The discovery of potent and selective non-steroidal glucocorticoid receptor modulators, suitable for inhalation

Article abstract of DOI:10.1016/j.bmcl.2014.03.070

We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound collection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimization of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.

Full text of DOI:10.1016/j.bmcl.2014.03.070

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of potent and selective non-steroidal glucocorticoid
receptor modulators, suitable for inhalation
Karl Edman a, Ragnhild Ahlgren b, Malena Bengtsson b, Håkan Bladh b, Stefan Bäckström a, Jan Dahmén b,
Krister Henriksson b, Per Hillertz a, Vijakumar Hulikal b, Anders Jerre b, Liz Kinchin b, Charlotte Kåse b,
Matti Lepistö c, Irene Mile b, Stinabritt Nilsson b, Amir Smailagic c, John Taylor c, Ann Tjörnebo b,
Lisa Wissler a, Thomas Hansson c,
⇑
a Discovery Sciences, Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b AstraZeneca R&D Lund, Scheelevägen 1, SE-221 87 Lund, Sweden
c Respiratory, Inflammation and Autoimmunity, Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators
with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound col-
lection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimi-
zation of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo
models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 4 February 2014
Revised 21 March 2014
Accepted 24 March 2014
Available online xxxx
Keywords:
Glucocorticoid receptor
Non-steroidal selective glucocorticoid
receptor modulators
Inhalation
Indazoles
Glucocorticoids represent the most potent anti-inflammatory
drugs available today and allow successful treatment of several
chronic inflammatory and autoimmune diseases. This includes
the treatment of respiratory diseases, such as asthma and chronic
obstructive pulmonary disease (COPD), where inhaled glucocorti-
coids (IGCs) alone or in combination with bronchodilators are the
mainstay of clinical therapy. However, there is still a large unmet
clinical need in both of these diseases.1
Glucocorticoids exert their function through binding to the glu-
cocorticoid receptor (GR). Following ligand binding the cytoplas-
mic receptor translocates to the nucleus, where it regulates gene
transcription by both activating and repressive mechanisms. In
analogy with other nuclear hormone receptors, the GR serves as
an assembly point for transcription coregulators that can directly
modify chromatin structure and/or impact the activity of the gene
transcription apparatus.2 Modulation of receptor activity with non-
steroidal ligands, through differential coregulator recruitment can
be envisioned as a route to develop GR ligands with improved effi-
cacy and/or safety compared to conventional steroids. This vision is
supported by related work in the field of non-steroidal ligands act-
ing on the estrogen and androgen receptors.3
During the last decade non-steroidal GR ligands have been iden-
tified by large number of organizations.4 Two examples of com-
pounds reaching clinical studies for topical and oral
administration respectively are Mapracorat (1) and PF-04171327
(2) in Figure 1.5 Furthermore, the development of novel non-steroi-
dal GR agonists has been facilitated by increased understanding of
the structural biology of GR.6
We initiated a search for inhaled GR modulators for the treat-
ment of respiratory diseases. A high throughput screen of the
AstraZeneca compound collection using a competitive human GR
fluorescence polarization binding assay identified the sulfonamide
3 as a potent ligand (Fig. 2). This compound was attractive as it
could be synthesized in one step from commercially available
starting materials. Further profiling of functional cellular activity
using a reporter gene assay system, wherein gene transrepression
(TR) activity was determined in Chago K1 cells stably transfected
with a construct containing several TRE sites (AP-1 pathway) pre-
ceding a Lac Z reporter, revealed the compound to be a functional
antagonist. An initial key objective was therefore to introduce
functional agonism in this series.
⇑
Corresponding author. Tel.: +46 31 7763623; fax: +46 31 7763724.
E-mail address: thomas.g.hansson@astrazeneca.com (T. Hansson).
http://dx.doi.org/10.1016/j.bmcl.2014.03.070
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070

2
K. Edman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1
GR binding and activity in TRE reporter gene assay for selected compounds
OPO3H2
CF3
O
O
S
X
N
R
O
HO
CF3
H
N
H
N
H
N
O
Compd
X
R
GRa IC50 (nM)
TR agonistic modea
N
Eff.b (%)
2
F
1
IC50 (lM)
3
5
6
7
8
C
Phenyl
55
85
18
240
23
42
75
10,000
10,000
NA
NT
—
—
—
71
—
57
88
—
Figure 1. Mapracorat 1 and PF-04171327 2.
