Glucocorticoid receptor modulators informed by crystallography lead to a new rationale for receptor selectivity, function, and implications for structure-based design

Article abstract of DOI:10.1021/jm401616g

The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands. Compound 10, the archetype of a new f

Full text of DOI:10.1021/jm401616g

Article
pubs.acs.org/jmc
Glucocorticoid Receptor Modulators Informed by Crystallography
Lead to a New Rationale for Receptor Selectivity, Function, and
Implications for Structure-Based Design
Matthew W. Carson,† John G. Luz,‡ Chen Suen,†,§ Chahrzad Montrose,† Richard Zink,† Xiaoping Ruan,†
Christine Cheng,† Harlan Cole,† Mary D. Adrian,† Dan T. Kohlman,† Thomas Mabry,† Nancy Snyder,†
Brad Condon,‡ Milan Maletic,‡ David Clawson,† Anna Pustilnik,‡ and Michael J. Coghlan*,†
†Lilly Research Laboratories, A Division of Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana 46285, United States
‡Eli Lilly Biotechnology Center, 10300 Campus Point Drive, Suite 200, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for
inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands.
Compound 10, the archetype of a new family of dibenzoxepane and dibenzosuberane sulfonamides, is a potent anti-inflammatory
agent with selectivity for the GR versus other steroid receptors and a differentiated gene expression profile versus clinical
glucocorticoids (lower GR transactivation with comparable transrepression). A stereospecific synthesis of this chiral molecule
provides the unique topology needed for biological activity and structural biology. In vivo activity of 10 in acute and chronic
models of inflammation is equivalent to prednisolone. The crystal structure of compound 10 within the GR ligand binding
domain (LBD) unveils a novel binding conformation distinct from the classic model adopted by cognate ligands. The overall
conformation of the GR LBD/10 complex provides a new basis for binding, selectivity, and anti-inflammatory activity and a path
for further insights into structure-based ligand design.
nisms. The GR positively regulates transcription via TA, a
process by which the GR, bound to an endogenous ligand,
translocates from the cytoplasm into the nucleus where the
resulting dimeric GR/ligand complex acts as an agonist and
activates transcription by binding to glucocorticoid response
elements (GREs) in susceptible promoter regions. The GR/
ligand complex can also repress transcription, via the
monomeric GR/ligand complex binding directly to tran-
scription factors such AP-1 and NF-κB. The latter phenomenon
is the prevailing mechanism of action for anti-inflammatory and
immunosuppressant action.
INTRODUCTION
■
Modulation of the glucocorticoid receptor (GR) has long been
pursued as a favored target of medicinal and organic
chemistry.1,2 The discovery of cortisone and related steroidal
GR modulators in the middle of the 20th century was followed
by a decades-long effort prosecuting the steroid pharmaco-
phore.3 Ligands were sought that maintain anti-inflammatory
efficacy, demonstrate selectivity versus related steroid hormone
receptors, and reduce side effects associated with the
endogenous function of glucocorticoids (GCs). New structural
classes emerged, yet the goal of a novel functional GR ligand
remained elusive until the 1990s when new chemotypes were
discovered.4,5 This was largely an empirical pursuit until the
paradigm of differentiation of transrepression (TR) versus
transactivation (TA) emerged.6−8
Due to their potent anti-inflammatory properties, GR ligands
have found broad application in the treatment of inflammation
and autoimmune indications such as asthma, rheumatoid
arthritis, and allergy. Clinically approved steroidal GR agonists
The GR mediates a breadth of biological processes (e.g.,
inflammation, gluconeogenesis, immunity, and homeostasis/
development) through predominantly transcriptional mecha-
Received: October 18, 2013
Published: January 21, 2014
© 2014 American Chemical Society
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include cortisol, prednisolone (pred), dexamethasone (dex),
and fluticasone furoate. The side effects of steroidal GR
agonists, attributed to GR-mediated TA, instigated research
into the development of molecules that have an increased
preference for GR-mediated TR. Such GR ligands are termed
“dissociated” due to their discrimination between the pathways
of TA (agonism) and TR. Maintenance of anti-inflammatory
activity via TR of proinflammatory gene expression and
reduction of side effects of endocrine function by attenuating
agonism has been a preferred hypothesis for differentiation of
GR pharmacology.9−12 Yet, as the true complexity of GR-
mediated processes has become apparent, it has been suggested
that this hypothesis may be limited in its application as a
paradigm for drug design.13
Selectivity has also been aggressively sought as cross
reactivity with related steroid receptors the mineralocorticoid
receptor (MR), the androgen receptor (AR), the progesterone
receptor (PR), and the estrogen receptor (ER) would also
potentiate dramatic physiological liabilities.14 During these
experiments, a clear structural basis for functional activity and
selectivity has been the goal, yet the GR ligand binding domain
(LBD) is among the most difficult of the steroid nuclear
hormone receptors (NHRs) to crystallize. The first GR LBD
crystal structure was disclosed using dex (Chart 1), and
subsequently, the GR LBD binding conformations of more
steroidal GR agonists and one steroid antagonist emerged.15−17
The binding mode of a class of flexible trifluoromethyl carbinol
GR agonists (TFCs), for example, GSK 866, (Chart 1)16,18,19
was more recently described that occupies a novel channel
originally observed in the crystal structure of A ring fused
steroids such as deacyl cortivazol (Chart 1).16,18−20 Novel GR
ligands showing enhanced selectivity, differentiated function
(TA vs TR), and oral in vivo activity have been disclosed, yet
little structural information has emerged to influence ligand
design.9−12 We surmised that novel GR ligands could be
identified with differentiated function based on a unique
complex within the GR LBD. Herein, we characterize
compound 10 with several close analogues that represent the
first members of a novel class of potent, selective, and orally
active GR ligands,21−30 the crystal structure of compound 10
within the GR LBD, and the structural basis for TR activity and
selectivity versus the remaining steroid NHRs.
RESULTS
■
Scheme 1 details the synthesis of compound 10 that begins
with the protection of 2-iodophenol (1) as its MOM ether
(2)31,32 followed by palladium-mediated Sonogashira cou-
pling33 with 1-butyne to provide acetylene 3. Deprotection
followed by Mitsunobu coupling34 with 2-iodo-3-methoxyben-
zyl alcohol (5)35 affords alkynyl ether 6 that was subjected to a
recently reported23 stereospecific sequence of platinum-
catalyzed diboronation to intermediate 7 and cyclization to
the dibenzoxepane vinyl boronate (8). The synthesis of 10 was
completed by condensation of 8 with N-(3-iodophenyl)
methanesulfonamide (9) to deliver this highly congested
product in 13% overall yield for seven steps.
Chart 1. Steroid and TFC GR Ligands
Notably, this molecule is intrinsically strained, since the
coplanar substituents of the olefin must all adapt to the
demanding steric environment by rotating out of conjugation
with the double bond. The structural requirements of the
tricylic system and the need to accommodate the benzylidene
ring in close proximity to the methoxy substituent result in an
overall molecular structure with a subtle and unusual element of
planar chirality due to these steric interactions. The resulting
increase in overall molecular volume is believed to be key to the
successful realization of the GR LBD crystal structure versus
several less strained members of this family (Chart 2). Although
Scheme 1. Preparation of Dibenzoxepane 10
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a
Comparison of the m-sulfonamides shows binding affinity of
10 for the GR is slightly better than pred with improved
selectivity versus the MR. Binding affinity for the remaining
steroid hormone receptors is also depicted in Table 1 where 10
shows a strong preference for the GR. In vitro functional assays
were also performed for each of these receptors in agonist and
antagonist modes versus relevant reference standards (Table
S1, Supporting Information). In all cases, the functional activity
seen for 10 is highly selective (>100×) compared to the other
three potent GR ligands 11−13. This molecule is also
representative of differentiated function, since dibenzoxepane
members of this series have lower maximal efficacy versus
conventional glucocorticoids. Agonism of the GR is a measure
of TA, and in this assay, 10 shows differentiated efficacy versus
pred (53% for 10 versus 114% for pred at 10 μM, n = 10, see
Chart S1, Supporting Information).
