Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2010.11.054

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

Full text of DOI:10.1016/j.bmcl.2010.11.054

Bioorganic & Medicinal Chemistry Letters 21 (2011) 137–140
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimisation of a selective non-steroidal glucocorticoid
receptor antagonist
Angus R. Brown a, , Michael Bosies a, Helen Cameron a, John Clark a, Angela Cowley a, Mark Craighead a,
⇑
Moira A. Elmore a, Alistair Firth a, Richard Goodwin a, Susan Goutcher a, Emma Grant a, Morag Grassie a,
Simon J. A. Grove a, Niall M. Hamilton a, Hannah Hampson a, Alison Hillier a, Koc-Kan Ho c, Michael Kiczun a,
Celia Kingsbury c, Steven G. Kultgen c, Peter T. A. Littlewood a, Scott J. Lusher b, Susan MacDonald a,
Lorraine McIntosh a, Theresa McIntyre a, Ashvin Mistry a, J. Richard Morphy a, Olaf Nimz a,
Michael Ohlmeyer c, Jack Pick a, Zoran Rankovic a, Brad Sherborne a, Alasdair Smith a, Michael Speake a,
Gayle Spinks a, Fiona Thomson a, Lynn Watson a, Mark Weston a
a Merck Research Laboratories (MSD), Newhouse, Motherwell, Lanarkshire ML1 5SH, UK
b Merck Research Laboratories (MSD), PO Box 20, Oss 5340 BH, Netherlands
c Pharmacopeia, Inc., PO Box 5350, Princeton, NJ 08543-5350, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR
antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with
a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.
Ó 2010 Published by Elsevier Ltd.
Received 28 October 2010
Revised 9 November 2010
Accepted 9 November 2010
Available online 16 November 2010
Keywords:
Nuclear receptor
Glucocorticoid receptor
GR antagonist
Nuclear receptors are a class of soluble intra-cellular receptor
proteins which regulate the expression of specific genes in almost
all mammalian cells. Binding of the endogenous ligand to the nu-
clear receptor results in a conformational change in the receptor
which allows it to bind directly to specific DNA sequences and reg-
ulate transcription of the adjacent genes.1 Hence, nuclear receptors
and their endogenous ligands play key roles in a range of funda-
mental biological processes and as such are attractive targets for
drug discovery.2
OH
N
O
O
H
O
S
OH
HO
O
N
H
H
O
H
N
O
1
2
The glucocorticoid receptor (GR) is one member of this nuclear
receptor super-family which has proved a fruitful target for drug
discovery, resulting in a number of GR agonists being available as
marketed drugs.3 However, the therapeutic potential of GR antag-
onists remains less well developed. This is despite strong evidence
linking the ability to block the action of the endogenous glucocor-
ticoid steroid hormone cortisol 1 (Fig. 1), and the effect of this
blockade on the pathology of disease states characterised by
elevated cortisol levels (e.g. Cushing’s syndrome,4 hypertension,5
diabetes,6 glaucoma7 and depression7,8).
Figure 1. Structures of cortisol 1 and the HTS derived hit compound 2.
The non-selective glucocorticoid receptor/progesterone recep-
tor (PR) antagonist Mifepristone (RU-486) has been one of the most
studied GR antagonists discovered to date, but its clinical use con-
tinues to be compromised largely due to the poly-pharmacology
associated with is PR antagonist activity.4,9 We were therefore
interested in identifying more selective GR antagonists. Our main
rational from the outset was that the lack of selectivity of the ste-
roid derived ligands may be due, in part, to the steroid scaffold it-
self. Hence our initial goal was to identify a non-steroidal GR
antagonist, of which several were known in the literature, and then
⇑
Corresponding author. Tel.: +44 1698 736163; fax: +44 1698 736187.
E-mail address: angus.brown@merck.com (A.R. Brown).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.11.054

138
A. R. Brown et al. / Bioorg. Med. Chem. Lett. 21 (2011) 137–140
determine if such a ligand would prove easier to optimise in terms
of selectivity over related nuclear receptors.
were then purified by RP-HPLC, fully characterised and screened
for activity at GR and PR. This array of 160 analogues gave 93 ac-
tives (>50% inhibition, GR single point screening), 42 of which were
Screening of 3.87 million compounds by Pharmacopeia, using a
human GR fluorescence polarisation binding assay,10 resulted in
the identification of the heterobiaryl sulfonamide 2 (Fig. 1) as a po-
tent GR antagonist (GR binding affinity10 pEC50 8.4; GR antago-
nism11 pEC50 7.3).
active (<1 l
V) in our in vitro functional assay of GR antagonism.11
Alkyl sulfonamide derivatives were in general not tolerated (data
not shown), although a wide range of heteroaryl and aryl deriva-
tives such as 13 showed good potency at GR and retained selectiv-
ity over PR. Variation of the sulfonamide portion highlighted that
the N–H was essential for potency and subsequent attempts to re-
place the sulfonamide functionality with bioisosteric replacements
were not tolerated (data not shown).
