Optimization of drug-like properties of nonsteroidal glucocorticoid mimetics and identification of a clinical candidate

Article abstract of DOI:10.1021/ml500387y

A series of nonsteroidal dissociated glucocorticoid receptor agonists was optimized for drug-like properties such as cytochrome P450 inhibition, metabolic stability, aqueous solubility, and hERG ion channel inhibition. This effort culminated in the identification of the clinical candidate compound (R)-39.

Full text of DOI:10.1021/ml500387y

Letter
pubs.acs.org/acsmedchemlett
Optimization of Drug-Like Properties of Nonsteroidal Glucocorticoid
Mimetics and Identification of a Clinical Candidate
Christian Harcken,* Doris Riether, Pingrong Liu, Hossein Razavi, Usha Patel, Thomas Lee,
Todd Bosanac, Yancey Ward, Mark Ralph, Zhidong Chen, Donald Souza, Richard M. Nelson,
Alison Kukulka, Tazmeen N. Fadra-Khan, Ljiljana Zuvela-Jelaska, Mita Patel, David S. Thomson,
and Gerald H. Nabozny
Department of Medicinal Chemistry and Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc.,
900 Ridgebury Road, Ridgefield, Connecticut 06877, United States
S
* Supporting Information
ABSTRACT: A series of nonsteroidal “dissociated” glucocorticoid
receptor agonists was optimized for drug-like properties such as
cytochrome P450 inhibition, metabolic stability, aqueous solubility, and
hERG ion channel inhibition. This effort culminated in the identification of
the clinical candidate compound (R)-39.
KEYWORDS: Glucocorticoid mimetics, nonsteroidal glucocorticoids, glucocorticoid-induced osteoporosis, azaindoles,
anti-inflammatory agents, drug-like properties
ortisol and the related cortisone and corticosterone are
Csteroid hormones that are referred to as glucocorticoids
(GCs) and bind to the glucocorticoid receptor (GR), which
belongs to a large family of transcription factors, the
superfamily of nuclear hormone receptors. GCs play an
important role in the regulation of the immune system and
therefore are widely used in the treatment of inflammatory and
immune diseases such as rheumatoid arthritis, asthma, allergy,
and sepsis.1 Synthetic GCs, which differ from cortisol in their
pharmacokinetics and pharmacodynamics, have been created
with dexamethasone (1) and prednisolone (2) (Figure 1) being
among the most extensively used anti-inflammatory agents.
However, because of harmful dose-limiting side effects and the
occurrence of glucocorticoid resistance, the use of these drugs is
limited. Side effects include weight gain, hypertension, muscle
weakness, skin thinning, diabetes, and the most troublesome
GC-induced osteoporosis leading to a weakening of the
trabecular bone, which causes a significant increase in the risk
of spine, hip, and rib fracture.2,3
Upon binding of GCs to GR, a conformational change is
provoked leading to the release of GR from the chaperone
complexes and unmasking of nuclear localization signals
followed by translocation of the GR−ligand complex to the
nucleus.4,5 There, it is thought to directly and indirectly induce
the expression of a few hundred genes, which is largely cell-type
specific. The precise molecular mechanism is highly complex
and, despite an impressive amount of research, still only
partially understood. However, a simplistic hypothesis, which is
based on a series of experiments,6,7 has become broadly
accepted among researchers aiming at GCs with reduced side
effects. This hypothesis attributes the anti-inflammatory effects
of GCs to the inhibition of gene transcription, referred to as
transrepression, while making the activation of transcription,
called transactivation, responsible for the majority of side
effects. Mechanistically it was rationalized that transrepression
involves the GR−ligand complex indirectly in the transcription
process through its interaction in a monomeric form with
transcription factors such as NF-κB and AP-1 resulting in the
down-regulation of key cytokine inflammatory mediators such
as TNF-α, IL-1, IL-2, and IL-6. The transactivation pathway
directly involves homodimers of GR recognizing GR response
elements (GREs) on the DNA resulting in the transcription of
genes.8 While this hypothesis is experimentally poorly
Received: September 24, 2014
Accepted: November 17, 2014
Figure 1. Glucocorticoid receptor agonists.