Chart 2. Depictions of Compound 10 within the GR LBD
a
Individual substituents of the double bond adapt to form a highly
strained yet rigid three-dimensional structure to maintain coplanarity
of the olefin substituents.
Compounds with binding Ki values less than 1 nM were
tested in several TR models of anti-inflammatory activity. Of
these, the IL-1β induced inhibition of IL-6 in CCD-39SK cells
emerged as a strong indicator of in vivo efficacy.36 Table 2
shows members of this family with potency comparable to pred
in this assay.
the synthesis implies no enantioselectivity, attempted separa-
tion of the enantiomers was unsuccessful using over 20 chiral
solid phases under varied chromatographic conditions.
Calculations of inversion barrier between enantiomers indicate
a 70 kcal activation energy leading to the conclusion that
interconversion of these rigid isomers under physiological
conditions is unlikely.
Table 2. Inhibition of IL-1β Induced IL-6 in CCD-39SK
Cells
Analogues 11−19 were prepared following published
procedures.23,26−28 These are less strained compounds where
the substituents on the double bound are closer to coplanarity
(14 being an exception). Table 1 shows the binding data for
members of this series as well as the commonly used GR
clinical standards pred and dex. The structure−activity
relationship (SAR) clearly indicates a preferred position on
the benzylidene R1 group for GR binding. The meta-substituted
methylsulfonamides have attractive in vitro profiles in that they
are potent (Ki < 1 nM) GR ligands with varying degrees of
selectivity versus related steroid NHRs.
compd
IC50 [nM], (SEM), n
10
4.38 (0.384) 11
4.80 (0.561) 5
9.32 (0.746) 4
75.9 (24.0) 7
11
12
13
pred
7.26 (0.276) 10
Several ensuing TR assays showed this sulfonamide to be
equipotent and fully efficacious versus pred. For example,
reduction of lipopolysaccharide (LPS)-induced TNFα in U937
Table 1. Comparison of NHR Binding for Dibenzosuberane and Dibenzoxepane Analogues
a
binding Ki [nM]
compd
R1
R2
R3
X−Y
GR
AR
MR
PR
10
11
12
13
14
15
16
17
18
19
pred
dex
m-NHSO2Me
m-NHSO2Me
m-NHSO2Me
m-NHSO2Me
m-N(Et)SO2Me
o-NHSO2Me
p-NHSO2Me
m-SO2NHMe
H
Et
Et
Me
H
OMe
H
−CH2O−
−CH2O−
−CH2O−
−CH2CH2−
−CH2O−
−CH2CH2−
−CH2CH2−
−CH2CH2−
−CH2CH2−
−CH2CH2−
0.268 (0.026)
0.146 (0.053)2
0.257 (0.041)
0.226 (0.054)
>100
239 (51.0)
9.74 (3.81)2
5.98
6.10 (4.58)
0.967 (0.176)2
0.342 (0.0635)2
2.48 (1.79)
NT
26.8 (6.33)
15.5 (1.73)2
6.17
H
H
418 (150)
99.0 (40.7)
NT
b
Et
H
OEt
H
NT
>100
NT
NT
NT
H
H
>100
NT
NT
NT
H
H
>100
NT
NT
NT
H
H
>100
NT
NT
NT
m-NH2
H
H
>100
NT
NT
NT
1.14 (0.295)
0.690 (0.240)
2520 (474)
1410 (706)
0.290 (0.240)
0.360 (0.160)
>10 000
561 (196)
a
All assays are the mean of n > 3 with each experiment run in triplicate unless indicated by a single value or n after the SEM shown in parentheses.
NT = not tested.
b
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Figure 1. Acute evaluation of (a) compound 10 vs (b) prednisolone (pred) in the carrageenan-induced paw edema (CPE) assay. Compound 10
■
▼
shows in vivo effects reducing paw weight ( ) and IL-1β secretion ( ) comparable to that seen with pred.
cells37,38 was comparable between these two ligands (com-
pound 10 IC50 = 19.1 9.3 nM (n = 3) vs IC50 = 8.2 1.0 nM
(n = 12) for pred). The overall profile of in vitro selectivity and
TR activity led to compound 10 being selected for in vivo
assessment.
fundamental binding mode of this nonsteroidal ligand within
the GR LBD was lacking. The structural biology of
glucocorticoids and related steroids is fairly well understood,
yet the conformation of novel GR ligands within the GR LBD
has not evolved to a level where their design assures receptor
selectivity, anti-inflammatory function, and the potential to
optimize druglike properties. To date, the GR LBD complexes
of only two pharmacophores, steroids and trifluoromethyl
carbinols (TFCs),15,16,20,42 have emerged by way of X-ray
crystallography, and in these cases, the argument has been
made for ligands occupying a larger cavity leading to TR
activity and a rationale for the limited NHR selectivity seen
with these agents. We sought to define the corresponding
binding interactions for these sulfonamides compared with
known GR ligands beginning with steroidal reference standards.
The crystal structures of the GR LBD bound to dex as well as
to other anti-inflammatory steroids reveal the key interaction of
the steroid C-11 hydroxyl group with N564 of the GR LBD
that is essential for TR activity. A similar role for the hydroxyl
group of the TFCs is proposed based on subsequent structures
of TFC/GR LBD complexes. In the case of steroids and TFCs,
a hydroxyl group is employed to secure the key N564
interaction where additional β fluorine substitution can be
used to attenuate the O−H bond strength and modulate TR
potency.
Pharmacokinetics (PK) in the Sprague−Dawley rat summar-
ized in Table S2 (Supporting Information) show an acceptable
1
half-life (t / = 1.4 h iv), high bioavailability (F = 100%), a low
2
volume of distribution (Vd = 7 L/kg), and moderate clearance
(56 mL/(min·kg)) suggestive of acceptable intravenous (iv)
and oral (po) profiles. The relative equivalence in exposures in
both the iv and po arms of this study (AUC = 1487 562 and
1303 108 (ng·h)/mL, respectively) supports both routes of
administration, yet po dosing was used in subsequent in vivo
assays.
In vitro characterization led to evaluation in in vivo models of
anti-inflammatory activity. A pair of well-known assays,
carrageenan-induced paw edema (CPE)39 and collagen-induced
arthritis (CIA),40 were employed to assess the anti-inflamma-
tory effects of 10. Acute activity was measured in CPE where 10
shows improvements in overall inflammation as measured by
changes in paw weight and inhibition of stimulated
inflammatory cytokines such as IL-1β comparable to pred
(Figure 1).
Evaluation of multidose efficacy in CIA was then assessed in
therapeutic mode41 where rats were dosed orally with pred and
10 for two weeks after disease onset. By using this model,
compound 10 reduces signs and symptoms of disease on a time
course comparable to pred (Figure 2a). Measures of overall
change in paw edema by AUC, histological evaluation of bone
resorption, pannus, cartilage damage, and joint inflammation
show an improvement in 10-treated groups. In many cases, the
histology scores are superior to those seen in the pred-treated
positive control group (Figure 2).
