Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity

Article abstract of DOI:10.1002/cmdc.201600529

The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pK_i=6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pK_i=7.3. Two protein–ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.

Full text of DOI:10.1002/cmdc.201600529

Accepted Article
Title: Structure-based drug design of mineralocorticoid receptor
antagonists to explore oxosteroid receptor selectivity
Authors: Anneli Nordqvist, Gavin O’Mahony, Maria Fridén-Saxin,
Marlene Fredenwall, Anders Hogner, Kenneth L. Granberg,
Anna Aagaard, Stefan Bäckström, Anders Gunnarsson, Tim
Kaminski, Yafeng Xue, Anita Dellsén, Eva Hansson, Pia
Hansson, Ida Ivarsson, Ulla Karlsson, Krister Bamberg, Majlis
Hermansson, Jennie Georgsson, Bo Lindmark, and Karl
Edman
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201600529
Link to VoR: http://dx.doi.org/10.1002/cmdc.201600529
A Journal of
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Structure–based drug design of mineralocorticoid receptor
antagonists to explore oxosteroid receptor selectivity
Anneli Nordqvist,*[a] Gavin O’Mahony,[a] Maria Fridén-Saxin,[a] Marlene Fredenwall,[a] Anders Hogner,[a]
Kenneth L. Granberg,[a] Anna Aagaard,[b] Stefan Bäckström,[b] Anders Gunnarsson,[b] Tim Kaminski,[b]
Yafeng Xue,[b] Anita Dellsén,[b] Eva Hansson,[b] Pia Hansson,[b] Ida Ivarsson,[b] Ulla Karlsson,[b] Krister
Bamberg,[a] Majlis Hermansson,[a] Jennie Georgsson,[a] Bo Lindmark[a] and Karl Edman[b]
[a]
Dr. A. Nordqvist, Dr. G. O’Mahony, Dr. M. Fridén-Saxin, M. Fredenwall, Dr. A. Hogner, Dr. K.L. Granberg, Dr. K. Bamberg, M. Hermansson, Dr. J.
Georgsson and B. Lindmark,
Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit,
AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden.
E-mail: anneli.nordqvist@astrazeneca.com
[b]
Dr. A. Aagaard, Dr. S. Bäckström, Dr. A. Gunnarsson, Dr. T. Kaminski, Dr. Y. Xue, A. Dellsén, E. Hansson, P. Hansson, I. Ivarsson, U. Karlsson, and Dr. K.
Edman,
Discovery Sciences, Innovative Medicines and Early Development Biotech Unit,
AstraZeneca, Pepparedsleden 1, 431 83 Mölndal, Sweden.
Supporting information for this article is given via a link at the end of the document.
Abstract: The mineralocorticoid receptor (MR) is a nuclear hormone
receptor involved in regulation of body fluid and electrolyte
homeostasis. In this study we explore selectivity triggers for a series
of non-steroidal MR antagonists to improve selectivity over other
members of the oxosteroid receptor family. A biaryl sulfonamide
compound was identified in a high-throughput screening (HTS)
campaign. The compound bound to MR with a pKi=6.6, but displayed
poor selectivity over the glucocorticoid receptor (GR) and the
progesterone receptor (PR). Following X-ray crystallography of MR in
complex with the HTS hit, a compound library was designed that
explored an induced fit hypothesis that required movement of the
Met852 side-chain. An improvement in MR selectivity of 11–79-fold
over PR and 23–234-fold over GR was obtained. Given the U-shaped
binding conformation, macrocyclizations were explored yielding a
macrocycle that bound to MR with a pKi=7.3. Two protein–ligand X-
ray structures were determined confirming the hypothesized binding
mode for the designed compounds.
However, the use of both spironolactone and eplerenone is
contraindicated in diabetic patients with CKD due to the risk of
hyperkalemia. In addition, the use of spironolactone is further
hampered by sexual side effects that are caused by poor
selectivity over AR and PR.[10] Thus, there is a great interest in the
discovery and development of novel, selective MR antagonists
with improved efficacies and side effect profiles to treat CKD in
patients. A number of non-steroidal MR antagonists have been
reported in the literature[11] of which finerenone[12] has recently
completed phase II clinical trials (ARTS-HF and ARTS-DN) and
Bayer AG is now conducting two phase III trials in diabetic kidney
disease (FIGARO-DKD, FIDELIO-DKD).
We identified a biaryl sulfonamide compound (1) in a high-
throughput screening (HTS) campaign using an MR binding
assay. Subsequent testing in a functional assay demonstrated
that the compound was a full MR antagonist.
Introduction
The glucocorticoid receptor (GR), the mineralocorticoid
receptor (MR), the progesterone receptor (PR) and the androgen
receptor (AR) form the oxosteroid receptor family (NR3C1-4).[1]
They are ligand activated transcription factors with crucial roles in
a broad range of fundamental biological functions, ranging from
reproduction and development to cognition and stress response.
The challenge in generating subtype selective ligands lies in the
fact that the steroid receptors all bind cholesterol derivatives and
several structural features of the ligand binding pockets are
conserved across the receptor family. Therefore, designing a
selective compound requires detailed understanding of the
structure–activity relationship (SAR) across the receptor family.
