Non-steroidal dissociated glucocorticoid agonists: Indoles as A-ring mimetics and function-regulating pharmacophores

Article abstract of DOI:10.1016/j.bmcl.2011.09.018

We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.

Full text of DOI:10.1016/j.bmcl.2011.09.018

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6842–6851
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Non-steroidal dissociated glucocorticoid agonists: indoles as A-ring
mimetics and function-regulating pharmacophores
Raj Betageri a, , Thomas Gilmore a, Daniel Kuzmich a, Thomas M. Kirrane a, Jörg Bentzien a,
⇑
Dieter Wiedenmayer a, Younes Bekkali a, John Regan a, Angela Berry a, Bachir Latli a, Alison J. Kukulka a,
Tazmeen N. Fadra a, Richard M. Nelson a, Susan Goldrick b, Ljiljana Zuvela-Jelaska b, Don Souza b,
Josephine Pelletier b, Roger Dinallo c, Mark Panzenbeck c, Carol Torcellini c, Heewon Lee a, Edward Pack a,
Christian Harcken a, Gerald Nabozny b, David S. Thomson a
a Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA
b Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA
c Drug Discovery Support, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed
SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation
profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist
activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is
equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound
(R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 7 August 2011
Revised 1 September 2011
Accepted 6 September 2011
Available online 10 September 2011
Keywords:
Non-steroidal glucocorticoid agonist
Dissociated activity
Function-regulating pharmacophore
The glucocorticoid receptor (GR), a member of the nuclear
receptor family, is an attractive drug discovery target, and chemi-
cal entities that function as GR agonists and antagonists have ther-
apeutic potential. Research in the last decade has led to a better
understanding of molecular mechanisms that mediate functions
of the GR.1–7 Transrepression (TR) promotes downregulation of
target genes that encode cytokines, cell adhesion molecules and
enzymes, and TR is thought to be predominantly involved in medi-
ating the anti-inflammatory effects of the GR.8–10 Overactivation of
certain transcriptional genes, known as transactivation (TA), has
been implicated in the deleterious side effects of glucocorticoids
(GCs).11,12 The concept, ‘dissociated GR ligand’ has evolved out of
a suggestion that it is feasible to design GR ligands that display dif-
ferential effects on TR and TA pathways. A therapy based on such
dissociated GR agonists is anticipated to give the desired outcome
of ant-inflammatory effect similar to prednisolone but with de-
creased side effects. At a molecular level, designing a dissociated
GR ligand entails optimized positioning of function-regulating
pharmacophores (FRPs) in addition to achieving a suitable size
and shape of the ligand.
As a result, identifying non-steroidal dissociated GR agonists
has been a goal sought by drug discovery efforts for some time.
Several reports disclosing novel non-steroidal GR ligands from dif-
ferent structural classes have appeared in the literature.13–24 Re-
cent review articles25–27 summarize different chemotypes that
function as GR agonists with a dissociated profile.
Identification of trifluoromethylcarbinol group as a pharmaco-
phore essential for the agonist activity of GR ligands can be charac-
terized as one of the significant discoveries in the development of
GR modulators. Disclosure of ZK216438 (+ enantiomer) as a disso-
ciated GR ligand spurred extensive research activity that has led to
the discovery of multiple classes of GR agonists containing trifluo-
romethylcarbinol group. Summarized accounts of the activity pro-
files of these compounds can be found in the GR literature.26–29
Selected examples of trifluoromethylcarbinol containing GR
agonists are shown in Figure 1. In addition to identifying several
A- and D-ring mimics, these studies have provided insight into
the binding modes of various non-steroidal GR agonists. X-ray
structures of GR ligand binding domain (GR—LBD) co-crystals with
GSK130 and GSK231 have been solved which have helped in estab-
lishing the role of trifluoromethylcarbinol in these ligands. Hydro-
xyl group of trifluoromethylcarbinol moiety corresponds to the
steroidal 11-b-OH and the previously hypothesized hydrogen
bonding interaction with Asn564 is now confirmed by these
⇑
Corresponding author. Tel.: +1 203 798 5428.
E-mail address: raj.betageri@boehringer-ingelheim.com (R. Betageri).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.018

R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
6843
CF3
CF3
OH
Ar
OH
Cl
H
N
N
N
CF3
OH
N
H
N
O
N
N
N
F
O
O
H2N
O
Cl
O
O
F
ZK-216348
GSK1
H2N
O
CF3
OH
N
F
N
N
O
CF3
OH
N
N
CF3
OH
H
N
N
F
F
H
H
N
H2N
O
N
O
O
GSK2
GSK3
PF1
Figure 1. Known non-steriodal GR agonists containing trifluoromethylcarbinol pharmacophore.
