H
H
10
11
Table 1. GR and PR binding affinity with different C4-position linkers
X
Br
S
Ar
Ar
c
O
N
H
N
N
H
H
a
GR IC50 (nM)
Compound
X
PR IC50 (nM)
12
13
14
15
16
NH
O
>2000
>2000
360
>2000
>2000
460
Scheme 3. Reagents and conditions: (a) Pd(OAc)2 (0.1 mol%), (2-
biphenyl)dicyclohexylphosphine, KF, ArB(OH)2, toluene, THF, rt,
16 h, 41–77%; (b) N-bromosuccinimide, CH3CN, À20 °C, 30 min, 50–
75%; (c) K2CO3, DMSO, allyl mercaptan, rt, 16 h, 20–65%.
S
a Values are means of two experiments.

H. Takahashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1549–1552
1551
Table 2. GR and PR binding affinity with different C4-position linkers
Table 3. GR and PR binding affinity with different C6-position
substituents
R
S
S
Ar
O
O
N
N
H
H
N
H
11A
a
GR IC50 (nM) PR IC50 (nM)
Compound
R
a
GR IC50 (nM) PR IC50 (nM)
Compound Ar
2-MeO-phenyl
Phenyl
16
17
18
19
20
21
22
23
24
25
26
27
28
11A
Allyl
Propyl
360
1015
548
460
1600
16
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
360
>2000
>2000
>2000
>2000
>2000
>2000
>2000
1000
460
>2000
360
Cyclopentyl
Cyclohexyl
2-Methylbutyl
3-Methylbutyl
Phenyl
>2000
>2000
>2000
>2000
>2000
660
2-F-phenyl
371
303
2-Me-phenyl
2-EtO-phenyl
2-CF3-phenyl
2-NO2-phenyl
2-Ph-phenyl
3-MeO-phenyl
3-EtO-phenyl
4-MeO-phenyl
>2000
>2000
>2000
>2000
>2000
460
703
804
Benzyl
Phenethyl
>2000
194
800
>2000
>2000
620
4-Chlorophenyl
–C(O)CH3
–CH2C(O)OCH3
>2000
>2000
>2000
>2000
>2000
>2000
>2000
1400
>2000
>2000
>2000
>2000
>2000
2,3-Di-MeO-phenyl >2000
2,5-Di-MeO-phenyl >2000
2,6-Di-MeO-phenyl >2000
–CH2C(O)NHCH3 >2000
>2000
a Values are means of two experiments.
2-MeO-5-F-phenyl
2-Cl-3-OH-phenyl
190
129
960
a Values are means of two experiments.
alkyl groups were generally tolerated, and (iv) polar
substituents such as ketone, ester, amide were not
tolerated (Table 2). Surprisingly, phenethyl derivative
24 demonstrated improved GR binding affinity
(IC50 = 190 nM) and exhibited more than 10-fold
selectivity over PR binding (IC50 > 2000 nM). Overall,
replacement of the allyl group with a phenethyl group
resulted in a 2-fold improvement in GR binding affinity,
and a significant improvement in PR binding selectivity.
Additionally, both benzyl 23 and acetyl 26 derivatives
demonstrated higher binding affinity to PR than GR.
The SAR analysis at this position suggested that minor
modifications modulated both GR binding affinity and
PR selectivity. Lastly, simple methyl substituted deriva-
tive on C4-position 11A showed no binding affinity to
either GR or PR. These results suggest that it is required
to employ optimally sized substituent and precise
positioning for improved GR binding affinity.
3-hydroxy 43 disubstituted analogs displayed good GR
binding affinity and significant binding selectivity over
PR. Adding a fluorine group at the 5-position of the
2-methoxyphenyl ring resulted in a 2-fold increase in
GR binding affinity and moderate improvement of selec-
tivity profile. For substituents on the phenyl ring at the
C2-position, although a hydrogen-bonding motif or a
halogen atom was favored, precise positioning of hydro-
gen-bonding functional group was required in order to
achieve improved GR binding affinity. Overall the
SAR of this position displayed that minor modifications
resulted in a substantial loss of both GR and PR binding
affinity.