O
O
O
O
N
N
N
N
2,6-Dimethylphenyl
5-Isoquinoline
5-Quinoline
4-Indole
NA
0.96
NA
0.76
1.8
NA
NA
O
O
S
9
4-Indazole
N
H
10
11
12
5-Quinoline
6-Quinoline
6-Isoquinoline
3
—
Figure 2. HTS hit 3.
13
14
N
N
18
1.1
2.2
59
56
N
N
As a first step to improve the synthetic scope, a heteroatom was
160
introduced in the benzylic position in compound 3 to allow parallel
chemistry to be used in expansion of the series. A study of the
activity of various aryl ethers and anilines was then initiated, using
a synthetic route that enabled preparation of ethers (entries 5–8)
and amines (entries 9–14) as single enantiomers and exemplified
by the synthesis of compounds 7 and 9 in Scheme 1.
NT: not tested. NA: not active (<50% inhibition) at 10
lM.
a
Values are means of at least two experiments.
% Of dexamethasone at 10À6 M.
b
Compounds were tested for GR binding and cellular activity in
the TR reporter gene assay. For several compounds, affinity to the
receptor was retained or improved compared to compound 3
(Table 1). Interestingly, the attachment position to the heterocyc-
lics exhibited a steep SAR. For example, the quinoline (10) linked
through the 5-position has an affinity to GR of 75 nM whereas
affinity is completely abolished for the 6-linked quinoline deriva-
tive (11). This observation is supported by modeling studies in
which the elongated ligand binding pocket in the receptor strictly
dictates conditions for the width of compounds. From Table 1 it is
also clear that the positioning of aromatic heteroatoms plays an
important role for functional activity. While the 5-isoquinoline
(6) is more potent than the 5-quinoline (7) in the binding assay,
only the 5-quinoline is an agonist in the cellular TR assay. This
can be understood from the perspective that modeling studies
place the heterocyclic ring in the same region of the ligand binding
pocket as the A and B rings of conventional steroids. For optimal
interactions to the receptor, the heteroatom of the heterocyclic
ring must emulate the 3-keto oxygen of steroids to pick up key
interactions with Arg611, Glu570 and a conserved water mole-
cule.7 These interactions between helices 3 and 5 of GR have been
shown to be critical drivers for agonism.8
Next, the sulfonamide substituent was explored using a route
exemplified for the synthesis of the quinoline (20) and isoquinoline
(26) ethers in Scheme 2.
A range of sulfonamides of different sizes were examined,
exemplified by compounds 18–27 in Table 2. All substituted phe-
nyl sulfonamides (entries 18–25) showed inferior GR binding com-
pared to the corresponding mesityl sulfonamides. Modeling places
these substituents in an area corresponding to the steroid D-ring
17a position and the data confirm the critical role of this pocket
for binding affinity. The fact that the potent 1,2-diazoles (entries
26 and 27) are much larger than the original mesityl also illustrates
the tendency for flexibility in this region of the receptor, as previ-
ously reported.9 Since the 1,2-diazoles also exhibited a decrease in
lipophilicity (logD for matched pairs 6 and 26, were 4.1 and 3.5,
respectively), they were selected for further investigations
together with the more potent mesityl congener.
In an effort to emulate the phenyl-pyrazole A-ring substituents
in steroid derivatives, such as in cortivazole, a series of 1-aryl-1H-
indazoles were synthesised.10 Indazoles 30–42 and 47–52 (Table 3)
were prepared as exemplified by the synthesis of compounds 30
and 47 (Scheme 3).11 The O, C, S and SO2 linked 1-aryl-1H-inda-
zoles 29, 43–45 and the CF3 branched indazole ether 46, were syn-
thesized by different routes.12
Many compounds showed excellent potency in the binding and
TR assays. The activity of compounds in primary cells were studied
in human peripheral blood mononuclear cells (hPBMCs), where the
O
O
a
S
O
OH
N
S
O
release of tumor necrosis factor-a (TNF-a) in response to lipopoly-
H2N
H
O
4
7
9
saccharide (LPS) stimulation was used to test the anti-inflamma-
tory effects. As can be seen from data in Table 3, several of the
compounds were confirmed to be potent agonists in this assay.