We sought to crystallize this sulfonamide in complex within
the GR LBD mindful of the need for N564 and the associated α
helix 3 to adopt the proper conformation in active ligand
complexes. Unlike other more planar analogues evaluated as
candidates,21,22 the unique topology of 10 imparted sufficient
stability to the resulting complex to enable cocrystallization.
Structure of the GR LBD/Compound 10/D30 Complex.
The overall crystal structure of compound 10 bound to the GR
LBD (Figure 3, PDB accession code 4LSJ) is similar to that of
previously determined cocrystal structures of GR LBD/agonist
complexes (residues 522−777, F602Y, C638G) having an
average rms deviation of 1.2 Å for all Cα atoms when
superimposed upon the GR LBD dex bound structure
(1M2Z),15 excluding the α-1 helix (residues 559−777). The
F602Y and C638G mutants enhance protein solubility to
facilitate the GR LBD crystallization.15 As had been observed in
a previous GR LBD structures (3H52),43 α helix 1 forms a
domain swapping interaction, in this case with a symmetry
related molecule (Figure S1, Supporting Information).
Compound 10 is bound in the expected steroid ligand binding
site found at the bottom of the domain and formed by generally
PK in the rat measured at the conclusion of the CIA live
phase (day 15) is summarized in Table S2 (Supporting
Information). Dose proportional exposures were observed with
1
a Tmax of 1 h and t / values of 1.4−3.3 h depending on the dose.
2
The overall profile is consistent with the earlier rat PK data at 3
mpk indicating no differences due to rat strain or the collagen
adjuvant treatment (compare Tables S2 and S3, Supporting
Information).
Although this sulfonamide demonstrates hormone receptor
selectivity, TR activity, differentiated efficacy in GR agonist
mode, and strong in vivo efficacy, an understanding of the
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Figure 3. Structure of the GR LBD/D30/10 trimeric complex (PDB
accession 4LSJ). Ribbon diagram of the GR LBD (cyan) (residues
526−777) and the D30 peptide (orange) (residues 2−11) with 10
represented as sticks and colored by atom (C, yellow; N, blue; O, red).
Compound 10 is bound in the steroid binding pocket with the
methylsulfonamide pointing down.
Figure 2. Data from the collagen-induced arthritis (CIA) model.
Female Lewis rats were injected with collagen in the base of the tail,
and disease was allowed to develop for 14 d. Groups (n = 10) of rats
responding to the collagen challenge were selected and dosed daily
with 10 or pred over the course of the following 15 d. (a) Changes in
ankle diameter were measured throughout the study showing 10 dose
dependently reduced inflammation with a time course comparable to a
10 mpk dose of pred (red dotted line). (b) AUC of paw diameter
measures during live phase with statistical analysis. (c) Histology
scores of the affected hindpaws. Scores: 5 = response equivalent to
untreated collagen challenge group (vehicle control); 0 = response
shows no histological changes versus normal healthy joint. Data
indicate a strong response to 10 resulting in near normal joint
histology at the highest test dose. *p values < 0.01 by Students t test
versus the untreated vehicle control group.
Figure 4. Comparison of GR (cyan) LBD/10 crystal structure. (a)
Methylsulfonamide of compound 10 (colored by atom) forms
hydrogen bonds (2.5−3.5 Å) with the GR side chains of N546 and
T739. H bonds are denoted by dotted lines. M646 is folded back. (b)
The 10 tricycle interacts with the side chain of F623 through edge-to-
face π-stacking with the two flanking aromatic rings of the
dibenzoxepane.
́
hydrophobic residues contributed primarily by α helices 3, 6, 8,
and 11 (Figure 3). (Helices are numbered according to the
original estrogen receptor crystal structure.44) The cofactor
mimic D30 peptide45 is found in the coactivator binding site
with the disposition of its LXXLL motif being in agreement
with that of the canonical coactivator binding mode (see Figure
S2, Supporting Information).
LBD side-chain contacts are entirely hydrophobic, with the
exception being a pair of hydrogen bonds formed between the
10 methylsulfonamide and N564 of the GR LBD as well an
another H bond with T739 (2.7, 3.1, and 2.6 Å respectively)
(Figure 4a). The kinked tricyclic ring lies perpendicular to the
long axis of the domain in the horizontal plane with its concave
Compound 10 is oriented with the methylsulfonamide group
α to the tricyclic ring structure (Figure 4a). The ligand/GR
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surface facing the loop extending from the terminus of α helix
5. The two flanking aromatics and the central oxepane of the
tricycle participate via the concave surface of 10, in classic edge-
to-face π−π stacking interactions with the side chain phenyl of
F623 (Figure 4b). The convex surface interacts with the side
chains of M601, M604, and L732. The edge of the phenyl
moiety of the tricycle interacts hydrophobically with L566 and
is within hydrogen-bonding distance to the side-chain oxygen
of Q570. Additional hydrophobic and van der Waals
interactions are noted between the methoxyphenyl in 10 and
the M639 and L563 side chains, and both carbons ortho to the
sulfonamide nitrogen interact with the C736 and N564 side
chains as well as the Y735 phenyl ring.46
Dibenzoxepane 10 and dex occupy roughly similar volumes
within the steroid binding site and display comparable relative
orientations. The steroid core of dex partially overlaps with the
tricycle of 10, and the dihydroxyacetyl group of the dex D ring
points downward similar to the methylsulfonamide of 10
(Figure S4a, Supporting Information). The volume of the
ligand in the GR LBD for 10 and dex bound structures are 311
and 300 Å3, respectively. The A ring of dex lies nearly in the
same plane as one of the phenyl rings in the tricycle of 10;
however, the B, C, and D rings of dex slope downward toward
the bottom of the binding pocket as opposed to the tricycle of
10 that is enclosed within a region of hydrophobic side chains
at the top of the LBD. The most significant deviation between
the two binding modes occurs at the methoxyphenyl of 10 that
projects outward toward α helix 8 into space occupied by the
M646 side chain in the dex bound structure (compare Figures 4
and S4a, Supporting Information). The binding mode of 10
forces the rearrangement of the M646 side chain to
accommodate the methoxyphenyl moiety, resulting in an
upward shift of the M646 side chain by 6.4 Å. In addition, in
the dex X-ray structure, a hydrogen bond is formed between
the C-17 hydroxyl of the D ring and Q642 . In contrast, in the
10-bound structure, the Q642 side chain folds back and in
between α helices 8 and 11, a shift which is characterized by a
5.5 Å movement of the side-chain terminus. The only
interaction between 10 and the Q642 side chain is via a
long-range van der Waals contact (4.1 Å).
reference, 10 shows strong activity in the acute CPE model of
inflammation with respect to general anti-inflammatory
response (reduction of paw weight) as well as measurement
of specific cytokines (IL-1β). In a two week multidose study in
an established model of rheumatoid arthritis (CIA) run in
therapeutic mode, this dibenzoxepane has a comparable onset
of action, overall efficacy, and improved protection of bone and
joint degradation evaluated by histology. The overall PK profile
is attractive, and notably, it is comparable across rat strains with
no drastic effect from disease state. The overall in vivo efficacy
is comparable to pred.