© XXXX American Chemical Society
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Table 1. Profile of Previous Leads
GR IC50
a
IL-6 IC50 [nM] and
MMTV and OC CYP 1A2, 2D6, 2C9, 2C19, HLM (%Qh),
thermodyn. sol pH
6.8 [μg/mL]
patch clamp hERG
channel IC50 [μM]
ab
,
b
compd
[nM]
max. eff. [%]
max. eff.
3A4 IC50 [μM]
rat PK t1/2
c
c
3
4
2
4
4 (93%)
10%/68%
0%/32%
nd /nd /2/0.2/0.9
30/4/10/1/0.1
47/2 h
89/1 h
10
9
4.5
3.8
13 (72%)
a
b
IC50 values are the mean of at least two values, each determined from duplicate 11-point concentration response curves. Maximum efficacy at the
c
highest tested concentration compared to dexamethasone, defined at 100%; maximum concentration tested is 2 μM. Not determined.
Table 2. Substitution on Azaindoles
a
a
ab
,
compd (R1 = Me)
R
GR IC50 [nM]
PR IC50 [nM]
IL-6 IC50 [nM] and max. eff. [%]
CYP3A4 IC50 [μM] HLM (%Qh)
8
H
10
9
1900
38 (89%)
5 (93%)
0.1
0.1
2
76
72
81
78
93
53
66
39
73
31
93
91
9
3-Me
240
10
4-Me
11
22
510
26
7
1100
59 (91%)
170 (71%)
>2000
11
5-Me
>2000
>2000
>2000
1000
0.6
0.2
3
12 (R1 = OMe)
5,7-Me2
5-Phe
13
2 (96%)
14
5-NH2
37 (91%)
38 (87%)
3 (96%)
0.5
0.6
0.3
0.01
2
15
5-NMe2
5-OiPr
10
10
3
>2000
>2000
>2000
1700
16
17
5-(4-morpholinyl)
5-CONH2
5-SO2Me
3 (94%)
18
7
300 (54%)
>2000 (40%)
19 (R1 = OMe)
28
>2000
2
a
b
IC50 values are the mean of at least two values, each determined from duplicate 11-point concentration response curves. Maximum efficacy at the
highest tested concentration compared to dexamethasone, defined at 100%; maximum concentration tested is 2 μM.
supported and partially even contradicted,9 it served as an
appealing working model for drug discovery programs over the
past decade.10 Several companies have invested intense research
in the quest of identifying functionally selective, so-called
“dissociated” synthetic glucocorticoids with the goal of offering
a therapeutic advantage over currently marketed GCs.11−13
We have previously disclosed the identification of non-
steroidal GC mimetics,14,15 including a series of trifluorome-
thylcarbinol derived compounds.16−19 Therein we described
the identification and profile of azaindole compounds such as 3
and 4 that have demonstrated excellent nuclear receptor
selectivity (specifically over the progesterone and mineralo-
corticoid receptors), potent GR agonism (as indicated by their
in vitro suppression of IL-1 induced IL-6 production in human
foreskin fibroblasts), and excellent in vivo activity in chronic
disease models (indicated by their inhibition of collagen-
induced arthritis in mouse) (Figure 1). We described
compounds exemplified by 3, which showed reduced trans-
activation (as indicated in vitro by their reduced potency and
reduced maximum efficacy in an MMTV-promoter transfected
HeLa cell) and reduced incidence of metabolic side effects
upon chronic dosing in vivo (as indicated by reduced increase in
body fat, reduced triglyceride, and reduced insulin levels
compared to equi-efficacious doses of prednisolone in healthy
mice). Compounds exemplified by 4 showed reduced activation
of a bone relevant dissociation marker (such as suppression of
vitamin D stimulated osteocalcin production in MG-63
osteosarcoma cells) and reduced bone loss compared to
traditional GCs upon chronic dosing in healthy mice in vivo
(indicated by a reduced decrease in femur cortical thickness by
microCT compared to prednisolone). However, to enable the
evaluation of compounds with such dissociation clinically, a
compound with drug-like properties suitable for clinical testing
had to be identified. The optimization effort toward our clinical
candidate (R)-39 is reported in this letter.