X-ray crystal studies. Our previous report showed that replacing
the trifluoromethyl group with a benzyl or cyclohexyl moiety in
a GR ligand related to ZK216438 alters the functional profile from
agonist to an antagonist.32 Some of the dissociated GR ligands cur-
rently in the clinical trials are likely to be trifluoromethylcarbinol
derivatives which highlight their prominent role in the discovery
of non-steroidal glucocorticod mimetics.
HO
HO
O
H
O
H
11
F
OH
OH
HO
HO
17
D
H
H
A
H
O
O
Our quest for receptor selective, dissociated GR ligands utilized
ZK216348 as the starting point and the SAR studies led to the dis-
covery of compound 1 (Fig. 2).33,34 Compound 1 was reported as a
potent GR ligand but one that displayed poor selectivity over PR
and MR. In the in vitro assays 1 is a partial agonist and shows a dis-
sociated profile. The docking pose of a closely related analogue of
1, generated by docking that compound into the GR—LBD derived
from the GR—LBD dexamethasone co-crystal X-ray structure, sug-
gests that the left-hand side (LHS) phenyl ring of 1 and the A-ring
of dexamethasone overlay. In addition, SAR studies of 1 revealed
that subtle changes in the substitution patterns on the LHS phenyl
ring have a dramatic effect on the agonist activity, consistent with
literature data where steroid A-ring mimics are reported to play a
crucial role in influencing the agonist activity. We set out to ex-
plore different A-ring replacements in compound 1 as an initial
step towards identifying novel dissociated GR ligands. A quick sur-
vey of aromatic heterocyclic rings such as 2-benzofuran, 2-benzo-
thiophene, 2-benzimidazole and 2-indole identified compound 2
as a potent GR agonist.35 Compound 2 showed no detectable bind-
Dexamethasone
Prednisolone
CF3
R
F
HO
O
Cl
R =
N
N
N
H
N
H
NC
O
1
2
3
4
Figure 2. Steroidal and non-steroidal glucocorticoid agonists.
residues and the A-ring carbonyl are observed for dexamethasone.
The C-5 and C-6 carbon atoms of the indole ring are in proximity to
the Gln570/Arg611 residues but lacking a hydrogen bond acceptor
at either of these positions (R)-2 cannot participate in an interac-
tion similar to the A-ring carbonyl of dexamethasone. Instead, a
potential hydrogen bond between the indole NH and the Leu563
backbone carbonyl is implicated by the model (Fig. 3a). Adjacent
to the indole NH is the C-7 carbon and the presence of the iso-butyl
side chain of Leu566 is suggested in the surrounding binding re-
gion. The binding region at the C-4 position of the indole ring is
hydrophobic with the presence of the Ile608 and Phe623 side
chains. At the D-ring region, no key interactions between the 2-
methoxy-5-fluoro ring and the GR—LBD (Fig. 3b) are identified. A
design approach was formulated that utilizes our binding hypoth-
esis and exploits the scope of literature reports that describe small
structural changes to ligands can have significant effects on the
functional profile.41–44 We set out to examine the effect of substi-
tution by small groups such as F, CH3, CF3 and cyano on the indole
ing to AR at 1.1 lM but it lacks the desired nuclear receptor selec-
tivity over PR and MR (Table 1). In the in vitro transactivation assay
compound 2 is not dissociated. However, full agonist properties of
this ligand, as reflected by both the IC50 and % maximal efficacy in
the TR assays, make this an attractive candidate for further optimi-
zation.36 Accordingly, SAR studies were initiated aimed at improv-
ing the PR and MR selectivity and in vitro dissociation profile of the
GR agonist 2.
To gain insight into binding interactions of 2 with the GR—LBD,
(R)-2 was docked37–39 into the GR—LBD using the dexamethasone
GR—LBD co-crystal X-ray structure.40 The indole and 2-methoxy-5-
fluoro groups of (R)-2 and the A- and D-rings, respectively, of dexa-
methasone overlay (Fig. 3a and b). A common interaction for the
two ligands involves the Asn564 side chain, where the central
hydroxyl of (R)-2 and the 11-b-OH of dexamethasone overlay
and are suggested to hydrogen bond the Asn564 side chain. Key
interactions that engage the network of the Gln570/Arg611

6844
R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
Table 1
Data on the Enantiomers of 2
F
F
CF3
CF3
OH
OH
N
H
N
H
O
O
(R)-2
(S)-2
Compd
IC50 (nM)
MR
IC50 (nM),b (% efficacy)c
IL-6 inhibition
EC50 (nM),d (% efficacy)c
Aromatase induction
GR
PR
ARa
Dexamethsasone
Prednisolone
2
(R)-2
(S)-2
3
15
22
10
>2000
>2000
290
207
1200
33
44
383
190
1100
0.51 (100)
7 (96)
7 (87)
6 (87)
>2000 (20)
1.8 (100)
19 (92)
23 (93)
18 (98)
>2000 (20)
NDB at 2000 nM
NDB at 1100 nM
NDB at 1200 nM
>2000
a
NDB: no detectable binding.