In addition to the in vitro and in vivo properties of 10 in vivo
anti-inflammatory activity, receptor selectivity, and in vivo
function, it provides a unique insight into structural require-
ments for GR ligands. Crystal structures of functional GR
ligands in the LBD all have a hydrogen-bond interaction with
N564 in helix 3 as a unifying trait associated with TR and anti-
inflammatory activity. This mandatory interaction with N564 is
maintained via the sulfonamide group of 10 acting not only as
the noted hydrogen-bond donor but also as an acceptor
resulting in a stronger association with the portion of the GR
LBD needed for anti-inflammatory activity. The added
hydrogen bond with T739 provides additional stabilization of
the overall complex in the proper conformation.
With the key N564 interaction secure, the remainder of the
dibenzoxepane binding mode digresses substantially from those
seen with other chemotypes. In contrast to classic GR ligands,
interactions associated with polar groups in the steroid D ring
are absent for 10 that also does not form a “meta pocket” with a
larger binding volume seen with TFCs and steroids.
Compound 10 adopts a novel conformation involving
interactions largely due to hydrophobic and van der Waals
interactions resulting in a compact ligand/LBD complex similar
to the endogenous ligand cortisol in terms of the overall
volume, yet 10 is very different in its associated GR LBD
residues.
Compound 10 does not interact with the canonical fashion
with the trio of G/R/F residues found in all of the steroidal
hormone receptors that are engaged by carbonyl or hydroxyl
groups in the steroid A ring. In the GR/10 complex, GR F623
now adopts an altered conformation through a novel edge to
face interaction rather than the conventional π−π face to face
relationship seen for steroids thereby providing added
stabilization of this unique complex. The binding conformation
of 10 is much more distinct when compared to the TFCs
(compare Figures 4a and S4b, Supporting Information). The
TFC agonists (3E7C) take advantage of the plasticity of the GR
binding pocket that had been noted in the deacylcortivazol
crystal structure. A “meta” channel opens between α helices 3
and 6 allowing for the expansion of the steroid binding pocket,
accommodating the deacylcortivazol phenylpyrazole extension
of the steroid D ring. In contrast, 10 does not extend into any
part of the “meta” channel nor does the TFC scaffold extend
into the space occupied by the region containing the
methoxyphenyl of the dibenzoxepane that thus far appears to
be a unique aspect of 10 binding resulting in displacement of
the M646 side chain. (Figure 5). As in the GR/dex crystal
structure, the M646 side chain adopts an extended con-
formation in the GR/TFC complex that would not be
compatible with 10 binding.
DISCUSSION
■
These GR ligands have been prepared by two general routes
enabling a variey of analogues to be realized. Of these, the
platinum-mediated pinacol diboronate route has been most
reliable, especially when preparing sterically challenging
analogues. The synthesis proceeds by a stereospecific route,
yet for molecules such as 10 and 14, there is no expectation of
enantioselectivity. In the case of 10 and 14, significant barriers
to inteconversion are apparent, yet compounds lacking R3
substituents adopt a planar orientation with lower steric
concessions and a low barrier to interconversion. In vitro
SAR from Table 1 indicates a clear preference for the meta-
methysulfonamide containing an acidic proton capable of
forming the required hydrogen bond with N564 (compare
close analogues 10 and 14). The added methoxy group in 10
versus 11−13 improves selectivity versus the MR, and the
dibenzoxepanes emerge as partial agonists of the GR (compare
10−12 versus 13, dex, and pred in Table S1, Supporting
Information).
Dibenzoxepane 10 represents a novel ligand for the GR with
differentiated TR versus TA function whose in vivo activity is
similar to the reference steroid prednisolone. Compared to this
Many of the critical interactions between the GR and 10
appear to be potentially conserved in related NHRs. Among the
GR, PR, MR, AR, and ERα, the phenylalanine that occupies
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Supporting Information). No PR structure in the Protein Data
Bank displays the equivalent methionine in the folded back
conformation. However, in the MR/spironolactone crystal
structure (3VHU), the equivalent methionine is found in the
folded back position (Figure S5d, Supporting Information).
The residue equivalent to GR A605 is MR S811 whose freedom
of rotation may allow for the corresponding movement in the
M852 side chain seen in the MR/spironolactone (antagonist)
and the MR/aldosterone (agonist) crystal structure. One
implication of this, if movement of M646 is important to GR
binding and selectivity, is that the MR might be predicted to
bind with higher affinity to 10 than would the PR. In fact, this is
observed in binding studies (Table 1).
Selectivity versus other NHRs arises from subtle interactions
associated with 10 and the resulting disposition of M646 and
A605. By using this paradigm, it is possible to understand
relative cross reactivity between NHRs informed by the
interaction of these residues. The comparison of the MR and
PR in this fashion led to a prediction of NHR selectivity
supported by binding and functional data. The role of the
methoxy group in the topology of 10 is a key factor in the
binding configuration in that this group perturbed the overall
structure of the ligand sufficiently to provide a stabilized
complex within the LBD protein. The distinctive binding mode
of this unique GR ligand suggests that leveraging the noted
hydrophobic interactions associated with the novel F623 π−π
interactions, the disposition of M646 and A605 employed to
provide receptor selectivity, and the mandated N564
interactions enable strategies for ligand design and reveal
previously unexplored opportunities regarding functional
requirements of the GR LBD and its associated ligands.
Figure 5. Methoxyphenyl of 10 occupies a unique space within the GR
binding site. The crystal structure of GR LBD bound to 10 versus
other GR LBD crystal structures comparing the side-chain positions of
M646. The methoxyphenyl of 10 forces the M646 side chain to adopt
a unique conformation. (The GR LBD is colored cyan with the 10
ligand colored by atom.) The color scheme for the methionine side
chains in other GR LBD structures is as follows: dex, orange (1M2Z);
TFC, magenta (3E7C); fluticasone furoate, green (3CLD);
deacylcortivazol, purple (3BQD).
position 623 in the GR is structurally conserved (Figure 4b).
The critical GR amino acids whose side-chain polar atoms
interact with 10 are conserved in the PR and the MR (Figure
6).
CONCLUSIONS
■
In summary, we have provided evidence for the in vitro and in
vivo efficacy of a novel class of GR ligands. Key SAR features
include the essential meta-sulfonamide on the benzylidene ring
associated with TR and the observation that dibenzoxepanes
provide partial agonist activity related with differentiated
function. Of the molecules presented, compound 10 was
selected for in vivo evaluation where it showed comparable
efficacy and onset of action in acute and chronic anti-
inflammatory models versus the clinical reference standard
prednisolone. The unique topology of 10 enabled determi-
nation of the GR LBD/10 binding mode that departs from
canonical steroid binding interactions as well as those of recent
GR ligands. The critical N564 hydrogen bond associated with
anti-inflammatory activity is provided by the meta-sulfonamide
thereby explaining the noted SAR trend. Moreover the
remaining unusual interactions with F623 and side chains
from the α8 helices contribute to the novel binding modes and
the observed hormone receptor selectivity. The correlations
between the in vitro and in vivo efficacy as well as the structural
basis for many of the observed SAR trends in the context of the
novel crystal structure provides guidance enabling the discovery
of improved GR ligands.
Figure 6. Critical binding interactions are conserved between the GR,
PR, and MR. Crystal structures of the GR LBD (cyan), the PR
(1ZUC) (orange), and the MR (2A3I) (slate blue) are superimposed
and depicted as ribbon diagrams. Compound 10 and amino acid side
chains are represented as sticks and colored by atom. Ligand
interactions with polar atoms are represented by dashed lines. All
polar side chain atoms that interact with 10 are preserved.