Functional agonism is driven by a well defined structural state
of the receptor, where coactivators can be recruited to the binding
surface outlined by helix 3, 4 and 12 (Fig. 3).7 To better understand
the molecular details of the conformations induced by the sulfon-
amides, we determined the X-ray structure of the GR ligand bind-
ing domain (LBD) in complex with compound 30.13
The structure revealed that the compound makes multiple
interactions to helix 3, stabilizing it in an agonistic conformation
(Fig. 4A). Specifically the nitrogen of the sulfonamide linker makes
a direct interaction to the Od atom of Asn564. In addition, one of
O
O
S
b
O
N
N
H
N
O
O
S
H
N
c
NH
N
H
Scheme 1. Reagents and conditions: (a) 2-mesitylenesulfonyl chloride (2.1 equiv),
pyridine, rt, 16 h; (b) 5-hydroxyquinoline (1.7 equiv), DMF, CsCO3, rt, 16 h; (c) 4-
aminoindazole, NMP, 130 °C, 2 h.
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070

K. Edman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
O
c
O
O
N
N
N
O
15
a, b
OTs
OH
O
N
H2N
O
O
d
N
16
O
Cl
O
O
e
f
S
O
O
N
N
15
N
H2N
H
20
Cl
SO2Cl
NHNH2
N
g, h
N
N
O
O
17
S
O
N
i
N
H
26
N
N
e
O
16
H2N
Scheme 2. Reagents and conditions: (a) phthalic anhydride, toluene, DIEA, reflux, Dean–Stark, 2 h; (b) TsCl, pyridine, rt, 16 h; (c) 5-hydroxyquinoline (1.1 equiv), DMF, CsCO3,
100 °C, 2 h; (d) 5-hydroxyisoquinoline (1.1 equiv), DMF, CsCO3, 100 °C, 2 h; (e) NH2NH2 H2O, EtOH, reflux, 2 h; (f) 2,4-dichloro-6-methylbenzenesulfonyl chloride (1.2 equiv),
pyridine, rt, 16 h; (g) 2,4-pentanedione, DIEA, EtOH, reflux, 48 h; (h) HSO3Cl, CH2Cl2, 0 °C, 1 h, then reflux 2 h, then SOCl2, reflux, 2 h; (i) pyridine, rt, 16 h.
Table 2
Adjacent to Asn564, the backbone carbonyl of Leu563 is within
hydrogen bonding distance of the amine linker at the indazole 4
GR binding and activity in TR reporter gene assay for selected compounds
O
O
N
O
O
position (Fig. 4A). The importance of this interaction is emphasized
by ether congener 29, which lacks a donor at this position and has
no activity in the hPBMC assay (compared to 14 nM for 30). Inter-
estingly, compound 46 illustrates that the hPBMC activity can be
recovered if a trifluoromethyl is introduced adjacent to the sulfon-
amide nitrogen. It is plausible that the introduction of the electron
withdrawing group strengthens the ability of the sulfonamide
nitrogen to donate its hydrogen to Asn564.
S
O
N
S
O
R
N
R
N
H
H
25-27
18-24
Compd
R
GRa IC50 (nM)
TREa
IC50
M)
Eff.b (%)
(
l
18
19
20
2,4-Dimethylphenyl
2,4,6-Trichlorophenyl
2,4-Dichloro-6-
1600
80
220
NT
2.4
NA
—
64
—
Beyond the sulfonamide, the mesitylene tail enters hydrophobic
volume where helices 3, 7 and 11 meet (Fig. 4B). As predicted, this
volume overlaps with the 17
most highly potent steroid ligands. Compounds like 48, with a
large 17 substituent, highlight the tendency for flexibility of this
region. It is interesting to note that none of the natural ligands for
the steroid receptors extend into this volume. The intrinsic plastic-
ity of the receptor in this volume is likely coupled to the mecha-
nism of ligand entry into and exit from the ligand binding
domain.15 At the other end of compound 30, the indazole fragment
a substituent that can be found in
methylphenyl
21
22
23
24
25
2-Methoxyphenyl
3-Cyanophenyl
2-Cyanophenyl
Phenyl
2,4-Dimethylphenyl
Me
>10,000
1800
>10,000
>10,000
410
NT
NT
NT
NT
NT
—
—
—
—
—
a
N
26
32
NA
—
N
Me
makes a putative interaction to the N atom of Gln570 (Fig. 4A).