Introduction of a chiral methyl group 14 delivered our first
analogue with greater than 30 fold selectivity. The (R)-enantiomer
displayed greater potency and selectivity compared to the corre-
sponding (S)-enantiomer 15 and this preference for the (R)-stereo-
chemistry has proved consistent for all subsequent analogues
prepared to date. Chiral carbocyclic analogues were generally
prepared via a ligand-free aqueous Suzuki coupling, followed by
catch-and-release purification protocol (Scheme 3).12
Further in vitro profiling of 2 revealed poor selectivity at the
closely related progesterone receptor (PR) (PR bind pEC50 8.1; fold
selectivity GR vs PR ꢀ2).10 We therefore initiated a program aimed
at optimising the in vitro profile of this series with a focus on
improving selectivity over PR as our initial optimisation goal. The
compounds detailed in Table 1 were prepared according to the
general Schemes 1–4. Rapid analogue synthesis and in vitro profil-
ing quickly allowed us to build up a picture of the key SAR for this
novel heterobiaryl sulfonamide series exemplified by the progeni-
tor compound 2. Deletion of the terminal heteroaryl ring or varia-
tion of the biaryl connectivity to ortho or meta substitution was
not tolerated, and the benzylic linker between biaryl unit and sul-
fonamide functional group was optimal, with longer or shorter
chains resulting in loss of GR activity (data not shown).
Synthesis of 2 involved a two step sequence from commercially
available starting materials and therefore allowed the rapid syn-
thesis of a diverse set of close analogues. 4-Bromobenzylamine 3
was reacted with dimethylisoxazole sulfonyl chloride in the pres-
ence of base to provide the sulfonamide intermediate 4. We then
attempted to prepare a small one dimensional array by reacting
4 under standard Suzuki coupling conditions with 80 diverse
aryl/heteroaryl boronic acid derivatives (Scheme 1). After exten-
sive purification, the majority of compounds were isolated, albeit
in low yield in many cases. Single point screening identified 35 ac-
Whilst we had now demonstrated that good selectivity over PR
was achievable within this chemical series, the majority of selec-
tive analogues also suffered from low aqueous solubility (<1 mg/
L), poor microsome stability (Human and Rat intrinsic clearance,
CLi > 270 lL/min/mg protein) and low oral bioavailability (14, rat
F = 1%). Initial efforts at improving metabolic stability focused on
changes which would reduce the lipophilicity of the series (via
discrete introduction of heteroatoms), whilst at the same time
retaining the selectivity conferring features responsible for
selectivity over PR.
In general, we found that heterocycles were tolerated in the
terminal portion of the biaryl motif. In particular 3-pyridyl ana-
logues such as 16 were almost equipotent with the progenitor
compound 10. The microsome clearance of 16 was found to be high
tives (>50% binding inhibition at 10 lM) and these were profiled
further. The phenyl analogue 9 showed weak GR binding, but intro-
duction of an ortho substituent (Table 1, 10–12) dramatically
increased both the binding affinity for GR and also resulted in
improved selectivity via loss of potency at PR.