There is a structural basis for the observed steroid hormone
selectivity. The hydrogen bond formed by the GR LBD T739
and 10 would not form in the AR or ERα as this threonine is
replaced by leucine in both of these NHRs. Additionally, the
critical N564 is replaced by a threonine in the ERα (Figure S5a,
Supporting Information). The apparent conservation of critical
binding features between the GR, PR, and MR prompts
hypotheses about the cause of the noted selectivity of 10
toward the GR. This may be related to the freedom of rotation
imparted to the M646 side chain by the presence of an alanine
at position 605 in the GR. In the PR and AR, this position is
occupied by valine that sterically prevents the extent of
movement observed in the GR M646 side chain (Figure S5b,c,
EXPERIMENTAL SECTION
■
Materials and Methods. All reactions, unless specified, were
carried out under an atmosphere of nitrogen in oven-dried glassware.
Reagents and solvents were obtained from Aldrich Chemical, Acros
Organics, or Strem and used without further purification. 1-Iodo-2-
(methoxymethoxy)benzene (2) and (2-iodo-3-methoxy-phenyl)-
methanol (5) were prepared according to the literature procedure
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Journal of Medicinal Chemistry
Article
cited in the main paper. Thin layer chromatography was performed on
0.25 mm E. Merck silica gel 60 F254 plates and visualized under UV
light. Flash chromatography was performed using Agilent Super Flash
silica cartridges. Liquid chromatography−mass spectrometry (LC/
MS) analysis was carried out on an Agilent 1100 Series LC/MSD
using 5−100% acetonitrile/0.1% formic acid over 7.0 min. NMR
spectra were recorded on a Varian 400, and chemical shifts are
expressed in parts per million relative to solvent signals: CDCl3 (1H
7.26; 13C 77.0 ppm) or DMSO-d6 (1H 2.50; 13C 39.5 ppm). High-
resolution mass spectra (HRMS) were obtained by electrospray (ESI)
ionization. All compounds were refined to at least 95% purity via high-
performance liquid chromatography.
1-But-1-ynyl-2-(methoxymethoxy)benzene (3). A solution of 1-
iodo-2-(methoxymethoxy)benzene (2) (5.00 g, 18.9 mmol) in THF
(100 mL) was degassed for 10 min with nitrogen in a 250 mL pressure
flask prior to the addition of CuI (0.36 g, 1.9 mmol), PdCl2(PPh3)2
(0.66 g, 0.95 mmol), and triethylamine (13.2 mL, 94.7 mmol). The
resultant mixture was bubbled with nitrogen for 10 min and then with
1-butyne for 5 min; then, the flask was sealed, warmed to 50 °C, and
stirred overnight. The mixture was partitioned between EtOAc (200
mL) and water (100 mL). The aqueous phase was extracted with
EtOAc (100 mL). The combined organic phases were washed with
brine (200 mL), dried over Na2SO4, and concentrated under reduced
pressure to give a dark semisolid. Purification via flash chromatography
(9:1 hexanes/EtOAc) gave 2.49 g (69%) of the titled compound as a
tan oil: 1H NMR (DMSO-d6) δ 7.32 (dd, J = 8.0, 1.4 Hz, 1H), 7.23−
7.27 (m, 1H), 7.10 (dd, J = 8.0, 1.4 Hz, 1H), 6.92 (td, J = 7.2, 1.2 Hz,
1H), 5.21 (s, 2H), 3.40 (s, 3H), 2.42 (q, J = 7.5 Hz, 2H), 1.14 (t, J =
7.5 Hz, 3H); 13C NMR (DMSO-d6) δ 157.7, 133.5, 129.5, 122.2,
115.9, 114.3, 96.0, 94.9, 76.6, 56.1, 14.3, 13.1.
for 18 h. The reaction was allowed to cool to room temperature and
partitioned between EtOAc (100 mL) and water (50 mL). The layers
were separated, and the aqueous layer was extracted with EtOAc (50
mL). The combined organic layers were washed with water (5 × 50
mL) and then brine (50 mL). The organic layer was dried with
Na2SO4, filtered, and concentrated under reduced pressure.
Purification via flash chromatography (95:5 hexanes/EtOAc) gave
1.56 g (66%) of the titled compound as an off-white powder: 1H NMR
(DMSO-d6) δ 7.25 (t, J = 8.0 Hz, 1H), 7.09−7.14 (m, 1H), 7.04 (d, J
= 6.8 Hz, 1H), 6.88−6.92 (m, 3 H), 6.72 (d, J = 8.0 Hz, 1H), 4.97 (s,
2H), 3.83 (s, 3H), 1.95 (q, J = 7.6 Hz, 2H), 1.28 (s, 12 H), 1.10 (s,
12H), 0.85 (t, J = 7.6 Hz, 3H); 13C NMR (DMSO-d6) δ 157.4, 154.6,
140.6, 131.0, 129.4, 129.0, 127.4, 120.6, 120.2, 112.3, 110.4, 88.0, 83.1
(2C), 73.9, 56.5, 39.7−40.1 (m, 2C), 24.6−26.9 (m, 4C); HRMS
(ESI) m/z calcd for C30H43B2IO7 [M + H2O]+ = 664.2288, found
664.2481.
2-[(1E)-1-(10-Methoxy-6H-benzo[c][2]benzoxepin-11-ylidene)-
propyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (8). A 150 mL
screw-top vessel was charged with 6 (0.39 g, 0.99 mmol) and dioxane
(93 mL), and the solution was purged with nitrogen for 15 min.
Potassium phosphate (0.76 g, 3.5 mmol) was added in a single portion
followed by PdCl2dppf·CH2Cl2 (0.097 g, 0.12 mmol), and the mixture
was heated at 80 °C for 18 h. The reaction was allowed to cool to
room temperature and partitioned between EtOAc (150 mL) and
water (100 mL). The layers were separated, and the aqueous layer was
extracted with EtOAc (50 mL). The combined organic layers were
washed with water (5 × 50 mL) and then brine (50 mL). The organic
layer was dried with Na2SO4, filtered, and concentrated under reduced
pressure. Purification via flash chromatography (95:5 hexanes/EtOAc)
gave 0.39 g (86%) of the titled compound as an off-white powder: 1H
NMR (DMSO-d6) δ 7.19 (dd, J = 8.4, 7.6 Hz, 1H), 7.07−7.11 (m,
1H), 6.90−7.00 (m, 2H), 6.91 (dd, J = 8.0, 0.8 Hz, 1H), 6.80 (td, J =
8.0, 1.2 Hz, 1 H), 6.67 (td, J = 8.0, 1.2 Hz, 1 H), 5.52 (d, J = 12.0 Hz,
1H), 4.80 (d, J = 12.0 Hz, 1H), 3.66 (s, 3H), 2.22−2.31 (m, 2 H), 1.03
(s, 6H), 0.96 (s, 6H), 0.94 (t, J = 7.2 Hz, 3H); 13C NMR (THF-d5) δ
155.0, 154.8, 141.6, 136.7 (br, low intensity olefin C binding to B),
134.8, 133.6, 130.6, 128.4, 127.1, 125.4, 119.7, 118.9 (2C), 111.1, 82.3,
69.1, 54.8, 24.7, 23.8, 14.0; HRMS (ESI) m/z calcd for C24H31BO4 [M
+ H]+ = 393.2355, found 393.2348.
2-But-1-ynylphenol (4).
A solution of 1-but-1-ynyl-2-
(methoxymethoxy)benzene (3) (2.49 g, 13.1 mmol) and oxalic acid
(2.36 g, 26.2 mmol) in methanol (52 mL) and water (13 mL) was
stirred at 55 °C for 3 days in a 500 mL round-bottomed flask. The
reaction mixture was partially concentrated under reduced pressure.