MR is activated by the hormone aldosterone and plays a
crucial role in body fluid and electrolyte homeostasis. There are
currently two MR antagonists, eplerenone and spironolactone, in
clinical use for treatment of high blood pressure and heart
failure.[2-5] More recently, benefits of MR blockade have been
explored in chronic kidney disease (CKD) where spironolactone
on top of antihypertensive medication reduced proteinuria.[7-9]
Figure 1. HTS hit (1, magenta) and the design idea to expand the biaryl
sulfonamides towards the pocket that can be created by movement of the
Met852 side chain, as illustrated in the overlay of two complex structures
(slate blue: Met852 in HTS hit structure or cyan: Met852 in 3WFG[6]). This
pocket accommodates a phenyl substituent of the benzoxazinone ligand
(turquoise) in the complex structure by Hasui et al.[6] Lower right displays the
design idea to cyclize compound 1 based on the U-shaped binding
conformation.
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However, this class of ligands have reported activity for several
oxosteroid receptors.[13-16] By combining SAR exploration and X-
ray structure determination we exploited differences in the ligand
binding pockets to improve selectivity towards MR. In addition,
careful analysis of the compound binding pose inspired the design
of the first macrocyclic oxosteroid receptor antagonist. Finally,
using a nuclear translocation assay we discovered that these
compounds antagonize MR by blocking aldosterone-mediated
receptor translocation into the nucleus. The distinct profile of
these compounds may lead to them finding utility as tools to clarify
genomic versus non-genomic MR effects.[17]
of the ligand binding pocket, compound 1 interacts directly with
OD11 of Asn770 through a hydrogen bond with the sulfonamide
NH. In addition, one of the ligand’s sulfonamide oxygen atoms
interacts indirectly with both Asn770 ND21 and Thr945 via a water
molecule. Polar interactions with Asn770 are a key motif among
many MR antagonists and computational investigations[20] of the
effect on binding affinity by different hydrogen-bonding core
fragments has been performed. Beyond the sulfonamide,
compound 1 folds back upon itself to form an intramolecular
packing interaction between the two phenyl moieties.
To explore the structural drivers of the functional profile of
compound 1, we superimposed this structure onto the structure of
MR in complex with aldosterone[19] (Figure 2B). This comparison
highlights that in contrast to the isoxazole of compound 1, the 3-
keto moiety of aldosterone is within direct hydrogen bonding
distance to both Arg817 and Gln776 (3.1 Å and 2.9 Å,
respectively). In addition, in the aldosterone complex structure,
the water-mediated contacts to ND2 of Asn770 and to Thr945 by
compound 1 are replaced by direct contacts through the C19
hydroxyl group of aldosterone. As such, compound 1 appears to
have weaker interactions to both the gatekeeper residues in
helices three and five and to Asn770 in helix three, both of which
are important functional triggers for receptor activation.[19,21]
Looking across the steroid receptor family, the structure of
compound 1 superimposes well on the structure of closely related
isoxazole compound (2a) in PR (Figure 2C). Interestingly, 2a is a
partial agonist at PR,[14] which emphasizes how small changes in
amino acid sequence lead to fundamental differences in the
functional response of closely related receptors to a given ligand.
A key distinction between the two receptors is that Ser810 in MR
is replaced by a methionine (Met759) in PR. This change
increases the interaction surface available between helices three
and five in PR, and which is a strong agonist driver across the
steroid receptor family.[22] As such, the serine to methionine
change can potentially explain why an MR antagonist can be a
PR partial agonist. Indeed, the naturally occurring human MR
Results and Discussion
Hit identification
Compound 1 (Figure 1) was identified as an MR ligand in an
HTS campaign using a scintillation proximity assay (SPA) with
tritiated aldosterone. Single concentration response data of MR
binding was followed up in ten point concentration response to
confirm binding affinity. Functional activity as an MR antagonist
was determined using a reporter gene assay with a GAL4 DNA
binding domain (DBD)-MR ligand binding domain (LBD) fusion
protein as secondary orthogonal assay.[18] The HTS hit (1) bound
to MR with an affinity of pKi=6.6 and had a moderate lipophilic
ligand efficiency (LLE=pKi-logD7.4)of 3.4 (Table 1). The functional
activity of pIC50=5.9 in the reporter gene assay was in a similar
range as for eplerenone (pIC50=6.1, Table 1). However compound
1 displayed a poor selectivity profile with a higher binding affinity
to both PR (pKi=6.9) and GR (pKi=7.2, Table 2). These results are
in agreement with previous published data of this structure class
as MR, GR and PR ligands.[13-16]
To build a molecular understanding of how the biaryl
sulfonamides drive their functional response and how to optimize
both potency and selectivity further, we determined the X-ray
structure of the MR LBD in complex with compound 1 (Figure 2A,
Table 3). The structure revealed that the isoxazole binds near the
polar gatekeeper residues Arg817 and Gln776. At the other end
Ser810Leu mutation converts progesterone from
a partial
antagonist to a full agonist at MR and as a result this mutation
exacerbates hypertension in pregnant women.[23]
Figure 2. The structure of MR LBD (blue) in complex with compound 1 (magenta). Selected amino acid side chains are shown in orange and putative hydrogen
bonds are shown as dotted lines. A) Refined 2mFo-DFc electron density. Ser811 is observed in three distinct conformations. B) Compound 1 superimposed on the
structure of MR LBD (amino acid side chains in light green) in complex with aldosterone[19] (light green). C) Compound 1 superimposed on the structure of PR[14]
(amino acid side chains in light blue) in complex with isoxazole 2a (light blue).