We then initiated a further round of synthesis to investigate the
SAR of the sulfonamide region of compound 2. We prepared the
parent heterobiaryl benzylamine 5 (Scheme 2) along with three
other biaryl cores from the first array which gave improved selec-
tivity over PR (cores 6–8). With these four biaryl benzylamines in
hand we reacted each of these with 40 diverse sulfonyl chlorides
in the presence of base (Scheme 2). The resulting crude products
(Human intrinsic clearance, CLi = 237
lL/min/mg protein; Rat
intrinsic clearance, CLi = 251 L/min/mg protein), with the major
l
metabolite identified as the inactive O-dealkylated pyridone
derivative (data not shown). Attempts to block metabolism via
conversion to the corresponding difluoromethoxy derivative 17
gave a modest improvement in rodent microsome stability (intrin-
sic clearance, CLi = 79
microsome stability was not improved (intrinsic clearance,
CLi > 270 L/min/mg protein). Introduction of a nitrile group para
lL/min/mg protein), but measured human
l
to the site of metabolism 18 gave a dramatic improvement in
Table 1
Summary of the structure–activity relationships of the biaryl sulfonamide chemotype
R3
O
R2
O
S
N
H
R1
Compd
R1
R2
R3
Stereo
chemistry
C log P
GR10 pEC50
PR10 pEC50
Binding selectivity
PR/GR
2
9
2,5-Dimethylisoxazole
Phenyl
2-Methoxyphenyl
2-Methoxy-5-fluorophenyl
Naphthalene
2-Methoxy-5-fluorophenyl
2-Methoxy-5-fluorophenyl
2-Methoxy-5-fluorophenyl
2-Methoxy-3-pyridyl
2-Difluoromethoxy-3-pyridyl
2-Methoxy-5-cyano-3-pyridyl
2-Methoxy-5-fluorophenyl
2-Methoxy-3-pyridyl
2-Methoxy-5-cyano-3-pyridyl
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dichlorophenyl
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
2,5-Dimethylisoxazole
1-Methyl-3-(trifluoromethyl)-1H-pyrazole
1-Methyl-3-(trifluoromethyl)-1H-pyrazole
1-Methyl-3-(trifluoromethyl)-1H-pyrazole
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
—
—
—
—
—
—
R
S
R
R
R
2.15
4.23
3.59
3.87
5.41
5.75
4.18
4.18
3.30
3.75
2.88
3.79
2.91
2.48
8.4
6.0
7.5
7.9
8.1
8.7
8.3
7.6
7.6
8.0
8.2
8.4
7.9
7.9
8.1
<5
2
—
7
10
23
29
38
9
34
52
12
198
>800
>800
10
11
12
13
14
15
16
17
18
19
20
21
6.7
6.9
6.7
7.3
6.7
6.6
6.1
6.2
7.1
6.1
<5
R
R
R
<5

A. R. Brown et al. / Bioorg. Med. Chem. Lett. 21 (2011) 137–140
139
N
N
O
O
O
S
O
NH2
a
S
b
O
N
H
O
N
H
Br
R1
Br
3
4
80 analogues
Scheme 1. Reagents and conditions: (a) 3,5-dimethylisoxazole-4-sulfonyl chloride, DCM, DIEA, rt, quantitative; (b) boronic acid, Pd(PPh3)4, K2CO3, DMF, 80 °C.
O
S
R2
O
Table 2
Potency, selectivity and pharmacokinetic data for compound 21
NH2
N
H
a
Parameter
Value
R1
R1
GR; PR (binding potency pEC50
Binding selectivity PR/GR
Solubility in PBS, pH 7.4 (mg/L)
)
7.9; <5
>800
28.9
<12
F
Human microsome intrinsic clearance (lL/min/mg protein)
where R1
=
Rat microsome intrinsic clearance (
GR; MR (antagonism11 pEC50
AUCpo (ng/mL h)
lL/min/mg protein)
<12
O
)
7; <5
8143
42
O
O
N
0–7.5 h
Rat F (%), Wistar, iv 2 mg/kg; po 10 mg/kg
CLp (mL/min/kg)
5
6
7
8
9.8
Vss (L/kg)
0.7
Scheme 2. Reagents and conditions: (a) R2 sulfonyl chloride, DCM/DIEA, rt.
clearance (Human intrinsic clearance, CLi = 36
l
L/min/mg protein;
in situ generation of aryl triflates14 followed by Suzuki coupling
under microwave mediated conditions (Scheme 4)13 gave im-
proved yields in our hands. In addition, use of starting phenol 22
also allowed easy access to homochiral intermediates 24 via
asymmetric synthesis of amines from ketone 23 using the method
reported by Ellman.15
In summary, using an HTS campaign we have identified a novel,
non-steroidal, non-selective GR antagonist. We have successfully
optimised this heterobiaryl sulfonamide series to deliver a very
selective antagonist of the glucocorticoid receptor, with excellent
in vitro stability and acceptable oral bioavailability. Compound
21 was also shown to be efficacious in in vitro models of glucocor-
ticoid receptor antagonism and will be the subject of a separate
publication.