Transrepression activity—IL-6 inhibition in HFF cells.
% Maximal efficacy versus dexamethasone, see note 50.
b
c
d
Transactivation activity—induction of aromatase in HFF cells, see notes 52 and 53.
Figure 3. (a) (Left) and b (right): Docking poses for (R)-2 (yellow) in the GR—LBD co-complex structure with dexamethasone40 (orange). (a) Shows the region around the A-
ring of dexamethasone, (b) Shows the region around the D-ring. Selected amino acids exhibiting key interactions with the ligand are shown. Potential H-bonds are indicated
as dashed white lines.
ring as well as modifying the right-hand side (RHS) phenyl ring
with the expectation that these changes could provide analogues
of 2 with an improved profile. Results of these studies are the sub-
ject of this report.
corresponding triflates 6. Sonogashira coupling followed by
Fe-AcOH reduction yielded the key aminoalkyne intermediates 9.
Alternatively, 9 could be prepared by employing ortho-iodo/bromo
anilines 7 for the Sonogashira coupling. Cyclization of 9 to the
indole analogue was accomplished by heating the corresponding
trifluoroacetylated derivative with K2CO3 or Cs2CO3 in DMSO. Syn-
thesis of compounds 2 and 10–25 (see Tables 2–4) has been
described in detail elsewhere.35,45,46 The enantiomers of 2 were
obtained by chiral resolution on a chiral-OD 2 cm semi-prep
column eluting with 5% isopropanol in hexanes. The first eluting
peak was identified as the eutomer (R)-2 based on the GR
binding affinity of the two enantiomers (Table 1) and previous
stereochemical assignments established from X-ray crystallo-
graphic studies.47
A systematic exploration of the SAR required a general method
for the preparation of target molecules represented by 5. Figure 4
depicts the retro-synthetic strategy for 5 highlighting 5a as the
key intermediate, which could be prepared by Sonogashira
coupling of the alkyne 5c and an appropriate triflate or halogen
derivative 5b. The trifluoromethyl ketone 5d, prepared using previ-
ously established chemistry,33,34 could serve as the precursor for
5c. The synthesis is outlined in Scheme 1. The desired ortho-nitro-
phenols were obtained by treating the phenol precursors with
nitronium tetrafluoroborate, which were then converted to the
X
CF3
CF3
O
Y
X
CF3
X
HO
+
CF3
HO
N
R
HO
H
NH2
5
5a
5b
5c
5d
Figure 4. Retro-synthetic pathway for the preparation of target molecules 5.

R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
6845
O
CF3
S
OH
O
OH
a
b
O
X
N+
O
O
X
X
N+
O
O
6
8
c, d
X
CF3
X
c
e, f
F
Y
CF3
+
CF3
F
F
X
HO
N
H
NH2
HO
NH2
HO
MeO
MeO
MeO
9
7
8
Scheme 1. Reagents and conditions: (a) NO2+ BFÀ4 MeCN, 0 °C, 1 h; (b) (CF3SO2)2O, NEt3, CH2Cl2, 0 °C, 2 h; (c) Pd(PPh3)2Cl2 (0.05 equiv), CuI (0.1 equiv), NEt3, MeCN, rt, 1–15 h;
(d) Fe, CH3CO2H, 60 °C, 30 min; (e) (CF3CO2)2O, CH2Cl2, 0 °C; (f) anhydrous K2CO3, DMSO, 140 °C, microwave, 15 min.
Table 2
7-Substituted indoles
5
4
3
CF3
OH
6
F
N
7
R
H
O
Compd
R
IC50 (nM)
PR
IC50 (nM), (% efficacy)
IL-6 inhibition
EC50 (nM), (% efficacy)
Aromatase induction
GR
MR
2
H
F
CH3
CF3
CN
22
15
56
120
27
290
930
1450
1750
>2000
383
435
1035
1650
1420
7 (87)
36 (75)
>2000 (26)
>2000
>2000 (30)
23 (93)
473 (89)
>2000 (26)
>2000
10
11
12
13
>2000
Table 3
4-Substituted indoles
R
CF3
OH
F
N
H
O
Compd
R
IC50 (nM)
PR
IC50 (nM), (% efficacy)
IL-6 inhibition
EC50 (nM), (% efficacy)
Aromatase induction
GR
MR
2
H
22
30
160
5
290
350
>2000
440
1000
383
415
>2000
225
790
7 (87)
6 (91)
290 (70)
8 (91)
88 (90)
23 (93)
9 (110)
IP* (30)
21 (91)
IP (49)
14
15
16
17
CH3
CF3
CN
Et
28
*
Indeterminate potency as a result of no defined inflexion point in the dose response curve.
The dihydrobenzofuran (DBF) analogues 26–30 (Table 5) were
prepared following previously described procedures.33 The synthe-
sis of DBF analogue 33 was carried out according to Scheme 2.