1 Atom names are assigned according to PDB convention.
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Table 1. Binding affinity, functional activity and LLE of the profiled MR antagonists.
Compound
R1
R2
R3
pIC50
RepG hMR[a]
pKi
hMR[b]
LogD7.4
LLE[c]
1
CH3
-
CH3
-
phenyl
5.9 (122%)
6.6±0.04
3.2
0.8
2.5
2.5
2.8
-
3.4
6.1
5.1
4.1
4.7
-
Eplerenone
2a[14]
2b
-
6.1±0.1 (107%) 6.9±0.1
6.9±0.3 (110%) 7.7±0.1
5.9±0.1 (96%) 6.7±0.1
6.7±0.2 (91%) 7.5±0.04d
CH3
CH3
CH3
H
CH3
CH3
CH3
H
5-chloro-1,3-dimethyl-pyrazol-4-yl
1-methyl-cyclopropyl
3,5-dimethyl-isoxazol-4-yl
5-chloro-1,3-dimethyl-pyrazol-4-yl
5-chloro-1,3-dimethyl-pyrazol-4-yl
2c[13]
8a
<5.0
5.5±0.1
13a
CN
CH3
6.2±0.3 (107%) 7.4±0.2
6.8±0.2 (110%) 7.9±0.2
6.0±0.1 (107%)d 7.0±0.1
6.6±0.2 (109%) 7.3±0.4
2.6
4.9
1-(difluoromethyl)-3,5-dimethyl-1H-
pyrazol-4-yl
13b
19a
19b
CN
CH3
CN
CN
2.9
2.6
3.0
5.0
4.4
4.2
CH3
CH3
5-chloro-1,3-dimethyl-pyrazol-4-yl
1-(difluoromethyl)-3,5-dimethyl-1H-
pyrazol-4-yl
25a
25b
25c
25d
25e
25f
3-chloro-phenoxymethyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
6.1±0.1 (116%) 7.5±0.3
6.7±0.1 (118%) 7.8±0.3
6.7±0.1 (104%) 8.2±0.1
6.3±0.1 (119%) 7.8±0.3d
6.5±0.2 (109%) 7.7±0.3d
6.5±0.1 (120%) 7.2±0.2
6.3±0.1 (127%) 7.3±0.1
6.3±0.1 (114%) 7.2±0.2d
6.7±0.1 (108%) 7.2±0.2
7.0±0.1 (107%) 7.7±0.1
>2.9
>3.0
3.8
<4.6
<4.8
4.4
4.0
<3.8
-
phenoxymethyl
3,5-difluoro-phenoxymethyl
2,3-difluoro-phenoxymethyl
4-methyl-phenoxymethyl
4-chloro-3-fluoro-phenoxymethyl
3-fluoro-5-methyl-phenoxymethyl
2-fluoro-5-methyl-phenoxymethyl
3-methoxy-phenoxymethyl
2-naphthyloxymethyl
3.8
>3.9
-
25g
25h
25i
>3.5
>3.9
3.8
<3.8
<3.3
3.5
4.1
-
25j
3.6
25k
25l
3-acetamidophenoxymethyl
4-sulfamoylphenoxymethyl
NAd,e
NAd,e
<5.0
<5.0
3.0
2.2
-
([1-methyl-1H-imidazol-2-
yl]methoxy)methyl
25m
CH3
CH3
1-methyl-cyclopropyl
NAd,e
NAd,e
<5.0
<5.0
1.5
-
25n
25o
30
(oxetan-3-yloxy)methyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1-methyl-cyclopropyl
1.8
2.0
>3
-
([1-fluorocyclobutyl]methoxy)-methyl CH3
benzyl CH3
6.2±0.1 (104%) 7.0±0.1
6.4±0.1 (123%) 7.0±0.2
5.0
<4.0
[a] Human MR reporter gene pIC50 is reported as mean±SD (n ≥ 3, unless noted). Efficacy in percent is reported in parenthesis. [b] Human MR binding affinity pKi
is reported as mean±SD (n ≥ 3, unless noted). [c] LLE=pKi-logD7.4, [d] n=2, [e] NA=not active
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3,5-dimethylpyrazole
chlorodifluoroacetic acid and Cs2CO3, to install the desired
pyrazole difluoromethyl substituent.
intermediate,
using
sodium
R2=
2a
2b
2c
a
c
The 3-cyanoisoxazole regioisomers were prepared in a
largely similar fashion, but with the functional group conversions
in a different order.[27] Reaction of 9 with hydroxylamine at 75°C
afforded 3-carboxyisoxazole 14. Conversion of ester 14 to nitrile
16, via the corresponding primary amide 15,[28] was performed
before introduction of the 4-aryl moiety. Iodination of 16 with NIS
followed by Suzuki cross-coupling with an N-BOC-protected
boronic acid afforded biaryl 17. Deprotection under standard
conditions followed by reaction with the requisite sulfonyl chloride
afforded sulfonamide 19a. The difluoromethyl moiety of 19b was
installed on the N-unsubstituted pyrazole as for 13b, using
chlorodifluoroacetic acid and Cs2CO3 in DMF.