Rat intrinsic clearance, CLi = 19 L/min/mg protein), although this
l
change had an adverse effect on selectivity over PR. After re-visit-
ing further heterocyclic variants of the sulfonamide region we
were able to identify the 1-methyl-3-(trifluoromethyl)-1H-pyra-
zole sulfonamide derivative 19 with much improved selectivity
which we rationalised to be due, in part, to this substituted hetero-
aryl ring (Table 1). Combining this new heterocyclic R2 portion
with the terminal biaryl 2-methoxy-3-pyridyl unit furnished ana-
logue 20 which, although slightly less potent at GR and rapidly
cleared, proved to be very selective over PR. Synthesis of the 5-ni-
trile analogue 21 (Scheme 4)13 provided a potent, selective GR
antagonist with an attractive in vivo PK profile (Table 2). For intro-
ducing heterocycles as the terminal portion of the biaryl motif,
CH3
H
CH3
H
CH3
O
S
R2
O
H
a
NH2
b
NH2
N
H
Br
R1
R1
Scheme 3. Reagents and conditions: (a) aryl boronic acid, Pd(OAc)2, water, microwave irradiation, 200 °C, 5 min followed by SCX purification; (b) aryl/heteroaryl sulfonyl
chloride, DCM, DIEA, rt.12
F
F
CH3
CH3
N
O
H
F
CH3
N
O
O
S
NH2
N
CH3
c
a
b
H
N
N
H
O
HO
N
24
23
N
O
22
N
O
21
N
O
Scheme 4. Reagents and conditions: (a) (i) N-phenyl-bis-(trifluoromethane-sulfonimide), K2CO3, THF, microwave irradiation 120 °C, 6 min; (ii) boronic acid, Pd(PPh3)4
(2 mol % w/w), NMP, microwave irradiation 120 °C, 10 min; (b) (i) R-(+)-tert-butanesulfinamide, DCM, titanium tetraethoxide, microwave irradiation 120 °C for 10 min; (ii)
NaBH4, ‘wet’ THF, MeOH; (c) 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-sulfonyl chloride, triethylamine, DCM, rt.13

140
A. R. Brown et al. / Bioorg. Med. Chem. Lett. 21 (2011) 137–140
solution of (R)-1-(50-fluoro-20-methoxy-biphenyl-4-yl)-ethylamine (20 mg,
References and notes
0.082 mmol) in dichloromethane (1 ml) and the resulting solution shaken at
room temperature overnight. Purification by preparative LCMS and removal of
solvent under reduced pressure gave 19 (8.5 mg, 40%): 1H NMR (400 MHz,
CDCl3): d 7.40, 7.16 (a/b, 4H), 7.05–6.88 (m, 3H), 4.89 (m, 1H), 4.60 (t, 1H), 3.80
(s, 3H), 3.72 (s, 3H), 1.52 (d, 3H) ppm; MS (ESI) m/z: 480 [M+Na]+.
1. Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz, G.; Umesono,
K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83,
835.
2. Schweitzer, A.; Knauer, S. K.; Stauber, R. H. Expert Opin. Ther. Patents 2008, 18,
861.
3. http://www.drugs.com/drug-class/glucocorticoids.html.
4. Schteingart, D. E. Expert Opin. Emerg. Drugs 2009, 14, 661.
5. Hu, X.; Bolten, C. W. Drug Dev. Res. 2006, 67, 871.
6. Tomlinson, J. W.; Stewart, P. M. Best. Pract. Res. Clin. Endocrinol. Metab. 2007, 21,
607.
13. The phenol 22 (2.0 mmol), N-phenyl-bis-(trifluoromethane-sulfonimide)
(2.0 mmol, 710 mg), K2CO3 (6.0 mmol, 830 mg), and 3.0 ml THF were heated
in the microwave at 120 °C for 6 min. Boronic acid (2.0 mmol), Pd(PPh3)4
(2 mol % w/w) and NMP (1 ml) added the vessel resealed and heated for a
further 10 min at 120 °C. The crude reaction mixture was partitioned between
ethyl acetate (50 ml) and saturated aqueous NaHCO3 (100 ml) washed with
saturated aqueous NaHCO3 (100 ml ꢁ 3), dried (MgSO4) and evaporated to give
the essentially pure biaryl ketone 23 in quantitative yield. The crude biaryl
ketone (2 mmol) and R-(+)-tert-butanesulfinamide (2 equiv) was dissolved in
DCM (3 ml) and to this was added titanium tetraethoxide (purity: 85–90%,
0.46 ml, 2 mmol). The reaction was then sealed and heated in the microwave at
120 °C for 10 min. The reaction was then cooled (to reduce internal pressure!),
decapped and poured into a suspension of NaBH4 (4 equiv) in wet THF (50 ml),
MeOH (20 ml) was then added. The reaction was almost instantaneous and the
mixture was then poured into brine (100 ml) with rapid stirring. The resulting
suspension was then filtered through Celite, washed with ethyl acetate and the
filtrate separated. The combined organic layers were then dried (MgSO4) and
evaporated to yield the crude product. The pure (R,R)-diastereomer was then
isolated by chromatography (SiO2, ethyl acetate) in 40–50% overall yield based
on the original amount of phenol. (S)-N-((R)-1-(4-(5-Cyano-2-methoxypyridin-
3-yl)phenyl)ethyl)-2-methylpropane-2-sulfinamide (22.60 mmol, 8.08 g) was
dissolved in methanol (200 ml) and hydrogen chloride (67.8 mmol, 33.9 ml)