Table 1 shows the drug-like properties of previously
identified and described compounds 3 and 4. Clearly, the
compounds are at high risk of drug−drug interactions due to
their potent cytochrome P450 (CYP) inhibition across multiple
isoforms. Preliminary dose projections showed a potential for
high clinical dose requirements due to their overall high
clearance and low bioavailability.18 The safety margins of these
compounds are limited due to potent inhibition of the human
ether-a-go-go potassium channel (hERG channel), which is
involved in cardiac repolarization. Lastly, the aqueous solubility
of these compounds is low, potentially contributing to low
bioavailability and complicating clinical formulation develop-
ment.
The 6-azaindole moiety had previously been identified as a
preferred steroid A-ring mimetic that allowed combining the
desired dissociated biological profile with acceptable PK
properties.16 However, it was assumed that this moiety is also
primarily responsible for the potent CYP inhibition and hERG
channel inhibition observed. The effect of substitution on the
azaindole ring system had not been examined previously. It was
hypothesized that substitution could have an effect on the
ability of the azaindoles to function as a nucleophilic ligand for
CYP enzymes. A limited exploration of substitution on the A-
ring mimetic had been undertaken on the related indole17 and
diazaindole18 ring systems; however, no significant trends with
B
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regard to reducing CYP inhibition had been observed. A more
systematic investigation was called for.
Table 3. 5-(4-Morpholinyl)-6-azaindoles
All analogues with modified substitution on the azaindole
were prepared in combination with the 2-methyl-5-fluorophen-
yl D-ring mimetic (except in two cases where the approximately
equivalent 2-methoxy-5-fluorophenyl moiety was used). Sub-
stitution in the 3-position of the azaindole as in 9 reduced NR
selectivity (PR selectivity factor was approximately 25-fold)
compared to the comparator 8 (Table 2). Small substitution in
the 4-position such as methyl in 10 appeared tolerated and
demonstrated some reduction in CYP3A4 inhibition. Larger
substituents in the 4-position were not tolerated (data not
shown).
Small substituents in the 5-position led to a significant
reduction in agonist potency (IL-6 IC50 170 nM in 11).
Additional 7-substitution abolished even the GR binding
affinity (GR IC50 510 nM in 12). Furthermore, flanking of
the nucleophilic azaindole nitrogen with methyl groups on both
sides as in 12 provided very little improvement in the CYP
profile.
GR IC50 IL-6 IC50 [nM] and CYP3A4
HLM
a
ab
,
compd
R
[nM]
max. eff. [%]
IC50 [μM] (%Qh)
20
21
22
23
24
25
26
27
28
29
30
H
8
17 (90%)
18 (89%)
48 (64%)
2 (95%)
53 (80%)
4 (95%)
2 (95%)
7 (95%)
56 (82%)
60 (81%)
0.1
0.1
0.1
0.2
4
33
29
nd
47
68
36
62
67
60
65
52
3-F
9
c
4-F
14
2
2-OMe-5-F
3-OH
24
19
10
21
46
62
170
2-CONH2
2-SO2Me
2-SO2NH2
3-CONH2
3-SO2Me
3-SO2NH2
3
0.01
0.5
2
0.7
2
Large substituents in the 5-position exemplified by phenyl-
substituted 13 were well tolerated. Compounds with large A-
ring substitution are presumed to access an induced A-ring
>2000 (37%)
a
IC50 values are the mean of at least two values, each determined from
b
pocket;21 they typically show steroid-like potency (IL-6 IC50
2
duplicate 11-point concentration response curves. Maximum efficacy
at the highest tested concentration compared to dexamethasone,
defined at 100%; maximum concentration tested is 2 μM. Not
nM for 13 vs 1 nM for dexamethasone), full agonist activity
(96% vs 100% for dexamethasone), and little or no dissociation.