The aqueous mixture was extracted with diethyl ether (2 × 100 mL).
The organic layers were combined, washed with brine (100 mL), dried
over Na2SO4, and concentrated under reduced pressure. Purification
via flash chromatography (9:1 hexanes/EtOAc) gave 1.49 g (78%) of
the titled compound as a tan oil: 1H NMR (DMSO-d6) δ 9.62 (s, 1H),
7.20 (dd, J = 7.2, 1.4 Hz, 1H), 7.08−7.12 (m, 1H), 6.83 (dd, J = 8.0,
1.4 Hz, 1H), 6.71 (td, J = 7.2, 1.2 Hz, 1H), 2.39 (q, J = 7.6 Hz, 2H),
1.14 (t, J = 7.6 Hz, 3H); 13C NMR (DMSO-d6) δ 158.6, 133.3, 129.4,
119.3, 115.8, 111.1, 95.4, 77.2, 14.4, 13.1; HRMS (ESI) m/z calcd for
C10H11O [M + H]+ = 147.0812, found 147.0804.
N-[3-[(1Z)-1-(10-Methoxy-6H-benzo[c][1]benzoxepin-11-
ylidene)propyl]phenyl] methanesulfonamide (10). A 50 mL micro-
wave vial was charged with 7 (0.16 g, 0.41 mmol), N-(3-
iodophenyl)methanesulfonamide (9) (0.19 g, 0.61 mmol), 3,5-
dimethoxyphenol (0.32 g, 2.0 mmol), potassium hydroxide (0.40 g,
6.1 mmol), water (1.6 mL), and dioxane (4.1 mL), and the solution
was purged with nitrogen for 15 min. Pd(PPh3)4 (0.047 g, 0.041
mmol) was added in a single portion, the reaction vessel was sealed,
and the mixture was heated at 80 °C for 18 h. The reaction was
allowed to cool to room temperature and partitioned between EtOAc
(50 mL) and 5 N sodium hydroxide (20 mL). The layers were
separated, and the organic layer was washed with water (30 mL) and
then brine (30 mL). The organic layer was dried with Na2SO4, filtered,
and concentrated under reduced pressure. Purification via flash
chromatography (4:1 hexanes/EtOAc) gave 131 mg (74%) of the
1-[(2-But-1-ynylphenoxy)methyl]-2-iodo-3-methoxy-benzene (6).
Trin-butylphosphine (4.0 g, 19 mmol) was added dropwise via syringe
to a solution of 2-but-1-ynylphenol (4) (1.0 g, 6.8 mmol), 1-iodo-2-
(methoxymethoxy)benzene (5) (1.7 g, 6.4 mmol) in benzene (32 mL)
at 0 °C. ADDP (2.5 g, 9.7 mmol) was added in one portion to give a
thick slurry that was allowed to warm to room temperature and then
warmed to 50 °C for 18 h. The mixture was diluted with EtOAc (150
mL) and washed with 5 N NaOH (2 × 100 mL) and then brine (100
mL). The organic layer was dried over Na2SO4 and concentrated
under reduced pressure. Purification via flash chromatography (95:5
hexanes/EtOAc) gave 1.53 g (61%) of the titled compound as a
1
titled compound as a clear crystalline solid: H NMR (DMSO-d6) δ
9.43 (s, 1H), 7.19 (dd, J = 8.0, 0.4 Hz, 1H), 7.04−7.15 (m, 3H), 6.91
(dd, J = 9.2, 0.8 Hz, 1H), 6.84−6.92 (m, 3H), 6.75−6.77 (m, 1H),
6.80 (dd, J = 8.0, 1.2 Hz, 1 H), 6.67 (d, J = 7.6, 1 H), 5.74 (d, J = 12.0
Hz, 1H), 4.94 (d, J = 12.0 Hz, 1H), 3.30 (s, 3H), 2.76−2.85 (m, 2H),
2.60 (s, 3H), (s, 3 H), 0.79 (t, J = 7.4 Hz, 3H); 13C NMR (DMSO-d6)
δ 156.9, 155.9, 145.0, 143.6, 139.5, 136.1, 134.6, 133.3, 131.0, 130.4,
129.6, 129.4, 126.7, 125.7, 121.8, 121.5, 121.0, 120.7, 119.4, 113.0,
71.3, 56.2, 39.6, 28.6, 14.3; HRMS (ESI) m/z calcd for C25H26NO4S
[M + H]+ = 436.1584, found 436.1576.
1
crystalline sold: H NMR (DMSO-d6) δ 7.19−7.38 (m, 4H), 6.90−
7.01 (m, 3H), 6.71 (m, 1H), 5.08 (s, 2H), 3.78 (s, 3H), 2.42 (q, J = 7.6
Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H); 13C NMR (DMSO-d6) δ 158.8,
158.1, 141.0, 133.4, 129.8, 129.7, 121.6, 121.0, 113.3, 113.5, 111.2,
96.4, 89.7, 76.7, 74.4, 57.0, 14.3, 13.1; HRMS (ESI) m/z calcd for
C18H18IO2 [M + H]+ = 393.0353, found 393.0347.
2-[(1Z)-1-[[2-[(2-Iodo-3-methoxy-phenyl)methoxy]phenyl]-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene]propyl]-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane (7). A 75 mL screw-top
vessel was charged with 1-[(2-but-1-ynylphenoxy)methyl]-2-iodo-3-
methoxy-benzene (6) (1.44 g, 3.67 mmol) and DMF (30 mL), and the
solution was purged with nitrogen for 15 min. Bis(pinacolato)diboron
(1.28 g, 4.77 mmol) was added in a single portion followed by
Pt(PPh3)4 (0.461 g, 0.367 mmol), and the mixture was heated at 80 °C
The chemistry described above as well as previously reported
procedures26−28 affords the following products:
N-[3-[(1Z)-1-(10-Ethoxy-6H-benzo[c][1]benzoxepin-11-ylidene)-
propyl]phenyl]-N-ethyl-methanesulfonamide (14). 1H NMR (400
MHz, DMSO-d6): δ (ppm) 0.70 (t, J = 7.2 Hz, 3H), 0.79 (t, J = 7.25
Hz, 3H), 1.13 (t, J = 7.0 Hz, 3H), 2.50−2.62 (m, 1H), 2.72 (s, 3H),
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2.76−2.88 (m, 1H), 3.25−3.38 (m, 2H), 3.38−3.50 (m, 1H), 3.66−
3.77 (m, 1H), 4.94 (d, J = 12 Hz, 1H), 5.80 (d, J = 12 Hz, 1H), 6.63
(d, J = 8.13 Hz, 1H), 6.73 (dd, J = 8.24/1.21 Hz, 1H), 6.78−6.82 (m,
1H), 6.87 (dt, J = 7.06/1.21 Hz, 1H), 6.95 (d, J = 7.03 Hz, 1H), 7.03−
7.16 (m, 4H), 7.16−7.20 (m, 1H), 7.20−7.26 (m, 1H). MS [M + H]+
calcd: 477.6, found: 477.8; [M + Na]+ found: 499.6.
N-(2-((10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-
methyl)phenyl)methanesulfonamide (15). 1H NMR (400 MHz,
DMSO-d6): δ (ppm) 2.80−3.04 (m, 2H), 3.07 (s, 3H), 3.28−3.48 (m,
2H), 6.64 (dd, J = 1.5, 7.9 Hz, 1H), 6.87−6.95 (m, 3H), 7.00 (s, 1H),
7.11−7.16 (m, 3H), 7.20−7.25 (m, 2H), 7.28 (dd, J = 1.0, 8.0 Hz,
2H), 7.60−7.62 (m, 1H), 9.39 (1H, bs). MS [M + H]+ calcd: 376.49,
found: 375.8; [M + Na]+ found 397.8.