(2 M in ether) added. Stirred at rt for 1 h then solvent removed. Solid residue
dissolved in methanol and passed through 3 ꢁ 20 g SCX columns. Elution with
2 M ammonia in methanol followed by evaporation of volatiles gave 24 (5.43 g,
95%) as an oil. To an ice-cold solution of 24 (19.19 mmol, 4.86 g) in DCM
(90 ml) was added triethylamine (67.2 mmol, 9.36 ml, 6.80 g) in one portion,
7. Clark, R. D. Curr. Top. Med. Chem. 2008, 8, 813.
8. Fitzsimons, C. P.; van Hooijdonk, L. W. A.; Morrow, J. A.; Peeters, B. W. M. M.;
Hamilton, N.; Craighead, M.; Vreugdenhil, E. Mini-Rev. Med. Chem. 2009, 9, 249.
9. Johanssen, S.; Allolio, B. Eur. J. Endocrinol. 2007, 157, 561.
10. Binding affinity of GR antagonists for recombinant human GR and PR:
compound affinity was determined via competition ligand binding
experiments using GR and PR fluorescence polarisation (FP) binding assay
kits (Panvera). This assay utilises the binding of a proprietary fluorescent
ligand to partially purified recombinant baculovirus expressed human
receptor. EC50 values were generated by analysis of the displacement curves
by
a minimum sum of squares method. All data are the means of 3–4
independent experiments.
11. The effect of GR antagonists on GR, PR and MR mediated functional responses
in CHO cells expressing recombinant human receptor: CHO cells were stably
transfected with human GR, PR or chimeric MR/GR (MR ligand binding domain,
GR DNA binding domain) and the MMTV-LUC luciferase reporter plasmid.
Antagonism of GR functional responses by test compounds was determined via
inhibition of cortisol (20 nM)-stimulated luciferase activity. Antagonism of PR
was determined by inhibition of progesterone (5 nM)-stimulated luciferase
activity. Antagonism of MR was determined by inhibition of aldosterone
(1 nM)-stimulated luciferase activity. EC50 values were generated by analysis
of concentration–response curves by a minimum sum of squares method. All
data are the means of 3–4 independent experiments.
12. Palladium acetate (56 mg, 0.25 mmol) was added to a mixture of 5-fluoro-2-
methoxyphenylboronic acid (850 mg, 5.0 mmol) and (R)-1-(4-bromo-
phenyl)ethylamine (500 mg, 2.5 mmol) in water (20 ml). This mixture was
heated for 5 min at 200 °C in a Smithcreator microwave oven and then diluted
with methanol (200 ml). The mixture was purified on a SCX column (20 g)
using 2 M ammonia in methanol to elute the intermediate amine. Evaporation
of solvents under reduced pressure gave (R)-1-(50-fluoro-20-methoxy-
followed over 15 min by
a solution of 1-methyl-3-(trifluoromethyl)-1H-
pyrazole-4-sulfonyl chloride (6.14 g) in DCM (30 ml). The reaction was
stirred at ambient temperature for 6.5 h then washed with 2 N HCl (ꢁ2),
Na2CO3, brine and dried over Na2SO4. Evaporation of volatiles and purification
over silica (DCM/MeOH 99.5:0.5 then 99:1) gave a foam (8.8 g) that was
recrystallised from ether/heptane to yield 21 as a colourless solid, (8.45 g,
95%): 1H NMR (400 MHz, DMSO): d 8.70 (s, 1H), 8.40 (s, 1H), 8.21 (s, 1H), 8.17
(s, 1H), 7.51, 7.35 (a/b, 4H), 4.45 (m, 1H), 3.99 (s, 3H), 3.83 (s, 3H), 1.36 (d, 3H)
ppm; MS (ESI) m/z: 466 [M+H]+.
14. Bengtson, A.; Hallberg, A.; Larhed, M. Org. Lett. 2002, 4, 1231.
15. Robak, M.-A. T.; Herbage, M. A.; Ellman, J. A. Chem. Rev. 2010, 110, 3600.
biphenyl-4-yl)-ethylamine as
Triethylamine (41.1 l, 0.295 mmol) and 1-methyl-3-(trifluoromethyl)-1H-
pyrazole-4-sulfonyl chloride (24.5 mg, 0.0984 mmol) were added to
a
gum (580 mg, 2.37 mmol, 96.6%).
l
a