Compounds 31 and 32 were prepared following a similar proce-
dure. Thus, the intermediate diol 34a prepared by following the
previously described procedures35,45 was converted to the corre-
sponding acetonide. Halogen-metal exchange using n-BuLi
followed by treatment with dimethyldisulfide and deprotection
of the acetonide gave the sulfide 34b. Cleavage of the vicinal
diol and concomitant oxidation of the sulfide to give the

6846
R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
Table 4
5- and 6-Substituted indoles
R
CF3
OH
F
N
H
O
Compd
R
IC50 (nM)
PR
IC50 (nM), (% efficacy)
IL-6 inhibition
EC50 (nM), (% efficacy)
Aromatase induction
EC50 (nM), (% efficacy)
MMTV induction
GR
MR
Dexamethsasone
Prednisolone
2
—
—
H
5-F
5-CH3
5-CF3
5-CN
5-CN
6-F
6-CH3
6-CF3
6-CN
3
>2000
>2000
290
33
44
0.51 (100)
7 (96)
7 (87)
1.8 (100)
19 (92)
23 (93)
94 (98)
230 (104)
640 (65)
28 (81)
6 (71)
17 (100)
16 (91)
—
—
—
—
—
15
22
24
66
290
8
383
405
1250
>2000
415
45
350
720
910
435
18
19
20
21
(R)-21
22
23
24
25
405
78 (84)
113 (86)
191 (77)
11 (89)
3 (90)
29 (94)
334 (60)
>2000
1800
>2000
815
155
265
1250
1600
395
1
36 (10)
30
38
77
14
33 (84)
976 (70)
>2000
—
—
—
—
14 (91)
113 (98)
Table 5
Substituted Dihydrobenzofuran Analogues
CF3
OH
R
N
H
O
Compd
R
IC50 (nM)
IC50 (nM), (% efficacy)
EC50 (nM), (% efficacy)
Aromatase induction
GR
PR
MR
IL-6 inhibition
26
27
28
29
30
31
H
F
CH3
Br
CN
19
8
10
32
15
6
225
255
145
415
710
>2000
400
265
240
585
405
610
10 (83)
21 (90)
7 (95)
16 (88)
104 (65)
100 (84)
311 (103)
125 (98)
16 (105)
45 (99)
356 (55)
SO2CH3
>2000* (20)
*
Data reflects MMTV Induction in HeLa, EC50 (nM), efficacy % versus dex.
methylsulfone-DBF-trifluoromethyl ketone 34 was carried out by a
two stage oxidation employing NaIO4 and RuCl3. Subsequent
elaboration of 34 gave the alkyne 35, which was subjected to
Sonogashira coupling with the iodo derivative 36, synthesized
according to literature protocols (Scheme 2). Cs2CO3 mediated
cyclization of the resulting intermediate 37 led to the racemic
6-cyano indole analogue 33. The chiral separation on a Chiralpak
AD-H preparative column using 20% isopropanol in hexanes
provided the eutomer (R)-33 as the second eluting peak.
Nuclear receptor binding affinity of the target compounds was
determined by their ability to compete for receptor binding with
tetramethylrhodamine labeled dexamethasone (GR, MR) or mife-
pristone (PR) or 3H-testosterone (AR).48 The transrepression poten-
tial was measured in human foreskin fibroblast (HFF) cells.
Inhibition of IL-1 stimulated IL-6 production and percent efficacy
was measured relative to dexamethasone, where inhibition by
dexamethasone was set to 100%.49,50
the clinic). Two factors could be important in this regard. The first
factor relies on identifying appropriate markers for evaluating
in vitro transactivation. Our transactivation assays employ the
MMTV promoter and aromatase51 as markers of gene activation.
The compounds were counter screened for the induction of
aromatase in HFF cells52 and the ability to activate the MMTV
promoter in HeLa cells transfected with a MMTV luciferase
construct.25,53–57 Comparisons with dexamethasone establish a
basis for identifying compounds with a dissociated profile. The sec-
ond factor relates to the required level of separation between the
transrepression and transactivation activities that could qualify
as in vitro dissociation. Broadly, dissociation can be defined as
weak potency or low % maximal efficacy or both in transactivation
assays compared to transrepression assays.
Since the racemic compound 2 and eutomer (R)-2 displayed
similar activity, SAR studies were performed using racemic
compounds. Initially, we prepared N-methyl and C-3 linked indole
analogues (data not shown) which showed no agonist activity in
the cellular assay while displaying good GR affinity. Introducing
CH3, CF3 and cyano groups at C-7 (Table 2, compounds 11, 12
and 13), which is proximal to the indole NH, has a similar effect
as good binding affinity did not translate into agonist activity.