N-(4-((10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-
methyl)phenyl)methanesulfonamide (16). 1H NMR (400 MHz,
DMSO-d6): δ (ppm) 2.80−2.97 (m, 2H), 2.94 (s, 3H), 2.25−2.40 (m,
2H), 6.72 (s, 1H), 6.90−6.95 (m, 1H), 6.94 (s, 4H), 7.03−7.11 (m,
2H), 7.17−7.20 (m, 2H), 7.23 (dt, J = 7.8/1.2 Hz, 1H), 7.34 (dd, J =
7.5/1.1 Hz, 1H), 7.44 (dd, J = 5.8/3.4 Hz, 1H), 9.70 (s, 1H). MS [M +
H]+ calcd: 376.49, found: 397.80 [M + Na]+.
The cells were pretreated with compounds (50 μL/well of 4X stock)
for 60 min. Finally, rhIL-1β at a final concentration of 1 ng/mL was
added. The cells were incubated at 37 °C in a CO2 incubator for 24 h.
A 100 μL amount of culture supernatant was transferred from each
well to the 96 wells and spun at 2000 rpm for 10 min at 4 °C in an
Eppendorf 5810R centrifuge to remove the cells. The flowthrough was
used directly for IL-6/cytokine/chemokine measurement. Each
compound was evaluated using 10 point half log dilutions with each
concentration representing the mean of triplicate assessment.
LPS-Stimulated TNFα in PMA-Differentiated U-937 Cells. U-937
cells were seeded onto 96-well cell culture plates at 100 000 cells/well,
200 μL/well of 20 nM PMA in media was added, and the cells were
incubated overnight (∼24 h) at 37 °C in a 5% CO2 incubator. The
medium was removed, and the cells were refed 200 μL/well of media
without PMA . The cells were treated with compounds (50 μL/well of
4X stock) for 60 min. LPS (50 μL/well of 400 ng/mL stock) was then
added, and the cells were incubated at 37 °C in a 5% CO2 incubator
for 24 h. Secreted TNFα was then measured using 25 μL/well of
filtrate via EIA/RIA and quantified. Each compound was evaluated
using 10 point half log dilutions with each concentration representing
the mean of triplicate assessment.
In Vivo Assays. Pharmacokinetics. Cannulated male Sprague−
Dawley rats were fasted overnight prior to receiving 3 mpk doses of
compound 10 at t = 0. (iv formulation: 20% microemulsion, 80% DI
water; po formulation: CMC/tween in DI water) Approximately 0.2
mL of blood was collected via cannula in heparin-coated collection
tubes at t = .08, 0.25, 0.5, 1, 2, 5, 8, 12, and 24 h postdose, and the
plasma isolated after centrifugation was diluted and analyzed for parent
compound by LC/MS-MS. (For compound 10, the upper and lower
limits of quantitation were 3.9 and 4000 ng/mL respectively.) These
data were then used to calculate values for exposure, half-life, volume
of distribution, clearance, and bioavailability .
5-Benzylidene-10,11-dihydro-5H-dibenzo[a,d][7]annulene (18).
1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.78−3.00 (m, 2H),
3.24−3.46 (m, 2H), 6.81 (s, 1H), 6.88 (dd, J = 7.5/1.2 Hz, 1H), 6.98−
7.05 (m, 3H), 7.06−7.16 (m, 4H), 7.18−7.24 (m, 3H), 7.34 (dd, J =
7.5/1.2 Hz, 1H), 7.46 (dd, J = 5.7/3.3 Hz, 1H). MS [M − H]− calcd:
281.38, found: 280.80 [M − 1]−.
Compounds 11, 12, 13, 17, and 19 have been previously
characterized.26−28
Binding Assays. Cell lysates from human embryonic kidney
HEK293 cells overexpressing human MR, GR, AR, ER, or PR and 3H-
3
3
aldosterone for the MR, H-dexamethasone for the GR, H-estradiol
3
for the ER, and H-methyltrienolone for the AR and PR are used for
Carrageeanan-Induced Paw Edema (CPE).39 Groups (n = 5) of
male Sprague−Dawley rats were dosed at t = 0 with 10 or pred
followed by carrageenan injection into the rear footpad of the mouse
at t = 2 h. At t = 5 h, the animals were euthanized, and the amputated
hindpaws were weighed as a measure of overall anti-inflammatory
activity. Paw exudate was extracted and evaluated for expression of IL-
1β in response to the carrageenan stimulus by enzyme-linked
immunosorbent assay (kit: rIL-1β: RLB00, R&D). Error bars
represent the SEM of mean values for each dose group
Collagen-Induced Arthritis (CIA). CIA was assayed using published
protocols.41 Female Lewis rats were injected with collagen in the base
of the tail, and disease was allowed to develop for 14 d. Groups (n =
10) of rats responding to the collagen challenge were selected and
dosed daily with 10 or pred over the course of the following 15 d.
Histology scores from Figure 2c were provided by BoulderBiopath
(http://bolderbiopath.com/) after blinded samples from CIA study
animals in all dose groups were sent for analysis. A p value of less than
0.01 by Students t test versus the untreated vehicle control group is
deemed statistically significant. PK assessment was done at the end of
study (day 15) following the rat PK protocol similar to that cited
above except blood samples were collected via tail the vein of the
Lewis rat.
Protein Expression, Purification, and Crystallization. Re-
combinant GR LBD (residues 522−777, F602Y, C638G) was
expressed in Escherichia coli as a cleavable N-terminally his-tagged
SMT-fusion and fermented in ZYP-5052 media in the presence of 0.05
mM C_1. A 250 mL amount of cells (BL21DE3 Codon Plus RIL,
Stratagene) was grown in 2 L flasks at 37 °C for 3−5 h after a 20-fold
dilution from a saturated culture after which the temperature was
reduced to 22 °C to induce protein expression. The cells were
harvested by centrifugation and then stored at −80 °C until
processing. Cells were lysed by sonication in a buffer containing
0.02 M Tris pH 8.5, 0.5 M NaCl, 25 mM imidazole, 5 mM β-ME, 0.2
M NDSB-256, 0.2% β-octylglucoside, 10% glycerol, protease inhibitors
(complete EDTA-free, Roche), and turbonuclease (Accelagen).
Recombinant GR LBD was purified from the clarified cell lysate
using Ni-NTA agarose (Qiagen) in batch mode. The Ni-NTA resin
was collected in a drip column and washed with lysis buffer. Protein
receptor−ligand competition binding assays to determine Ki values.
Competing test compounds are added from 0.01 nM to 10 μM. The
data are used to calculate an estimated IC50 and percentage inhibition
at 10 μM. The IC50 values for compounds are converted to Ki using
the Cheng−Prushoff equation. Values shown are the mean values of at
least three experiments each run in triplicate.
In Vitro Functional Assays (Agonist/Antagonist). Human
embryonic kidney HEK 293 cells were transfected with receptors and
reporter gene plasmids. The reporter plasmid containing two copies of
probasin ARE (androgen response element 5′-GGTTCTTGGAG-
TACT-3′) and the TK promoter upstream of the luciferase reporter
cDNA was transfected with a plasmid constitutively expressing hAR
using the viral CMV promoter. The reporter plasmid containing two
copies of GRE (glucocorticoid response element 5′-TGTACAG-
GATGTTCT-3′) and the TK promoter upstream of the luciferase
reporter cDNA was transfected with a plasmid constitutively
expressing either hGR, hMR, or hPR, using the viral CMV promoter.