With this opportunity for substitution in the 5-position we
planned to assess the impact of electron donating and electron
withdrawing groups (EWG) and further test the tolerance for
polar substituents based on our overall goal to address not only
the CYP and hERG channel inhibition, but also to increase
metabolic stability and increase solubility. Small electron donor
groups like amino in 14 or dimethylamino in 15 maintained a
similar profile compared to the unsubstituted azaindole.
Increased potency was achieved by substitution with a larger
donor group such as isopropoxy in 16 or 4-morpholinyl in 17.
None of these compounds show sufficient reduction of CYP
inhibition; however, it was noted that these donor-substituted
azaindole compounds typically showed improved metabolic
stability. Electron-withdrawing groups like carboxamido in 18
or methylsulfonyl in 19 diminish the nucleophilicity of the
azaindole nitrogen the most and result in a significantly
improved CYP profile (CYP3A4 IC50 18 μM for 18); however,
these compounds lose most of their GR agonist activity. None
of the substitutions on the azaindole ring system appeared to be
helpful in reducing CYP3A4 inhibition while maintaining an
overall acceptable profile. The hERG channel inhibition and
solubility profile of these compounds was not improved
significantly either (e.g., 13 hERG channel IC50 3.8 μM; 17
solubility 5.3 μg/mL at pH 6.8). Therefore, we turned next to
an exploration of substitution on the D-ring mimetic phenyl
ring.
c
determined.
mimetic in 23 had previously been demonstrated to be
approximately equivalent to the standard 2-methyl-5-fluoro-
phenyl group. It was known that phenolic hydroxyl groups are
tolerated in the 2-position (see 3 in Table 1); however, a
hydroxyl group in the 3-position as in 24 caused a significant
reduction of the maximum agonist efficacy. Despite this fact, we
were eager to test alternative polar substituents of different
electronic nature in an effort to address key liabilities. This
effort paid off as EWG-substitution in the 2-position with
hydrogen donor/acceptor properties such as carboxamido in
25, methylsulfonyl in 26, and sulfonylamido in 27 were very
well tolerated and provided compounds as potent and
efficacious as the 2-methoxy-5-fluorophenyl and 2-methyl-5-
fluorophenyl analogue discussed above. These EWG-substi-
tuted D-ring mimetics are in fact more potent and efficacious
than those standard moieties which only becomes apparent in
combination with weaker A-ring mimetics (see below). The
same EWG-substitution in the 3-position of the D-ring mimetic
such as in 28, 29, and 30 resulted in reduced potency and
efficacy in good agreement with the SAR understanding.
It was noted that the improved metabolic stability of the
donor-substituted azaindoles was maintained. All compounds in
Table 3 (with the exception of 23) showed good NR selectivity
with a PR and MR IC50 > 2000 nM. As expected, additional
polar substituents helped reduce hERG channel inhibition and
improve aqueous solubility (e.g., 26 hERG channel IC50 20 μM;
29 solubility 47 μg/mL at pH 6.8). Most importantly, the
CYP3A4 inhibition IC50 values for all compounds in Table 3
were ranging from 0.01 to 4 μM. Despite the absence of any
obvious CYP3A4 inhibition SAR trends, this supported the
further investigation of the series.