Thus, blocking the indole NH (N-Me analogue) or introducing steric
Achieving full therapeutic potential of synthetic GC mimetics is
only accomplished by reducing TA activity in addition to enhancing
nuclear receptor selectivity and agonist activity. A successful clin-
ical outcome would then depend upon establishing an in vitro/
in vivo (clinical) correlation to determine what level of in vitro dis-
sociation is necessary to achieve the desired dissociation in vivo (in

R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
6847
H2N
O
I
I
I
a
b
c
O
N+
N+
O
O
CN
O
CN
HO
NH2
CN
CF3
N
H
CN
O
36
CF3
O
CF3
e
O
O
d
O
HO
CF3
CF3
O
O
O
O
Br
Br
O
O
CF3
O
O
g, h and i
OH
j
k
f
CF3
OH
HO
HO
S
O
Br
S
34a
34b
34
O
NC
O
CF3
OH
O
NC
CF3
O
l
m
CF3
OH
O
N
H
NH
CF3
OH
36
S
O
S
O
O
S
O
O
O
35
37
33
Scheme 2. Reagents and conditions: (a) NaNO2, NaI, 30% H2SO4, DMSO, 0 °C; (b) SnCl2, EtOH, 90 °C, 3 h; (c) (CF3CO2)2O, CH2Cl2, 0 °C; (d) THF, À65 °C, then
methylallylmagnesium chloride (1.3 equiv), 4 h, rt, 12 h, 61%; (e) ClCH2CH2Cl, AlCl3 at 0 °C, then rt, 12 h, 30%;(f) LiAlH4, THF, 48 h; (g) 2,2-dimethoxypropane,
p-toluenesulfonic acid; (h) n-BuLi, -78 °C, (MeS)2, 2 h; (i) methanolic HCl, rt; (j) NaIO4, RuCl3, MeCN, rt, 5 h; (k) Al, HgCl2, THF, propargyl bromide, 40 °C, 1 h, 3 h, rt; (l) 36
(1 equiv), Pd(PPh3)2Cl2 (0.05 equiv), CuI (0.1 equiv), NEt3, THF, 0 °C to rt, 2 h; (m) Cs2CO3, DMSO, 100 °C, 3 h.
demand at the indole NH (7-substituted analogues) is detrimental
for the agonist activity. These results confirm the importance of the
indole NH—Leu563 interaction for the agonist activity, which is
suggested by the docking studies ( Fig. 3a). A fluorine atom, a
hydrogen atom isostere, does not have the same effect at C-7 and
compound 10 displays good GR binding and agonist activity. These
results reinforce the often observed lack of correlation between GR
binding affinity and agonist activity.58–60 7-substituted indoles
(10–13) are more selective over PR than 2. In the aromatase assay,
7-F compound 10, the lone C-7 substituted agonist, is deemed dis-
sociated with a 10-fold separation of EC50/IC50. Although the
in vitro profile reflects receptor selectivity and dissociation, com-
pound 10 was not advanced to in vivo studies because of poor
PK properties (data not shown).
Substitutions at carbon C-4 of the indole ring, which is on the
opposite side of the indole NH, are not detrimental for agonist
activity and the compounds display IC50 in the range 6–290 nM
(14–17, Table 3) and maximal efficacies greater than 70%. Increas-
ing the size of the substitution from methyl, ethyl and CF3 results
in incremental reductions of the agonist potency (compare 14, 17
and 15) suggesting a limitation in the size of the binding pocket
near the C-4 position of the indole. The cyano analogue 16 displays
90-fold PR selectivity, and potent agonist activity unlike the C-7 cy-
ano analogue 13. The cyano analogue 16 is not dissociated in the
aromatase assay. The 4-CF3 and 4-ethyl analogues (15 and 17)
show maximal efficacies of 30% and 49%, respectively, and are clas-
sified as dissociated in the aromatase assay. But the 30-fold GR/PR
selectivity for the ethyl analogue 17 was considered less than
optimal.