Additionally, we prepared compounds that combined
the optimal C4- and C6-position substituents (Table
4). These combination molecules contained the C4-
position phenethyl and the C6 2-methoxy-5-fluorophe-
nyl, to give derivative 44, or the C6 2-chloro-3-
hydroxyphenyl to give derivative 45. Compound 44
Subsequently, we explored the effects of varying the sub-
stitution pattern on the phenyl ring at the C6-position of
the dihydroquinoline core (Table 3). We focused on test-
ing the steric and electronic effects of substituents on the
phenyl ring. Shifting the methoxy group on the phenyl
ring from the ortho-position 16 (GR IC50 = 360 nM) to
the meta-position 36 (GR IC50 = 1000 nM) resulted in
an approximately 3-fold loss in GR binding potency,
while maintaining PR binding affinity resulting in 36
being a moderately PR selective ligand. The 4-methoxyl
phenyl analog 38 displayed a significant loss in both GR
and PR binding affinity. When the 2-methoxy group was
replaced with hydrogen 29, fluorine 30, methyl 31,
ethoxy 32, trifluoromethyl 33, nitro 34, and phenyl 35,
those compounds all demonstrated a loss of GR binding
affinity. These results suggested that there is a size limi-
tation for substituents on these positions. Neither did
the di-methoxy phenyl analogs 39, 40, and 41. On the
other hand, both 2-methoxy-5-fluoro 42 and 2-chloro-
Table 4. GR, PR, ER, and MR binding affinity of compounds with the
best substituent combinations at C4- and C6-positions
F
S
S
OH
O
Cl
N
N
H
H
44
45
Compound GR
PR
ER
MR
IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
a
44
45
84
220
>2000
1400
>2000
>2000
>2000
>2000
a Values are means of two experiments.

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H. Takahashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1549–1552
5. (a) Elmore, S. W.; Coghlan, M. J.; Anderson, D. D.; Pratt,
J. K.; Green, B. E.; Wang, A. X.; Stashko, M. A.; Lin, C.
W.; Tyree, C. M.; Miner, J. N.; Jacobson, P. B.; Wilcox,
D. M.; Lane, B. C. J. Med. Chem. 2001, 44, 4481; (b)
Kym, P. R.; Kort, M. E.; Coghlan, M. J.; Moore, J. L.;
Tang, R.; Ratajczyk, J. D.; Larson, D. P.; Elmore, S. W.;
Pratt, J. K.; Stashko, M. A.; Falls, H. D.; Lin, C. W.;
Nakane, M.; Miller, L.; Tyree, C. M.; Miner, J. N.;
Jacobson, P. B.; Wilcox, D. M.; Nguyen, P.; Lane, B. C. J.
Med. Chem. 2003, 46, 1016; (c) Coghlan, M. J.; Jacobson,
P. B.; Lane, B.; Nakane, M.; Lin, C. W.; Elmore, S. W.;
Kym, P. R.; Luly, J. R.; Carter, G. W.; Turner, R.; Tyree,
C. M.; Hu, J.; Elgort, M.; Rosen, J.; Miner, J. N. Mol.
Endocrinol. 2003, 17, 860.
displayed a 4-fold improvement in GR binding affinity
(IC50 = 84 nM) and a good selectivity profile across a pan-
el of nuclear hormone receptors. Overall, the SAR study
at the C4-position led to a 2-fold improvement in GR
binding affinity, specifically by replacing the allyl group
with a phenethyl group. The SAR study at the C6-posi-
tion also led to a 2-fold improvement of GR binding affin-
ity via the addition of a fluorine atom at the 5-position.
The combination of these two independent findings
resulted in a 4-fold improvement in GR binding affinity
and a significant improvement in the selectivity profile
(>20-fold) against PR. This additive result suggests that
the C4- and C6-positions are contributing independently
to the overall binding potency.