e
Me
N
The gatekeeper residues, Gln570 and Arg611, are arranged in the
open conformation to accommodate the p-fluorophenyl motif.16
This motif acts as a hydrophobic link between helices 3 and 5 at
the core of the receptor and makes a strong contribution to the
functional agonism observed for compounds with similar
moieties.8,17
This analysis indicates that the sulfonamide template makes
distinct interactions to the receptor relative to conventional steroid
ligands.7 Taken together with the fact that steroids are based upon
a more rigid scaffold, we hypothesized the sulfonamides would
induce a distinct structural state leading to a unique protein–pro-
tein interaction pattern. To investigate this further, we measured
the binding of the GR LBD to various coregulator peptides in the
presence of compounds 30 and 48 using surface plasma resonance
measurements.18 Figure 5 shows that the sulfonamides 30 and 48
have a different profile relative to the steroids dexamethasone and
fluticasone propionate.
N
27
97
NA
—
Me
NT: not tested. NA: not active (<50% inhibition) at 10
lM.
a
Values are means of at least two experiments.
% Of dexamethasone 10À6 M.
b
the sulfone oxygens is located within hydrogen bonding distance
to a putative water molecule, which is ideally placed to make fur-
ther interactions to both Thr739 and the Nd atom of Asn564. Inter-
actions to Asn564 are of particular importance as the Nd atom
makes a further hydrogen bond to the backbone carbonyl group
of Glu748, in the loop between helices 11 and 12, thus stabilizing
this region and directly contributing to agonism.14
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070

4
K. Edman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3
GR binding, activity in TR reporter gene assay and in PBMCs for selected compounds
N
N
N
N
Y
Y
O
O
O O
R2
R2
S
X
S
X
N
N
Z
Z
R1
R1
N
H
H
N
29-46
47-52
a
a
a
Compd
X
Y
R1
R2
Z
GR IC50 (nM)
TR IC50 (nM)
hPBMC IC50 (nM)
IC50
Eff.b (%)
IC50
Eff. (%)
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44c
45c
46c
47
48
49
50
51
52
O
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
iPr
Me
Me
Me
Me
Me
Me
CF3
Me
Me
Me
Me
Me
Me
C
C
N
C-Me
C
N
N
C
C-Me
C
C
C–F
C-OMe
C
C
C
C
C
C
N
C
C
N
N
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
F
F
F
H
H
H
H
Cl
F
Me
F
H
H
OMe
F
F
F
5.2
4.9
2.3
3.8
3.5
5.3
5.6
3.4
4.1
3.9
35
4.0
6.9
5.4
4.6
7.7
795
3.8
3.8
3.0
12
60
93
89
91
84
88
67
94
99
77
85
83
80
62
—
76
—
—
73
87
88
—
87
76
—
NA
14
23
8.8
1.6
8.1
2.0
13
23
12
2.1
28
NT
NT
12
NT
NT
36
NT
14
NT
13
NA
NT
—
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
CH2
S
1.5
2.3
6.6
1.5
6.9
2.6
3.7
14
4.5
12
6.1
41
NT
23
NA
NT
14
9.7
7.2
NT
16
73
62
55
82
63
84
72
69
58
61
69
—
—
37
—
—
76
—
SO2
O
F
F
F
NH
NH
NH
NH
NH
NH
60
—
68
—
OCF3
CN
H
4.7
5.7
23
43
NT
OMe
—
NT: not tested. NA: not active (<50% inhibition) at 10
l
M.
a
Values are means of at least two experiments.
b
% Of dexamethasone 10À6 M.
c
Racemic mixture.