carbonyl of the former residing in a space closer to the C-5 and
C-6 carbon atoms of the latter. Compound (R)-2 cannot interact
with the Arg611/Gln570 pair in a manner similar to dexametha-
sone as indole ring of the former lacks hydrogen bond acceptor
at either C-5 or C-6 position. Presence of hydrogen bond network
between the A-ring mimics of non-steroidal GR agonists and the
Arg611/Gln570 pair is established by the X-ray structures of GR—
LBD co-crystals with GSK130 and GSK231 (Fig. 1). Because of the
suggested proximity of the Arg611/Gln570 pair to the C-5 or C-6
positions of the indole ring in 2, it is expected that substitutions
at C-5 or C-6 positions to affect the activity profile of 2. While a
fluorine atom at C-5 or C-6 has little effect on the GR binding
affinity and agonist activity (Table 4), (18 and 22), introducing lipo-
philic groups such as CH3 and CF3 in the vicinity of Arg611/Gln570
pair is detrimental for the activity. The effect is more pronounced
at C-6. While the 6-CH3 and 6-CF3 analogues (23, 24) have GR bind-
ing affinity similar to 2, 6-CH3 compound 23 is a partial agonist
(IC50 334 nM, 60%) and 6-CF3 compound 24 is devoid of
agonist activity. These results perhaps suggest a disruption of the
agonist conformation of GR—LBD complex with 23 and 24. In con-
trast, both 5-CH3 and 5-CF3 analogues (19, 20, Table 4) display ago-
nist activity, but incremental loss in both the binding affinity and
agonist activity is observed on increasing the size of the substitu-
tion from CH3 to CF3. Introducing a hydrogen bond acceptor, cyano
group, at C-5 or C-6 is not detrimental and both 5-CN and 6-CN
analogues 21 and 25 display potent GR binding affinity and agonist
activity (Table 4). While the agonist activity of 21 and 25 is compa-
rable to that of the unsubstituted indole analogue 2, improvement
in selectivity over PR, MR is observed for both the compounds 21
and 25. 100-fold PR selectivity and 50-fold MR selectivity is
seen for 5-CN compound 21, which reflects a pronounced effect
As discussed earlier, our model suggests that the A-ring of dexa-
methasone and the indole ring of (R)-2 overlay with the A-ring

6848
R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
for 5-CN compared to 6-CN analogue 25 (ꢀ30-fold for PR and MR).
A hypothesis based on the amino acid residue differences in the li-
gand binding domains of GR, PR and MR could explain the ob-
served receptor selectivity data. The residue corresponding to
Cys622 (binding pose in Fig. 3a) in GR—LBD is Tyr777 in PR—LBD
and Tyr828 in MR—LBD. Interaction of the 5-CN with the Arg611/
Gln570 pair in PR and MR could lead to movement of the Tyr side
chain resulting in unfavorable interactions and loss in the binding
affinity for PR and MR. In the aromatase assay, the 5-CN compound
21 is not dissociated (28 nM, 81%) but the 6-CN indole 25 displays
an 8-fold higher EC50 (113 nM, 98%) than the IL-6 IC50 (14 nM,
91%). But the eutomer (R)-21 of the 5-CN compound exhibits a
maximal efficacy of 10% in the MMTV assay indicating a dissoci-
ated profile based on the % maximal efficacy.
Having identified the 5-CN analogue (R)-21 as a PR, MR selec-
tive GR agonist with a dissociated profile in the MMTV assay,
SAR was shifted to the RHS of the molecule 2 to assess the effect
of RHS modifications and identify a broader panel of dissociated
analogues of 2. For this purpose we initially decided to use the
unsubstituted indole ring at the LHS for the SAR studies. Docking
shows that (Fig. 3b), the phenyl ring of (R)-2 lies proximal to the
steroid D-ring domain suggesting opportunities for potential inter-
actions with Thr739. Our attention was drawn to the DBF ring,
since a dissociated GR agonist ZK216348 with a DBF moiety has
been reported in the literature ( Fig. 1).61,62 The unsubstituted
DBF compound 26 shows GR binding affinity, selectivity and ago-
nist activity similar to the 2-methoxy-5-fluoro analogue 2 (Table
5). The effect of substitution at a position para- to the oxygen atom
of the DBF ring was explored and the data is shown in Table 5.
Groups such as F, Br and CH3 do not alter the selectivity profile
(27–29). However, cyano (30) and methylsulfone (31) substitution
have a dramatic effect on GR/PR selectivity. While the cyano ana-
logue 30 is 50-fold selective, the methylsulfone compound 31
shows greater than 300-fold selectivity over PR, and 100-fold
selectivity over MR. The selectivity data can be rationalized using
our docking studies.
implicated. Some of the amino acid residue differences between
GR and PR in the D-ring domain could explain the observed GR/
PR selectivity. Two residues, Met639 and Gln642 are proximal to
the dihydrofuran ring (of DBF moiety) at the RHS. The correspond-
ing residues in PR are Phe794 and Leu797, respectively. As a result
the PR binding pocket is reduced in size and may not accommodate
both the dihydrofuran ring and the bulkier methylsulfone group.
Thus, PR binding is eroded leading to enhanced GR/PR selectivity.
In the transrepression assay, F, Br and CH3 analogues (27–29,
Table 5) show activity similar to the unsubstituted DBF analogue
26. The CN-DBF and methylsulfone-DBF analogues 30 and 31,
respectively, display 10-fold loss in transrepression potency as
compared to 26. In contrast to the indole analogues, DBF analogues
show more separation in the TA and TR activity. Compounds 26, 27
and 30 show a 3–30-fold difference in EC50/IC50. The most striking
result is the dissociated profile of the methylsulfone-DBF 31 ana-
logue in the MMTV assay (EC50
2 lM, % maximum efficacy 20).