6. Pooley, C. L. F.; Edwards, J. P.; Goldman, M. E.;
Wang, M. W.; Marschke, K. B.; Crombie, D. L.;
Jones, T. K. J. Med. Chem. 1998, 41, 3461.
We tested the functional activity of these compounds
using an IL-1-induced IL-6 assay in human foreskin
fibroblasts.10 Although both compounds 44 and 45 did
not show inhibition of IL-6 production (agonist activity)
in this assay, these compounds demonstrated their abil-
ity to prevent a response to dexamethasone induced-GR
transactivation of an MMTV reporter gene in HeLa
cells.11 In the latter assay, 44 and 45 demonstrated an
IC50 = 45 and 260 nM, respectively. These data suggest
that these ligands are selective GR antagonists.
7. Bellina, F.; Carpita, A.; Rossi, R. Synthesis 2004, 15, 2419.
8. Wolfe, J. P.; Singer, R. A.; Yang, B. H.; Buchwald, S. L.
J. Am. Chem. Soc. 1999, 122, 9550.
9. Fluorescence polarization competitive binding assays were
performed to quantitate the ability of test compounds to
displace ligands from GR, MR, ER, and PR in solution.
Binding reactions were assembled in 96-well microplates.
Baculovirus lysate containing either GR or MR was
incubated with 5 nM tetramethyl-rhodamine conjugate of
dexamethasone, and test compound dilutions in an assay
buffer containing 10 mM TES, 50 mM KC1, 20 mM
sodium molybdate, 1.5 mM EDTA, 0.04% w/v CHAPS,
10% v/v glycerol, and 1 mM DTT, pH 7.4. For the PR
assay, baculovirus lysate containing PR was incubated
with 5 nM tetramethyl-rhodamine conjugate of RU486
and the test compound dilutions. The ER binding assay
was performed using the ER Competitor Assay kit from
Panvera (Invitrogen part number P2614). This assay uses
purified baculovirus-expressed human ER and fluorescein
conjugate of a proprietary ER ligand (FluormoneTM ES2).
IC50 values shown are means of a single experiment
done in duplicate 11-point concentration–effect curves.
Bekkkali, Y.; Gilmore, T.; Spero, D. M.; Takahashi, H.;
Thomson, D. S.; Wang, J. PCT Int. Appl.,
WO2004018429.
10. Human foreskin fibroblasts were stimulated with 1 ng/mL
recombinant human IL-1 in the presence of test com-
pound. After 24 h, the degree of GR agonist activity
(transrepression) was determined by measuring IL-6 in the
tissue culture media.
11. HeLa cells stably transfected with MMTV luciferase
construct were preincubated with 20 nM dexamethasone
for 15 min. After preincubation, cells were treated with the
compounds or their vehicle (0.2% DMSO) and incubated
for 6 h. After the incubation, the induction of luciferase
was measured. IC50 values are means of two experiments.
In summary, a novel and potent series of selective GR
ligands was identified. We found that variation of both
the substituents at the C4- and the C6-positions can
impact both GR binding affinity and GR/PR binding
selectivity. The most potent ligand identified, compound
44, is a novel, potent, and highly selective GR antagonist.
References and notes
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Disc. Dev. 2003, 6, 224.
3. Coghlan, M. J.; Elmore, S. W.; Kym, P. R.; Kort, M. E.
Ann. Rep. Med. Chem. 2002, 37, 167.
4. (a) Chu, J. W.; Matthias, D. F.; Belanoff, J.; Schatzberg,
A.; Hoffman, A. R.; Feldman, D. J. Clin. Endocrinol.
Metab. 2001, 86, 3568; (b) Gettys, T. W.; Watson, P. M.;
Taylor, I. L.; Collins, S. Int. J. Obes. 1997, 21, 865; (c)
Phillips, C. I.; Green, K.; Gore, S. M.; Cullen, P. M.;
Campbell, M. Lancet 1984, 1, 767; (d) Belanoff, J. K.;
Flores, B. H.; Kalezhan, M.; Sund, B.; Schatzberg, A. F.
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