A limited structure−actvity relationship (SAR) study of D-
ring substitution had been performed previously using the
unsubstituted 6-azaindole;16 however, to allow for greater
flexibility with regard to potency as well as metabolic stability,
more drastic changes on the D-Ring were examined using the
potent and metabolically stable 5-morpholinyl substituted 6-
azaindole. Some historic D-ring mimetics were re-examined:
The unsubstituted phenyl comparator 20 showed good agonist
efficacy (Table 3). 3-Fluoro substitution as in 21 had little to no
effect, whereas 4-fluoro substitution in 22 reduced agonist
potency and efficacy. The 2-methoxy-5-fluorophenyl D-ring
After this exploration of substitution on A-ring and D-ring
mimetic moieties it became apparent that substituents in both
positions have a major impact on CYP3A4 inhibition and
metabolic stability but also hERG channel inhibition and
C
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ACS Medicinal Chemistry Letters
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solubility. However, it was not obvious whether any
combination of residues could achieve the desired balance of
all parameters. From various combination molecules that were
made, the combination of the A-ring mimetics with weak
agonist potency (but the best CYP inhibition profile) with the
most potent (but also most CYP3A4 inhibiting) D-ring
mimetics showed some promise. Specifically, the 5-EWG
substituted 6-azaindoles (such as 18 and 19 in Table 2) as A-
ring mimetics were combined with 2-EWG substituted phenyl
D-ring mimetics (such as 25 and 26 in Table 3).
strictly limited: The methylamide 32 loses all GR activity. The
bis-methylsulfone 33 shows a similar improvement in the CYP
profile but is rapidly metabolized. The sulfonamide 34 loses
additional potency. Next, an additional D-ring substitution was
investigated to fine-tune the profile. An additional 4-fluoro
substituent in 35 maintained the profile of 31, an additional
chloro substitution in 36 increased the maximum efficacy, the
compound lost the dissociated profile (data not shown), and an
additional methyl group in 37 started to erode the metabolic
stability. The size of the A-ring sulfone was varied next. The
ethylsulfones 38 and 39 maintain potency, while with further
increased size, exemplified here by the isopropylsulfone 40 or
the conformationally restrained cyclic sulfone 41, a significant
loss of potency is caused.
The first combination of the methylsulfonyl azaindole with
the 2-carboxamidophenyl D-ring mimetic 31 provided a
breakthrough (Table 4): The compound showed acceptable
Table 4. 5-(Alkylsulfonyl)-6-azaindoles
All compounds in Table 4 show good NR selectivity with PR
and MR IC50 values > 2000 nM and no detectable inhibition at
that highest tested concentration.
Compounds 38 and 39 showed the best overall profile and
were selected for further studies. Both compounds were
resynthesized as pure enantiomers using a combination of the
racemic route described in the Supporting Information and the
previously published general asymmetric synthesis for this
scaffold.22 (R)-38 and (R)-39 were isolated with >99% ee. A
highly optimized, large-scale enantiopure synthesis of (R)-39
was published recently.23 The pure enantiomers showed an
approximately 2-fold improved potency against GR compared
to the racemic mixtures as expected (Table 5). The maximum
transrepression efficacy remained at 87% and 88%, respectively,
which translated into a desirable dissociation profile with
maximum effects at 2 μM of 27% and 33% for the MMTV
reporter gene assay (vs dexamethasone at 100%) and 44% and
39% for osteocalcin production (vs dexamethasone at 100%),
respectively. Both compounds showed significantly reduced
inhibition of CYP activity across isoforms (measured as
inhibition of conversion of reference drug substrates in
microsomes). The metabolic stability of (R)-39 is higher than
for (R)-38; however, their in vivo PK profile is similar with rat
PK half-lives of 5.8 and 4.2 h, respectively. The thermodynamic
equilibrium solubility of the most stable polymorph of the
compounds was still low with 10 and 5 μg/mL, respectively;
however, formulations with acceptable dissolution rates and
preclinical bioavailability were identified for (R)-39. Lastly, the
increased polar surface area of the molecules resulted in
reduced affinity to the hERG ion channel (IC50 > 30 μM).