R1=BOC
R1=H
-
2a 2c
3
4
5
b
Scheme 1. Synthesis varying the sulfonamide substituent. a) 4-Bromo-3,5-
dimethylisoxazole, PEPPSI-IPr, KOH, 1,4-dioxane, 48 h, 65°C, (67%); b)
HCl, MeOH, 48 h, rt, (93%); c) RSO2Cl, NEt3, CH2Cl2, overnight, rt, (58–
64%).
Table 2. Binding affinity for GR and PR and selectivity ratio for MR.
R=
Compound
pKi
pKi
Selectivity
Selectivity
8a
8b
8c
8d
8e
b
a
hGR[a]
hPR[b]
hGR/hMR[c]
hPR/hMR[d]
1
7.2±0.3
5.5±0.2
7.9±0.3
6.3±0.2
8.1±0.3
7.3±0.1
7.2±0.1
7.1±0.1
6.8±0.4
6.3±0.1
6.3±0.3
6.3±0.04
6.2±0.1
6.3±0.2
6.3±0.2
6.5±0.1
6.3±0.1
6.1±0.1
6.8±0.2
6.0±0.1
6.4±0.2
6.5±0.1
7.3±0.3
6.9±0.1
4.7±0.2
7.2±0.1
6.5±0.1
7.2±0.1
6.8±0.1
7.0±0.1
6.5±0.1
6.9±0.4
6.10.3
0.29
25
0.52
160
2.6
1.3
2.4
4.0
8.7
3.3
2.7
27
Eplerenone
2a[14]
2b
0.52
2.1
0.26
1.5
5.1
0.89
3.1
14
6
7
8a-8e
2c[13]
13a
13b
19a
19b
25a
25b
25c
25d
25e
25f
Scheme 2. Synthesis varying the isoxazole part. a) 5-Chloro-1,3-dimethyl-
1H-pyrazole-4-sulfonyl chloride, Et3N, CH2Cl2, 12 h, 25°C, (92%); RB(OH)2,
K3PO4, Pd(dppf)Cl2, THF/water, 16 h, 80°C, (10–74%).
Chemistry
The detailed SAR of the sulfonamide aryl group was
investigated utilizing parallel chemistry (Scheme 1). Suzuki cross-
coupling[24] of the BOC-protected [4-(aminomethyl)phenyl]boronic
acid (3) and 4-bromo-3,5-dimethylisoxazole with subsequent
removal of the BOC-group yielded benzylamine 5, which was
further reacted with selected sulfonyl chlorides to form biaryl
sulfonamides 2a–2c in 58–64% yield.
6.9±0.2
6.7±0.2
6.7±0.2
6.9±0.1
6.4±0.2
6.2±0.3
6.1±0.3
6.1±0.3
6.0±0.2
6.3±0.3
6.8±0.1
6.4±0.04
7.5±0.01
34
7.4
30
68
42
14
23
5.8
6.5
14
Variation and replacement of the isoxazole moiety was carried
out as shown in Scheme 2. Reaction of 4-iodobenzylamine (6)
with the requisite sulfonyl chloride afforded iodo-substituted
sulfonamide 7, and subsequent Suzuki cross-coupling with
selected boronic acids, using Pd(dppf)Cl2 and K3PO4 in aqueous
THF at 80°C, afforded biaryl compounds 8a–8e (Scheme 2).
The 3- or 5-cyano-substituted isoxazoles 13a,b and 19a,b
were synthesized from ethyl acetopyruvate 9 (Scheme 3). For the
5-cyanoisoxazole regioisomers 13a and 13b, the reaction of 9
with hydroxylamine[25,26] afforded isoxazole 10. Ligand-free direct
palladium-mediated arylation of 10 with 4-bromobenzonitrile
furnished biaryl compound 11. Conversion of the ester group of
11 to the corresponding primary amide using ammonium
hydroxide, followed by catalytic hydrogenation of the nitrile group
afforded primary amine 12. This was reacted with the requisite
sulfonyl chloride and the isoxazole 5-carboxamide moiety
subsequently converted to the desired nitrile using POCl3 to afford
compound 13a. Compound 13b was prepared in a similar fashion
from compound 12, with the addition of an alkylation step on the
7.4
6.7
8.4
13
25g
25h
25i
14
13
25j
7.6
10
41
25o
39a
39b
44
4.8
0.22
0.56
1.0
0.59
0.43
1.6
[a] Human GR binding affinity pKi is reported as mean±SD (n ≥ 3, unless noted).
[b] Human PR binding affinity pKi is reported as mean±SD (n ≥ 3, unless noted).