Br
NH2
a, b, c
N
N
Br
d
N
N
O
O
H
28
e
S
N
N
F
H
30
F
O
O
S
NH2
N
H
4
N
N
O
O
H
N
S
N
H
N
N
f, g, h
F
47
17
Scheme 3. Reagents and conditions: (a) HCl, H2O, rt, 1 h, then NaNO2, H2O, À5 °C, 25 min, then HBF4; (b) KOAc, 18-crown-6, CH2Cl2, rt, 1 h; (c) 4-fluorobenzeneboronic acid,
Cu(OAc)2, pyridine, CH2Cl2, rt, 16 h; (d) NH3 (aq), MeCN, rt, 2 h; (e) BINAP, Pd(dba)3, toluene, NaOtBu, Microwave (300 W, 110 °C, 15 min; (f) L-alaninamide hydrochloride,
pyridine, DIEA, rt, 16 h; (g) borane-THF, THF, rt, 16 h; (h) 28, BINAP, Pd(dba)3, toluene, NaOtBu, microwave (300 W, 110 °C, 15 min).
Guided by above results compounds 30 and 48 were selected
for further characterization in vitro and in vivo. To measure steroid
hormone receptor selectivity, the compounds were profiled in pro-
of compounds in rat and ensure species cross over in in vivo phar-
macodynamic models. Analysis of physicochemical and in vitro PK
properties (Table 4) indicated both compounds could be regarded
as suitable for inhalation and with properties similar to reference
IGCs. This included high hepatic clearance that was sought to
ensure minimal systemic exposure of active ligand.
The metabolism and metabolites were investigated in vitro
using human liver microsomes and human hepatocytes. A major
metabolic pathway was observed to be N-dealkylation via oxida-
gesterone, mineralcorticoid, androgen and estrogen (ERa and ERb)
receptor binding assays. Both compounds showed little affinity to
any of the receptors tested and selectivity over progesterone and
mineralcorticoid receptors are improved compared to reference
inhaled steroidal glucocorticoids.19 Release of TNF
a
from LPS stim-
ulated rat PBMCs was used to establish anti-inflammatory effects
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070

K. Edman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Figure 5. The binding of various coregulator peptides to the GR LBD in complex
with compounds 30, compound 48, dexamethasone and fluticasone propionate.
compound 48. From Figure 6 it can be seen that compound 30 has
high lung tissue retention, about 50% of the dose remain in the lung
24 h after dosing. In contrast, the more polar compound 48 rapidly
disappeared from the lung tissue with only minor amounts of the
dose remaining in the lung 4 h after dosing. In a separate study the
blood concentration–time curves after i.t. instillation of both com-
pounds were determined. The result is in line with the results from
the study of lung tissue levels, showing more rapid systemic
absorption for compound 48 resulting in higher Cmax compared
to compound 30 (Fig. 7).
Figure 3. The global arrangement of GR in complex with 30. The Tif-2 coactivator
The efficacy of compound 30 was evaluated in a rat LPS model
of acute inflammation.22 In this model compound 30 was instilled
intratracheally 2 h prior to challenge with LPS, and experiments
terminated 4 h after challenge. The end points measured in this
model were total number of leukocytes in the broncho-alveolar
lavage (BAL) fluids, and increase in lung weight (edema).
peptide (yellow) binds in the groove outlined by helices 3, 4 and 12.
tion in the
a-methylene position leading compound cleavage and
formation of several fragments, including 4-aminoindazoles.21
Generally, compounds in this series showed little CYP P450 inhibi-
tion and among the more interesting compounds CYP inhibition
was low. Furthermore, the risk of cytochrome P450 related drug-
drug interactions for inhaled drugs is reduced because of the low
doses typically administered, leading to low systemic exposure.
Compounds were further characterized in vivo in pharmacoki-
netic models. Compound 48 was administrated iv and as predicted
from in vitro data, in vivo clearance was high. The lung concentra-
tions and blood PK after instillation by intra tracheal route were
determined for compounds 30 and 48. A key driver for lung reten-
tion of neutral compounds is the rate of dissolution, which is linked
to solubility. Compound 30 is more lipophilic and less soluble than
Compound 30 at doses of 30 and 100 lg/kg significantly inhib-
ited inflammatory cell influx in animals treated with LPS, by 90%
and 85%, respectively. The compound also inhibited lung edema
in a dose-dependent manner. Fluticasone propionate dosed at
same doses as compound 30 was used as reference (Figs. 8 and 9).
Cigarette smoke is the main risk factor for developing chronic
obstructive pulmonary disease (COPD) in humans. Using a mouse
model of inflammation, female Balb/C mice were exposed to ciga-
rette smoke twice daily for four days to induce a local inflamma-
tory response in the lung, reflected by an increase in the number
Figure 4. (A) Refined 2mFo-DFc electron density of GR in complex with 30. The dotted lines represent putative hydrogen bonds. (B) Detailed view of the mesitylene tail.