For unknown reasons, a previously not observed species
selectivity (reduced mouse functional transrepression potency
assessed as inhibition of TNF-stimulated IL-6 production in
mouse RAW cells, (R)-39 IC50 600 nM) of this subseries of
compounds precluded a pharmacological evaluation in standard
preclinical in vivo mouse models. However, the translation of in
vitro dissociation markers to preclinical in vivo parameters in
mouse models had been previously established with tool
compounds from this series and was expected to apply to these
a
IC50 values are the mean of at least two values, each determined from
b
duplicate 11-point concentration response curves. Maximum efficacy
at the highest tested concentration compared to dexamethasone,
defined at 100%; maximum concentration tested is 2 μM. Not
determined.
c
agonist potency and efficacy (IL-6 IC50 80 nM with 86% max.
efficacy) while not showing any CYP3A4 inhibition up to 30
μM and good metabolic stability (27% Qh). It is assumed that
the high polar surface area plays a key role in reducing the
affinity to CYP enzymes as an inhibitor and as a metabolic
substrate beyond what would have been expected if the
properties of the A-ring and D-ring moieties had simply been
additive. Curiously, these compounds with high polar surface
area on both ends of the molecule show somewhat diminished
GR binding (e.g., GR IC50 140 nM vs IL-6 IC50 80 nM for 31).
Available space in the 2-position on the D-ring mimetic is
Table 5. Profile of Enantiopure Preferred Compounds
GR IC50
a
IL-6 IC50 [nM] and MMTV and OC CYP 1A2, 2D6, 2C9, 2C19, HLM (%Qh),
thermodyn. sol pH
6.8 [μg/mL]
patch clamp hERG
channel IC50 (μM)
ab
,
b
compd
[nM]
max. eff. [%]
max. eff.
3A4 IC50 [μM]
rat PK t1/2
(R)-38
(R)-39
95
55
43 (87%)
23 (88%)
27%/44%
33%/39%
>50/>50/12/40/34
>50/41/12/9/8
69/5.8 h
11/4.2 h
10
5
>30
>30
a
b
IC50 values are the mean of at least two values, each determined from duplicate 11-point concentration response curves. Maximum efficacy at the
highest tested concentration compared to dexamethasone, defined at 100%; maximum concentration tested is 2 μM.
D
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optimized compounds.18 (R)-39 was tested in a 9-day type II
collagen-induced arthritis model in rats at 3, 10, and 30 mg/kg
qd po in 30% cremophor to guide human efficacious
concentration projections. Animals treated with the low dose
(3 mg/kg) of (R)-39 had nonsignificant decreases for all
measured histology parameters (ankle inflammation, pannus
formation, cartilage damage, and bone resorption). Pannus and
bone resorption were near significant (25%, p = 0.07). Mid-
dose (10 mg/kg) animals had significantly decreased pannus
and bone resorption (33%) as well as summed scores (27%),
while all parameters were significantly decreased (87−96%) in
the high dose (30 mg/kg) group (data not shown). The ED50
value for the summed scores was 14 mg/kg. No side effect
related parameters were feasible to be evaluated in this shorter
duration model.
glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorti-
coid response element; hERG, human ether-a-go-go related
gene; HLM, human liver microsome; IL-1,2,6, interleukin 1,2,6;
MMTV, mouse mammary tumor virus; MR, mineralocorticoid
receptor; NF-κB, nuclear factor kappa-light-chain-enhancer of
activated B cells; PR, progesterone receptor; Qh, hepatic blood
flow; TA, transactivation; TNF-α, tumor necrosis factor alpha;
TR, transrepression
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EXPERIMENTAL PROCEDURES
Synthesis. All compounds (except 9) described herein have been
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ASSOCIATED CONTENT
* Supporting Information
Assay descriptions, compound purity information, analytical
data for (R)-38 and (R)-39, and synthetic schemes detailing
the preparation of all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel. 203-778-7924. E-mail christian.harcken@boehringer-
ingelheim.com.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
AP-1, Activator protein 1; CYP, cytochrome P450; EWG,
electron-withdrawing group; FBS, fetal bovine serum; GC,
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