[c] GR selectivity is reported as the ratio of hGR Ki over hMR Ki. [d] PR selectivity
is reported as the ratio of hPR Ki over hMR Ki. [e] n=2
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,
R1=
13a 19a
c
d
e or
e, f
,
R1=
13b 19b
a,b
10
11
12
and
13a
13b
g
9
i
l or m
j
R2=COOEt
14
15
16
h
R2=CONH
R3=BOC
R3=H
17
18
2
k
and
19b
19a
Scheme 3. Synthesis of 3- or 5-cyano substituted isoxazoles. a) NH2OH, EtOH, 15 min, -5°C, then 3 h, 25°C, (91%); b) H2SO4, EtOH, 10 h, 75°C, (74%); c) 4-
Bromobenzonitrile, PdCl2, KOAc, DMA, 20 h, 130°C, (37%); d-i) NH4OH, THF, 15 h, 20°C, d-ii) Pd/C, HCl, MeOH, H2 (g), 15 h, 25°C, (35% from 11); e-i) R1SO2Cl,
Et3N, CH2Cl2, 5–20 h, 25°C e-ii) POCl3, pyridine, ‒20°C, then 5 h, 25°C, (19%); f) Chlorodifluoroacetic acid sodium salt, Cs2CO3, DMF, 5 h, 100°C, (3% from
12); g) NH2OH·HCl, NaHCO3, EtOH, 15 h, 75°C, (85%); h) NH4OH, THF, 15 h, 25°C, (70%); i) POCl3, pyridine, ‒20°C, then 15 h, 25°C, (quantitative yield); j-i)
NIS, TFA, 3 h, 65°C, j-ii) (4-((BOC-amino)methyl)phenyl)boronic acid, PdCl2(PPh3)2, NaHCO3, DMF, 6 h, 95°C, (21% from 16); k) TFA, CH2Cl2, 4 h, 25°C, (73%);
l) 5-Chloro-1,3-dimethyl-1H-pyrazole-4-sulfonyl chloride, NEt3, CH2Cl2, 20 h, 25°C (23%); m-i) 3,5-Dimethyl-1H-pyrazole-4-sulfonyl chloride, NEt3, CH2Cl2, 5 h,
25°C; m-ii) Chlorodifluoroacetic acid sodium salt, Cs2CO3, DMF, 5 h, 100°C (17% from 18).
An ether library exploring the Met852 pocket was designed and
1
O
O
synthesized (Scheme 4). The key library intermediate 24 was
prepared starting with the reduction of 3-methylisoxazole-5-
carboxylic acid (20) to the corresponding alcohol 21. Iodination
using NIS yielded aryl iodide 22. Boronic acid 23 was synthesized
from BOC-protected 4-(aminomethyl)phenylboronic acid (3). BOC
deprotection and reaction with 1-methylcyclopropane-1-sulfonyl
chloride gave boronic acid 23. Suzuki cross-coupling of aryl iodide
22 and boronic acid 23 provided the hydroxymethyl substituted
biaryl derivative which was directly mesylated to give 24. The
desired benzylic ethers 25a–25o were synthesized in a parallel
fashion from mesylate 24 by nucleophilic substitution with the
requisite alcohols.
R
N
N
N O
3
O H
b
d
OR
I
R1=COOH
R1=CH OH
20
21
22
a
2
NH
B(OH)
2
B(OH)
S
2
O
O
c
R3=Ms
24
e
-
R3=R
25a 25o
NH
S
5-Benzyl-substituted isoxazole (28, Scheme 5) was
synthesized as previously described.[29,30] Reaction of 28 with
NBS afforded bromide 29 which was cross-coupled with BOC-
protected 4-(aminomethyl)phenylboronic acid (3) using PEPPSI-
IPr as catalyst. TFA-mediated BOC removal and reaction of the
resulting primary amine with 1-methylcyclopropane-1-sulfonyl
chloride afforded sulfonamide 30.
Macrocycles 39a, 39b and 44 were synthesized using a ring-
closing metathesis (RCM) strategy as shown in Scheme 6. The
synthetic routes to all three macrocycles employed
bromoisoxazole 31[31] as starting point. Palladium-catalyzed
borylation of 31 followed by Suzuki cross-coupling with N-BOC-4-
NH
BOC
O
O
3
23
Scheme 4. Synthesis of alcohol library expanding from the isoxazole-5-
methyl. a) BH3Me2S, THF, 30 min, 0°C, then 16 h, rt, (quantitative yield); b)
NIS, TFA, 25°C then 30 min, 70°C, (57%); c-i) HCl, 1,4-dioxane, 4 h, 25°C,
(91%); c-ii) 1-Methylcyclopropane-1-sulfonyl chloride, NEt3, CH2Cl2, 16 h,
25°C, (80%); d-i) Pd(PPh3)4, Na2CO3, 1,4-dioxane, water, 4 h, 80°C, d-ii)
MsCl, NEt3, CH2Cl2, 0°C then 10 min, 25°C, (47%); e) ROH, NaH, DMF, 2 h,
60°C then 16 h, rt, (4–44%).
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5

10.1002/cmdc.201600529
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R1=Cl
26
27
bromobenzylamine afforded biaryl compound 32 which upon
BOC removal afforded primary amine 33. This was reacted with
3- or 4-bromobenzenesulfonyl chloride, and subsequent
deacetylation using ammonia in methanol afforded sulfonamides
34a and 34b respectively. Manganese(IV) oxide mediated
oxidation of primary alcohols 34a and 34b afforded aldehydes 35a
and 35b, which were converted to vinylarenes 36a and 36b under
standard Wittig conditions. Stille coupling of the bromoisoxazole
moiety of 36a and 36b with allyltributyltin afforded dienes 37a and
37b. These dienes then underwent RCM under high dilution
conditions at room temperature using second generation Grubbs’
catalyst to afford macrocycles 38a and 38b. The double bond was
reduced by catalytic hydrogenation to obtain the desired
macrocycles 39a and 39b.
a
R1=N(CH )OCH
3
3
b
d
c
28
29
For oxygen-linked macrocycle 44, deacetylation of 32
followed by Mitsunobu reaction with 3-vinylphenol afforded ether
41, which afforded benzylamine 42 upon removal of the BOC
protecting group. The second alkene moiety was introduced by
reaction of amine 42 with allyl sulfonyl chloride, affording diene 43.