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070

6
K. Edman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 4
Total cells in BAL
Physical Chemical properties and in vitro/vivo profile of 30 and 48
8.0
6.0
4.0
2.0
0.0
p<0.001
p<0.01
94%
p<0.05 p<0.01
p<0.01 p<0.01
84% 92%
p>0.05
37%
30
48
90%
85%
a
MR/PR/AR IC50
ERa/ERb IC50 (l
Rat PBMC IC50 (nM/% efficacy)
(
M)
l
M)
>1/0.77/>1
>5/>5
0.9/87
>5/4.5/>1
>5/>5
4.5/83
3.7
1.5/22
0.005
>200/33
>200/47
a
LogD7.4
4.1
Solubilityb
(
l
M)
<0.2/<0.43
<0.003
>200/13
78/18
>20
10
7.9
NA
NT
Hu PPB Fu% (fraction unbound%)
Rat Micsc/Heps Clint
d
d
Hu Micsc/Hu Heps Clint
CYP2D6, CYP1A2 CYP2C19 IC50
CYP3A4 IC50
l
M
>20 lM
lM
22
17
8.7
l
M
CYP2C9 IC50
l
M
lM
hERG20
(lM)
µ
( g/kg BW)
100
10
30
FP
100
10
30
LPS/
Saline/
Sham
Rat (iv) PK Cl/t /Vdse
78/6.4/15
½
Sham
Compound 30
NA: not active (<25% inhibition) at 50
l
M.
Figure 8. Inhibition of leukocyte influx following i.t. administration of compound
30 and fluticasone propionate (FP) in the acute LPS model.
a
MR assay in SPA format and PR, AR, ER
a
and ERb assays in FP format.
b
Thermodynamic solubility of crystalline/amorphous compound in 0.1 M phos-
phate buffer pH 7.4 at 25 °C for 24 h.
c
Microsome metabolism intrinsic clearance Clint
Hepatocyte metabolism intrinsic clearance Clint (l
Blood PK after iv administration; Cl (mL/min/kg), t (h), Vss (L/kg).
(
l
L/min/mg).
d
L/min/106 cells).
e
½
Lung Wet Weight
1.5
p<0.001
p<0.01
56%
42%
p<0.01 p<0.01 p>0.05 p<0.05
p<0.01
64%
1.4
1.3
1.2
1.1
1.0
32%
116%
78%
Figure 6. Lung tissue levels of compound 30 and compound 48 after i.t. admin-
istration to the lung.
µ
( g/kg BW)
100
10
30
100
10
30
LPS/
Saline/
Sham
Sham
FP
Compound 30
Figure 9. Inhibition of lung edema following i.t. administration of compound 30
and fluticasone propionate (FP) in the acute LPS model.
Neutrophils in BAL
0.5
p<0.05
38%
p>0.05 p>0.05
23% 0%
p>0.05 p<0.05
36%
p<0.001
0%
0.4
0.3
0.2
0.1
0.0
Figure 7. Blood levels of compound 30 and compound 48 after i.t. administration of
2 lmol/kg to the lung.
µ
(
g/kg BW)
10
30
100
30
FP
300
of neutrophils in BAL fluid.23 Compound 30 was tested in the
mouse cigarette smoke model. Mice were exposed to cigarette
smoke for 50 min, twice daily, for four days. Animals were pre-
treated with compound 30, 30 min prior to the first cigarette
smoke exposure on each day (4 days treatment in total). 24 h after
last exposure mice were terminated and the number of neutrophils
in the BAL fluids determined.
Air/
CS/
Naive
Sham Sham
BUD
Compound 30
Figure 10. Inhibition of neutrophil influx following i.t. administration of compound
30, fluticasone propionate (FP) and budesonide (BUD) in the acute cigarette smoke
model.
Compound 30 significantly inhibited neutrophil influx into BAL
fluid of mice exposed to cigarette smoke for four days at a dose of
30 lg/kg (Fig. 10). Fluticasone propionate and budesonide was
models including the acute Tobacco Smoke model. Structure stud-
ies of receptor–ligand binding provided insight into functional
drivers of agonism and cofactor affinity profiles revealed a differ-
ential binding mode compared to reference steroidal glucocorti-
coids. The ligands were shown to have PK properties (high
clearance and solubility range) suitable for further optimisation
as inhaled GR modulators.
used as references in this study.