Cells were transfected in DMEM media with 5% charcoal-stripped
fetal bovine serum (FBS). After an overnight incubation, cells were
trypsinized, plated in 96 well plates, incubated for 4h, and then
exposed, to 0.01 nM to 10 μM of test compounds in half log dilutions.
In the antagonist assays, low concentrations of agonist for each
respective receptor are added to the media (0.25 nM dexamethasone
for the GR, 0.3 nM methyltrienolone for the AR, 0.05 nM
promegestrone or R5020 for the PR, and 0.05 nM aldosterone for
the MR). After 24 h incubation with the compounds, cells are lysed,
and the luciferase activity is determined. Data are fitted to a four
parameter-fit logistics to determine EC50 values. The percentage
efficacy is determined versus maximum stimulation obtained with 100
nM methyltrienolone for the AR assay, with 30 nM promegestrone for
the PR assay, 30 nM aldosterone for the MR assay, and 100 nM
dexamethasone for the GR assay.
TR Assays. Il-1β Stimulated IL-6 in CCD-39SK Cells. CCD-39SK
cells were seeded onto 96-well cell culture plates at 20 000 cells/well in
modified Eagle MEM media containing 2% charcoal/dextran-treated
FBS and incubated at 37 °C in a 5% CO2 incubator. Compound
diluents in 96-well master plate were used with dexamethasone at 0.4
μM and 0.4% DMSO as positive and negative controls, respectively.
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was eluted with 0.02 M Tris pH 8.5, 0.5 M NaCl, 0.25 M imidazole, 5
mM β-ME and 10% glycerol. ULP1 protease (0.5%) was added to the
eluted sample, and then, it was dialyzed overnight at 4 °C against 0.02
M Tris pH 8.0, 0.5 M NaCl, 10% glycerol, 0.2% β-octylglucoside, and
5 mM β-ME. The sample was reapplied to the Ni-NTA (His-TRAP
HP, GE Healthcare), and the flowthrough was collected and
concentrated for further purification by gel filtration (Sephadex 200,
GE Healthcare) in 0.01 M Tris pH 8.5, 0.15 M NaCl, 0.2% β-
octylglucoside, and 2 mM DTT. Before final concentration to 6 mg/
mL, D30 peptide (HSSRLWELLMEAT) was added to a 3-fold molar
excess relative to the protein, and 10 was added to one molar
equivalent. The trimeric GR LBD/C_1/D30 complex crystallized in
the space group P21212 at 21 °C by vapor diffusion against 0.1 M bis-
Tris pH 5.5, 0.3 M Mg(COOH)2 using 1:1 drop ratio (0.3 μL).
Crystals were cryoprotected using 25% ethylene glycol and cryocooled
using liquid N2.
Data Collection and Refinement. X-ray diffraction data were
collected at the LRL-CAT beamline at the Advance Photon Source
(Chicago, Illinois) (λ = 0.97931 Å). The data were processed using
XDS. The crystal structure was determined by molecular replacement
(one copy of the trimeric complex/au) using GR bound to
dexamethasone as the search model (PHASER) and refined
(BUSTER) at 2.35 Å resolution, after several rounds of model
building (COOT), to an Rwork of 17.9% and an Rfree of 23.6% (Table
3). Residues 526−777 of GR LBD (chain A) are visible in the crystal
structure except for a loop comprised by residues 703−710 for which
representative electron density was lacking. Side chains for the
following residues (chain A) are truncated due to incomplete electron
density: K44, N759, and K777. Representative electron density is
missing for residues 1, 12, and 13 of the D30 peptide (chain B) as well
as for the side chain of R4. For C621 and L636, alternate side chain
conformations are present. Unambiguous electron density was present
for two copies of the ligand, one in the steroid binding site and a
second in the crystal packing interface (Figure S3b, Supporting
Information). There are 56 water molecules modeled into the
structure. There are no residues in the disallowed region of the
Ramachandran plot.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed in vitro comparisons of compound 10 versus reference
standard steroids in binding and functional assays are shown in
Table S1; PK data at the conclusion of the CIA live phase and
normal Sprague−Dawley rats are summarized in Tables S2 and
S3, respectively; additional information regarding the X-ray
conformation of the 10/GR LBD complex including electron
density maps are provided in Figures S1−3; Figures S4 and S5
show crystal structures of dex and TFC in the GR LBD and of
the mechanism of GR selectivity for 10. This material is
available free of charge via the Internet at http://pubs.acs.org.
Table 3. X-ray Collection Data and Refinement Statistics
Accession Codes
The coordinates for the structure of 10 within the GR LBD
have been deposited in the Protein Data Bank under accession
code 4LSJ.
Crystal Parameters
space group
Cell Dimensions (Å)
a
P21212
AUTHOR INFORMATION
Corresponding Author
*Phone: (317) 651-1620; e-mail: coghlan_michael@lilly.com.
39.0
139.4
48.1
■
b
c
Angles (deg)
α
Present Address
§Chen Suen: chen.suen@nih.gov.
90
β
90
Author Contributions
γ
90
M.J.C., J.L., C.S., and M.C. conceived and designed the
experiments. D.T.K., T.M., N.S., B.C., M.M., D.C., and A.P.
performed the experiments. A.E.S. collected human specimens.
K.S.C., C.S.H., and J.P.H. analyzed the data. M.C., J.L., and
M.J.C. wrote the paper. All authors have given approval to the
final version of the manuscript.
resolution (Å)
completeness (%)
Rsym (%)
99−2.35
99.9 (100)
10.6 (49.2)
13.9 (4)
6.9 (6.8)
0.97931
mean I/σ(I)
redundancy
wavelength (Å)
Refinement
resolution range
reflections
Rwork
Notes
The authors declare no competing financial interest.
2.35−25.36 (2.35−2.57)
11 492
ACKNOWLEDGMENTS
17.9% (18.4%)
23.6% (24.0%)
■
Rfree
The authors gratefully acknowledge the staff at the LRL-CAT
beamline at APS, Stephen Wasserman, Sonali Sojitra, John
Koss, and Laura Morisco, for data collection. Use of the
Advanced Photon Source at Argonne National Laboratory was
supported by the U.S. Department of Energy, Office of Science,
Office of Basic Energy Sciences, under contract no. DE-AC02-
06CH11357. We are also indebted to V. Joe Klimkowski at Eli
Lilly for the calculations of the binding volumes of the GR
ligands and the activation energy between enantiomeric forms
of 10.
rms dev. bonds
lengths (Å)
angles (deg)
Total No. of Residues
chain A
0.01
1.15
244
10
chain B
total no. protein atoms
heteroatoms
ligand atoms
total no. waters
Average B (Å2)
peptide
2145
118
62
56
ABBREVIATIONS USED
■
GR, glucocorticoid receptor; GRE, glucocorticoid response
element; MR, mineralocorticoid receptor; AR, androgen
receptor; PR, progesterone receptor; ER, estrogen receptor;
NHR, nuclear hormone receptor; TR, transrepression; TA,
transactivation; dex, dexamethasone; pred, prednisolone; LBD,
chain A
34.9
52.0
25.3
38.7
chain B
ligand
water
858
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Journal of Medicinal Chemistry
Article
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ligand binding domain; TFC, trifluoromethylcarbinol; CPE,
carrageenan induced paw edema; CIA, collagen induced
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