The MMTV and nuclear receptor selectivity data for 31 in combina-
tion with the results for the DBF compounds 26, 27 and 30 suggest-
ing a trend towards separation in the TA (aromatase data) and TR
activity prompted us to choose methylsulfone-DBF as the RHS for
further modifications, despite the lack of aromatase data for 31.
These results highlight the methylsulfone-DBF as an FRP mimick-
ing the steroidal D-ring.
With the goal of generating optimal compounds for in vivo eval-
uation, we prepared compounds combining the FRPs at the LHS
and RHS of the molecule that gave better nuclear receptor selectiv-
ity and dissociation. Based on the results discussed earlier, methyl-
sulfone-DBF RHS and both the 5-CN and 6-CN indole LHS were
employed to prepare 32 and 33. Compounds 32 and 33 display
high GR affinity and selectivity over PR, MR and both the cyano
compounds are 2-fold more potent in the IL-6 transrepression as-
say compared to the methylsulfone-DBF unsubstituted indole 31
(Table 6). The 6-Cyano compound 33 was resolved on a chiral col-
umn and the data for the enantiomers (S)-33 and (R)-33 is shown
in Table 6. Both compounds 32 and 33 transactivate poorly in the
Figure 5a and b show an overlay of the (R)-enantiomers of 31
and dexamethasone. In the A-ring region, no difference in the
interactions with the GR—LBD is observed between (R)-31 and
(R)-2 (Figs. 3a and 5a). However in the GR—LBD/(R)-31 binding
pose (Fig. 5b), the methylsulfone oxygen atoms of (R)-31 lie in a
space closer to the C-18 carbonyl of dexamethasone and potential
hydrogen bonding to the side chain hydroxyl of Thr739 is
MMTV assay (EC50 2 lM, % maximum efficacy <10). A smaller 2–3-
fold separation in the EC50/IC50 is seen in the aromatase assay and
both analogues maintain potent agonist properties. The eutomer
(R)-33 of the 6-CN analogue with more than 100-fold PR, MR and
AR selectivity, an IL-6 IC50 of 28 nM (% maximum efficacy 88)
and full dissociation in the MMTV assay and a weak dissociation
in the aromatase assay was chosen for in vivo profiling.
Figure 5. (a) (Left) and (b) (right): Docking poses for (R)-31 (cyan) in the GR—LBD co-complex structure with dexamethasone40 (orange). (a) Shows the region around the A-
ring of dexamethasone, (b) Shows the region around the D-ring. Selected amino acids exhibiting key interactions with the ligand are shown. Potential H-bonds are indicated
as dashed white lines.

R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
6849
Table 6
Data on the Compounds 32, 33, (S)-33 and (R)-33
R1
R2
CF3
OH
SO2CH3
N
H
O
R1 = CN; R2 =H, 32
R1 = H; R2 =CN, 33
(S)-33
NC
SO2CH3
SO2CH3
CF3
OH
N
H
O
O
(R)-33
NC
CF3
OH
N
H
Compd
IC50 (nM)
IC50 (nM) (% efficacy)
IL-6 inhibition
EC50 (nM), (% efficacy)
Aromatase induction
EC50 (nM), (% efficacy)
GR
PR
MR
AR
MMTV induction
32
33
(S)-33
(R)-33
5
5
405
2
>2000
>2000
1400
745
1450
430
405
230
49 (85)
39 (89)
—
80 (63)
125 (98)
—
>2000 (4)
>2000 (10)
—
NDB at 2000 nM
28 (88)
120 (74)
>2000 (10)
(R)-33 was evaluated for pharmacokinetic properties in mice
and rats. Modest bioavailability in mice and rats (26% and 51%,
respectively) was observed with an acceptable half-life after oral
dosing (Table 7).
Anti-inflammatory properties of a select panel of compounds,
that includes the in vitro dissociated compounds 32, 33 and (R)-
33, were determined in an LPS-stimulated mouse model of TNF-
production. Test compounds were administered orally in Cremo-
phor RH 1 h prior to LPS challenge and plasma TNF- levels were
measured 1 h after the challenge. Table 8 summarizes the data.
The unsubstituted indole (R)-2 did not show efficacy and the DBF
analogues (R)-29, 32 and 33 displayed ED50 less than 10 mg/kg.
a
Table 7
Pharmacokinetic data of (R)-33 in B10.RIII mice and Sprague-Dawley rats
a
Mousea
Ratb
iv CL (ml/min/kg)
VSS (L/kg)
t1/2 (hr)
po cmax (ng/ml)
po AUCinf (hr ng/ml)
F (%)
4
0.9
2.5
6187
32729
26
51
7.7
1.7
955
4850
51
(R)-33 completely inhibited TNF-a production at 3 mg/kg. With
an ED50 of less than 3 mg/kg (R)-33 represents a dissociated GR
agonist displaying an anti-inflammatory efficacy equal to prednis-
olone in the LPS model.
a
Dosed at 1 mg/kg i.v. in a PEG400/water (70:30) vehicle and at 30 mg/kg po as a
(R)-33 was also tested in a mouse model of collagen induced
arthritis (CIA), a chronic model of inflammatory polyarthritis
which shares many features with human rheumatoid arthritis.63
suspension in 30% Cremophor, mean of 3 mice.