In summary, we have successfully identified a series of highly
potent and selective non-steroidal glucocorticoid receptor modula-
tors. These were shown to have activity in pharmacodynamic
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070

K. Edman et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
7
H.; Alarcon, M.; Calles, K.; Engström, O.; Harlan, J.; Muchmore, S.; Ramqvist, A.;
Thorell, S.; Öhman, L.; Greer, J.; Gustafsson, J.-Å.; Carlstedt-Duke, J.; Carlquist,
M. J. Biol. Chem. 2003, 278, 22748.
Acknowledgments
The authors would like to thank Dr. Göran Carlström for NMR
support; Tina Jellesmark Jensen for GR LBD protein expression sup-
port; Ann-Helene Jansson and Charlotte Brange for in vivo study
support. We also thank Dr. Mark Furber for proofreading this
manuscript.
8. Zhang, J.; Simisky, J.; Tsai, F. T. F.; Geller, D. S. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 2707.
9. Biggadike, K.; Bledsoe, R. K.; Hassel, A. M.; Kirk, B. E.; McLay, I. M.; Shewchuk, L.
M.; Stewart, E. L. J. Med. Chem. 2008, 51, 3349.
10. (a) Hannah, J.; Kelly, K.; Patchett, A. A.; Steelman, S. L.; Morgan, E. R. J. Med.
Chem. 1975, 18, 168; (b) Shah, N.; Scanlan, T. S. Bioorg. Med. Chem. Lett. 2004,
14, 5199; (c) Ali, A.; Thompson, C. F.; Balkovec, J. M.; Graham, D. W.;
Hammond, M. L.; Quraishi, N.; Tata, J. R.; Einstein, M.; Ge, L.; Harris, G.; Kelly, T.
M.; Mazur, P.; Pandit, S.; Santoro, J.; Sitlani, A.; Wang, C.; Williamson, J.; Miller,
D. K.; Thompson, C. M.; Zaller, D. M.; Forrest, M. J.; Carballo-Jane, E.; Luell, S. J.
Med. Chem. 2004, 47, 2441.
11. Compound 39 was prepared analogous to compound 30, but starting from (S)
2-amino-3-methyl-1-butanol instead of (S) 2-amino-1-propanol.
12. Supplementary material 1: Synthetic schemes of the O, C, S and SO2 linked 1-
aryl-1H-indazoles 29, 43–45 and the CF3 branched indazole ether 46.
13. The experimental details are described in the attached Supplementary material
2: Materials and Methods: Structure determination. The coordinates for the
complex of GR LBD with compound 30 have been deposited in the PDB with
accession code 4csj.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
03.070.
References and notes
1. Barnes, P. J. J. Allergy Clin. Immunol. 2013, 131, 636.
14. Bledsoe, R. K.; Madauss, K. P.; Holt, J. A.; Apolito, C. J.; Lambert, M. H.; Pearce, K.
H.; Stanley, T. B.; Stewart, E. L.; Trump, R. P.; Willson, T. M.; Williams, S. P. J.
Biol. Chem. 2005, 35, 31283.
15. Edman, K.; Hogner, A.; Hussein, A.; Aagaard, A.; Bäckström, S.; Bodström, C.;
Wissler, L.; Jellesmark-Jensen, T.; Cavalin, A.; Nilsson, E.; Lepistö, M.; Guallar, V.
Manuscript in preparation.
16. Suino-Powell, K.; Xu, Y.; Zhang, C.; Tao, Y.; Tolbert, W. D.; Simons, S. S.; Xu, H. E.
Mol. Cell. Biol. 1915, 2008, 28.
17. Yoshikawa, M.; Yamamoto, K.; Shimizu, N.; Yamada, S.; Morimoto, C.; Tanaka,
H. Mol. Endocrinol. 2005, 19, 1110.
2. (a) Simons, S. S., Jr. Curr. Opin. Pharmacol. 2010, 10, 613; (b) Watson, P. J.; Fairall,
L.; Schwabe, J. W. R. Mol. Cell. Endocrinol. 2012, 348, 440.