RCM with second generation Grubbs’ catalyst in DCM under high
dilution conditions formed the macrocyclic ring, with catalytic
hydrogenation of the double bond thus formed affording desired
macrocycle 44.
30
Scheme 5. Synthesis of 5-benzyl substituted oxazole 30. a) N,O-
Dimethylhydroxylamine*HCl, pyridine, CH2Cl2, 0 °C, then, 4 h, rt, (quant.); b)
Propan-2-one oxime, BuLi, THF, 0°C, then H2SO4, water/THF, 1 h, ∆,
(quant.); c) NBS, AcOH, overnight, ∆, (86%); d-i) (4-((BOC-
amino)methyl)phenyl)boronic acid, PEPPSI-IPr, KOH, 1,4-dioxane,
overnight, 65°C, d-ii) TFA, CH2Cl2, overnight, rt; d-iii) 1-Methylcyclopropane-
1-sulfonyl chloride, NEt3, CH2Cl2, overnight, rt, (2% from 29).
O N
O N
O N
O N
HO
O
O
O
j
l
m
O
S
O
4
4
S
R HN
R HN
N
N
H
O
H
O
R4=BOC
R4=BOC
41
42
40
43
44
k
R4=H*HCl
i
O N
O N
O N
AcO
3
O N
R
AcO
a
c
g
Br
O
O
31
S
HN
N
H
S N
H
2
O
1
R
O
R
R1=BOC
R1=H
,
R2=CH OH,R3=Br
R2=CHO,R3=Br
and
h
34a b
38a
38b
32
33
2
d
e
f
b
,
35a b
,
R2=CHCH ,R3=Br
36a b
2
,
R2=CHCH ,R3=CH CHCH
37a b
2 2 2
O N
R2 = meta
a
b
R2 = para
S N
H
O
O
and
39a
39b
Scheme 6. Synthesis of macrocycles 39a, 39b and 44. a-i) Pinacolborane, Pd(MeCN)2Cl2, SPhos, toluene, water, 20 h, 90°C; a-ii) N-BOC-4-bromobenzylamine,
Pd2(dba)3, Xantphos, NaHCO3, toluene, 12 h, 90°C (46% from 31), b) 4 M HCl in THF, EtOAc, 12 h, rt, (83%); c-i) 3- or 4-Bromobenzenesulfonyl chloride,
pyridine, 12 h, 25°C, c-ii) NH3, MeOH, 12–20 h, 25°C, (34a 51%, 34b 60% from 33); d) MnO2, DCM, 24 h, 25–30°C, (35a 85%, 35b 58%; e) Ph3PCH3Br, tBuOK,
THF, 3–5 h, 0–5°C, (36a 41%, 36b 38%); f) Allyltributyltin, PdCl2(dppf), DMF, 7–10 h, 90°C, (37a 46%, 37b 22%; g) Grubbs II catalyst, DCM, 6 h, 20–25°C,
(38a 3%, 38b 12%); h) 1 atm H2 (g), Pd/C, EtOAc, 1 h, 20–25°C, (39a 58%, 39b 71%); i) NH3, MeOH, 20 h, 25°C, (92%); j) 3-Vinylphenol, DEAD, PPh3, THF, 5
h, 5°C, (52%); k) 4 M HCl in THF, 4 h, 25°C, (73% crude); l) Allylsulfonyl chloride, pyridine, 5 h, 0–25°C, (60%); m) Grubbs II catalyst, DCM, 5 h, 25°C, then 1
atm H2 (g), Pd/C, EtOAc, 1 h, 30°C, (12%).
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10.1002/cmdc.201600529
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binding affinity to compound 1, but with a reduced logD7.4 resulting
in an improved LLE. Compound 2b possessed similar MR
potency to eplerenone (Table 1). Small substituted heterocycles
such as in compounds 2a and 2c resulted in a 10-fold higher
binding affinity and improved functional activity with an increased
LLE compared to the HTS hit (1), but the selectivity over GR and
PR was still poor (Table 2).
Table 3. Data collection and refinement statistics.
Crystal
Compound
Compound
25d
Compound
44
1
Data collection
The dimethyl substitution pattern on the isoxazole ring was
sensitive to modification. Removing the methyl groups on the
isoxazole rendered a compound (8a) with ~100-fold lower binding
affinity than compound 2a (Table 1). Replacement of the
isoxazole ring was also investigated. Replacement of the 3,5-
dimethylisoxazol-4-yl moiety with either a 1,4-dimethyl-pyrazol-5-
yl (8b) or a 3,5-dimethyl-pyrazol-4-yl (8c) moiety led to a 4-fold
and 10-fold decrease in binding affinity respectively (Table 4).