Dosed at 2 mg/kg i.v. in a PEG400/water (70:30) vehicle and at 30 mg/kg po in a
PEG400/H2O/Tween (80/18/2) vehicle, mean of 3 rats.
b
Table 8
In vitro activities of indoles
Compd
IC50 (nM)
GR
IC50 (nM) (% efficacy)
IL-6 inhibition in HFF
EC50 (nM), (% efficacy)
Aromatase Induction in HFF
Inhibition ofTNF-
a
in mice (%)(n = 8) 3 and 10 mg/kg
Pred.
(R)-2
(R)-29
32
33
(R)-33
15
10
12
5
5
2
7 (96)
6 (87)
7 (92)
49 (85)
39 (89)
28 (88)
19 (92)
18 (98)
14 (120)
80 (63)
125 (98)
120 (74)
84
35
61
77
66
88
—
—
NE*
66
97
*
No efficacy.

6850
R. Betageri et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6842–6851
on both the A- and D-ring regions of molecule 2 (LHS and RHS
respectively), agonist activity is more influenced by A-ring substi-
tutions. 5-CN and 6-CN substituted indole rings and a RHS methyl-
sulfone-DBF moiety have been identified as function-regulating
pharmacophores that provide the beneficial properties for the GR
ligand 2. Highlighting the potent anti-inflammatory properties of
indole analogues is compound (R)-33 which is equipotent to pred-
nisolone in a LPS-TNF model of inflammation. Furthermore in the
mouse CIA model, (R)-33 slowed the progression of disease with
an efficacy of 45% at 30 mg/kg. We have identified indole ana-
logues of 2 that show a dissociated profile based on % maximal effi-
cacy or EC50 or both. To assess the significance of the observed
in vitro dissociation, further in vivo evaluation of the compounds
may be required. The results presented in the current studies open
opportunities for further explorations on the non-steroidal GR ago-
nist 2, the results of which will be communicated in the future.
Figure 6. Collagen Induced Arthritis Model: (R)-33. Data are given as mean of 10
animals per group; Statistical analysis of AUCs was performed with the Mann-
Whitney test: ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 compared to vehicle (30% Crem-
ophor RH).
Acknowledgments
We thank Doris Riether, Derek Cogan and Steven Brunette for
reviewing the manuscript. Thanks are also due to Eugene Hickey
for his assistance in generating the docking poses.
Table 9
Side effect profile of (R)-33 and prednisolone
% Change relative to vehicle control
References and notes
Prednisolone
3 mg/kg 30 mg/kg
(R)-33 30 mg/kg
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48. GR, PR and MR binding assays measured competition between test compound
and fluorescently labeled probes for binding to recombinant human receptors
in cell lysates made from baculovirus-infected insect cells. 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. The DMSO concentration in all binding reactions was 1% (v/v) and binding
reactions were incubated for one hour at room temperature before measuring
fluorescence polarization signal. The signal produced by maximal inhibition
(background signal) was determined in wells containing 2 mM reference
inhibitor for each receptor: dexamethasone for GR and MR, RU-486 for PR.
63. B10.RIII mice were immunized with porcine type II collagen and complete
Freund’s adjuvant then monitored for signs of arthritis. Animals were enrolled
into the study at the first signs of disease and treated daily with compound,
prednisolone or vehicle for a period of 5 weeks. The animals were monitored
regularly and arthritis severity was assessed through clinical scoring of all four
paws based on a score of 1–4. The clinical score per paw was summed to give a
maximal severity score of 16 for each animal. (R)-33 were evaluated in the
model at once daily oral doses of 30 and 10 mg/kg for a period of 5 weeks
versus prednisolone at 30, 10 and 3 mg/kg. At termination (day 35), the animal
was anesthetized with 50 mg/kg (10 mg/ml) Ketamine/15 mg/kg (2 mg/ml)
Zylazine. A whole body non-invasive density scan was performed to assess
body fat content. (PIXImus densitometer, Lunar Corp.). Serum samples were
collected for biomarker analysis. Triglyceride levels were determined with the
Wako L-Type TG-H kit (Wako Diagnostics, Richmond, VA). Free fatty acids in
serum were measured using the Wako NEFA-C Microtiter Procedure (Wako
Diagnostics, Richmond, VA, Catalog #994-75409). For the serum insulin assay,
the Ultra Sensitive Rat Insulin ELISA Kit (Crystal Chem Inc., Chicago, IL, Catalog
#90060) and Mouse Insulin Standard 1.28 ng (Catalog #90090) was used.