3. (a) McDonnell, D. P.; Wardell, S. E. Curr. Opin. Pharmacol. 2010, 10, 1; (b)
Houtman, R.; de Leeuw, R.; Rondaij, M.; Melchers, D.; Verwoerd, D.;
Ruijtenbeek, R.; Martens, J. W. M.; Neefjes, J.; Michalides, R. Mol. Cancer Ther.
2012, 11, 805; (c) Tran, C.; Ouk, S.; Clegg, N. J.; Chen, Y.; Watson, P. A.; Arora, V.;
Wongvipat, J.; Smith-Jones, P. M.; Yoo, D.; Kwon, A.; Wasielewska, T.; Welsbie,
D.; Chen, C.; Higano, C. S.; Beer, T. M.; Hung, D. T.; Scher, H. I.; Jung, M.;
Sawyers, C. L. Science 2009, 324, 787.
4. (a) Takahashi, H.; Razavi, H.; Thomson, D. Curr. Top. Med. Chem. 2008, 8, 521;
(b) Schäcke, H.; Berger, M.; Hansson, T. G.; McKerrecher, D.; Rehwinkel, H.
Expert Opin. Ther. Pat. 2008, 18, 339; (c) Regan, J.; Razavi, H.; Thomson, D. Annu.
Rep. Med. Chem. 2008, 43, 141; (d) Kuzmich, D.; Bentzien, J.; Betageri, R.;
DiSalvo, D.; Fadra-Khan, T.; Harcken, C.; Kukulka, A.; Nabozny, G.; Nelson, R.;
Pack, E.; Souza, D.; Thomson, D. Bioorg. Med. Chem. Lett. 2013, 23, 6640. and
references cited therein.
5. (a) Schäcke, H.; Zollner, T. M.; Döcke, W. D.; Rehwinkel, H.; Jaroch, S.; Skuballa,
W.; Neuhaus, R.; May, E.; Zügel, U.; Asadullah, K. Br. J. Pharmacol. 2009, 158,
1088; (b) Xiao, H.; Du, S.; Tunca, C.; Braden, T.; Long, K. R.; Lee, J.; Webb, E. G.;
Dietz, J. D.; Hummert, S.; Rouw, S.; Hegde, S. G.; Webber, R. K.; Obukowicz, M.
G. Endocrinology 2011, 152, 3123.
6. Veleiro, A. S.; Alvarez, L. D.; Eduardo, S. L.; Burton, G. ChemMedChem 2010, 5,
649.
7. (a) Bledsoe, R. K.; Stanley, T. B.; Delves, C. J.; McKee, D. D.; Consler, T. G.; Park, D.
J.; Stewart, E. L.; Wilson, T. M.; Lambert, M. H.; Moore, J. T.; Pearce, K. H.; Xu, H.
E. Cell 2002, 110, 93; (b) Kauppi, B.; Jakob, C.; Färnegardh, M.; Yang, J.; Ahola,
18. Supplementary material 3, Materials and Methods: Surface Plasmon Resonance
Experiments.
19. Salter, M.; Biggadike, K.; Matthews, J. L.; West, M. R.; Haase, M. V.; Farrow, S.
N.; Uings, I. J.; Gray, D. W. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007, 293, L660.
20. Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Harmer, A.;
Henthorn, D. C.; Ding, M.; Davis, A.; Small, B. G.; Heapy, C. G.; Abi-Gerges, N.;
Paulsson, F.; Jacobson, L.; Schroeder, K.; Neagle, B.; Albertson, N.; Hammond, T.
G.; Sullivan, M.; Sullivan, E.; Valentin, J.-P.; Pollard, C. E. J. Pharmacol. Toxicol.
Methods 2006, 54, 189.
21. Formation of 4-aminoindazoles posed an issue due to their potential risk of
genotoxicity. Handling of this issue will be presented in future reports from our
group.
22. Jansson, A.-H.; Eriksson, C.; Wang, X. Vascul. Pharmacol. 2005, 43, 101.
23. Önnervik, P.-O.; Lindahl, M.; Svitacheva, N.; Stämpfli, M.; Thim, K.; Smailagic,
A.; Virtala, R.; Taylor, J. D. Inflamm. Res. 2010, 59, 817.
Please cite this article in press as: Edman, K.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.03.070