Replacement of the 3,5-dimethylisoxazole by 3-cyanophenyl (8d)
also led to about a 10-fold loss in binding affinity, in spite of
docking studies in crystal structure 5L7E indicating potential
interactions between the nitrile group of compound 8d and
gatekeeper residues Gln776 and Arg817. Such an interaction has
been observed for ligands at other oxosteroid receptors.[33,34] The
introduction of a fluorine substituent ortho to the nitrile (8e) led to
regained binding affinity and functional activity, but at the cost of
an increased logD7.4 (logD7.4=3.1, Table 4) compared to
compound 2a (logD7.4=2.5, Table 1).
PDB ID
5L7E
5L7G
5L7H
Wavelength (Å)
(Beamline at ESRF)
0.971
(ID23-1)
0.976
(ID29)
0.972
(ID23-1)
Space group
P212121
P212121
P212121
48.62
77.82
78.65
48.63
77.70
78.31
48.67
77.89
78.62
Cell parameter (Å)
Solvent content (%)
Resolutiona (Å)
I/Iσa
43.31
42.99
43.39
1.86–50.0
(1.86–1.93)
2.01–8.63
(2.01–2.08)
48.67–1.84
(1.84–1.90)
15.1 (1.8)
99.0 (91.4)
5.7 (2.7)
11.4 (1.6)
99.6 (98.6)
6.2 (6.3)
12.2 (1.5)
100.0 (100.0)
6.5 (6.2)
Completenessa (%)
Redundancya
Structure refinement
The preference for the positioning of the isoxazole oxygen and
nitrogen in the binding pocket as given in the X-ray structure of
compound 1 with MR (Figure 2A) was supported by the
regioisomeric pairs 13a, 19a and 13b, 19b. A 5-cyano-3-methyl-
isoxazol-4-yl (13a and 13b) moiety led to 3-4-fold higher binding
affinity compared to the 3-cyano-5-methyl-isoxazol-4-yl moiety
(19a and 19b). In the complex structure of compound 1, the 5-
position of the isoxazole points towards an area that is occupied
by amino acid side chains (of Met852, Ser811 and Leu938) while
the 3-position is placed close to the helix three backbone. The 3-
cyano regioisomers (19a and 19b) would cause a steric clash with
helix three, preventing the preferred orientation of the isoxazole
ring, thus disturbing the key polar interactions with the gatekeeper
residues Gln776 and Arg817, and resulting in a loss of binding
affinity. We propose that the nitrile substituents of 13a,b and
19a,b preferentially occupy the area near Met852, Ser811 and
Leu938 on steric grounds, and that the higher binding affinity of
the 5-substituted congeners (13a and 13b) reflects the preferred
orientation of the isoxazole. Compound 13b also displayed an
improved selectivity over GR and PR (Table 4) compared to the
HTS hit (1). Docking studies suggest that the nitrile of 13b
interacts directly with MR Ser811, which is an amino acid that
differs between MR compared to GR (Ala605) and PR (Val760).
In addition, Ser810 in MR is replaced respectively by Met604 and
Met759 in GR and PR, leading to an overall more hydrophilic
region in the MR receptor compared to that in GR or PR.
41.35–1.86
(1.86–1.94)
41.32–2.01
(2.01–2.12)
42.27–1.84
(1.84–1.92)
Resolutiona (Å)
Number of
25453 (2610)
4.67
20269 (2869)
5.13
26595 (2943)
5.05
reflections useda
Fraction of test set
for calculating
Rfree (%)
No. of reflections in
the test seta
1287 (122)
1040 (161)
1343 (153)
0.180 / 0.197
(0.230 /
0.277)
0.207 / 0.243
(0.229 /
0.257)
0.194 / 0.224
(0.247 /
0.284)
a
R factor: Rwork /Rfree
R.m.s.d.
Bond lengths (Å)
Bond angles (°)
0.010
0.96
0.010
1.00
0.010
0.97
Estimated
(Luzzati plot / DPI by
free R[32]) (Å)
error
0.191 / 0.112
28.47 / 25.52
0.293 / 0.163
35.30 / 33.11
0.218 / 0.124
32.12 / 38.25
B
values (mean B
value / Wilson plot)
(Å2)
Number of atoms --
final model
Protein
Water
2142
158
24
2136
87
2181
181
28
Ligand
31
Supported by the increased selectivity over GR and PR of
compound 13b we explored if protein-ligand interactions in other
areas of the MR LBD could be beneficial to gaining additional
oxosteroid receptor selectivity. Using the X-ray structure of MR in
complex with compound 1 and a previously published MR
structure in complex with a benzoxazinone ligand (PDBcode
3WFG)[6] it was hypothesized that it would be possible to extend
the ligand from the isoxazole 5-methyl substituent towards the
area around Met852 which is occupied by a phenyl ring of the
ligand in 3WFG (Figure 1). We hypothesized that targeting this
[a] Outer shell values are shown in parenthesis.
Structure–activity relationship
Initial SAR exploration focused on modifications of the
sulfonamide substituent and modifications and replacement of the
isoxazole
moiety.
The
1-methyl-cyclopropylsulfonamide
substituent of 2b provided a compound with similar potency and
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10.1002/cmdc.201600529
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Table 4. Binding affinity, functional activity and LLE of the profiled MR
to 68 for compound 25c and 25c the had a better selectivity ratio
over GR compared to eplerenone. The selectivity over PR was
improved 58-fold from a PR/MR binding affinity ratio of 0.52 for
compound 1 to 30 for 25c. The overall best selectivity over PR
was observed with compound 25j (Table 2).
antagonists.
pIC50
pKi
hMR[b]
LogD7.4
LLE[c]
O
H
N
N
N
S
RepG
hMR [a]
R
O
Cl
The most potent compounds in the library were the benzylic
ethers and where the phenyl ring of the benzylic group had
smaller substituents, such as halogens, methyl or methoxy.
However, larger groups such as 2-naphthyloxymethyl (25j) also
provided potent compounds (pIC50=7.0). Compounds with polar
phenyl substituents such as 25k or 25l or with heterocycles such
as the imidazol-2-methyleneyl (25m) were inactive. Compound
25n with an oxetane ring instead of the phenyl ring was also
inactive, but examples where the phenyl could be replaced by a
non-aromatic systems were identified, here exemplified by
compound 25o (pKi=7.0).
The structure of MR LBD in complex with 25d verified that the
proposed isoxazole orientation of compound 1 was retained
(Figure 3). In addition, the structure confirmed our hypothesis that
the sidechain of Met852 would move to accommodate the 2,3-
difluorophenoxymethyl moiety (Figure 3). Differences between
the oxosteroid receptors can be found in residues in contact with
the ligand and with Met852 that is required to move to
accommodate the phenyl ether substituent, and we believe that
this, at least partially, can rationalize the general selectivity
increase of the ether linked compounds (25a–25j and 25o) over
compound 2b. None of the biaryl sulfonamide compounds
reported in Table 2 displayed binding affinity to rat AR.
R=
1,4-dimethyl-1H-pyrazol-
5-yl-
6.4±0.2
(108%)
8b
8c
8d
7.0±0.1
6.5±0.1
6.6±0.1
2.4
2.4
3.1
4.6
4.1
3.5
3,5-dimethyl-1H-pyrazol-
4-yl-
6.0±0.1
(116%)
6.1±0.1
(102%)
3-cyanophenyl-
6.6±0.03
(98%)
8e
3-cyano-2-fluorophenyl-
7.2±0.2
6.1±0.1
3.1
3.3
4.2
2.9
39a
5.6±0.1
(100%)
39b
5.7±0.01
(100%)
6.1±0.2
7.3±0.3
3.2
3.0
3.0
4.5
44
6.6±0.4
(104%)
The binding pose of compound 25d positions the 5-
benzyloxymethyl isoxazole substituent in close spatial proximity
to the 1-methyl-cyclopropyl attached to the sulfonamide functional
group at the other end of the molecule. Based upon this
observation, the possibility to cyclize the compounds was
explored (Figure 1). For compound 1, the U-shaped binding pose
(Figure 2A) places the isoxazole 5-methyl carbon atom either
3.7 Å or 4.0 Å distant from the para or closest meta carbon atom
of the sulfonamide phenyl substituent respectively, suggesting
that a two atom linker would be needed to complete the
macrocycle.
[a] Human MR reporter gene pIC50 is reported as mean±SD (n ≥ 3, unless
noted). Efficacy in percent is reported in parenthesis. [b] Human MR binding
affinity pKi is reported as mean±SD (n ≥ 3, unless noted). [c] LLE=pKi-logD7.4
,
[d] n=2
pocket could give selectivity for MR, both through direct polar
interactions with MR Ser811 and through secondary effects from
the required movement of the conserved Met852 side chain in an
area where the receptors otherwise differ. In the MR structure in
complex with compound 1, the sidechain of Met852 adopts a
similar position as that in the published X-ray structure of MR in
complex with aldosterone (PDBcode 2AA2),[19] whereas in public
MR antagonist structures (e.g. PDBcode 3WFG), the Met852
sidechain is shown to be moved by the ligand, creating an induced
fit pocket that can accommodate phenyl substituents.[6,35,36]
Molecular modeling suggested that a two atom linker
extending from the 5-position of the isoxazole would be of suitable
length to evaluate this hypothesis and a compound library of
ethers was prepared based on compound 2b. Overall, the ether
library (25a–25o) provided compounds with up to 34-fold
improvement in binding affinity (25c, pKi=8.2) and up to 12-fold
increase in functional activity (25j, pIC50=7.0) (Table 1) compared
to matched pair compound 2b. Although there was a general
increase in lipophilicity the improvement in potency was enough
to give improvements in LLE. More importantly, an improved
selectivity for MR over both GR and PR was observed (Table 2)
across the library (25a–25j and 25o). The best selectivity profile
was observed with compound 25c. The GR/MR binding affinity
ratio was improved 234 fold from a ratio of 0.29 for compound 1
Figure 3. Refined 2mFo-DFc electron density of MR LBD (blue) in complex
with compound 25d. Selected amino acid side chains are shown in orange
and putative hydrogen bonds are shown as dotted lines.
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8

10.1002/cmdc.201600529
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FULL PAPER
Based on compound 1, two macrocyclic compounds with a
three atom linker between either the meta or para position of the
phenyl attached to the sulfonamide and the isoxazole ring were
designed
to
explore
conformationally-restrained
biaryl
sulfonamides. Compared to parent compound 1, macrocycles
39a and 39b both had decreased binding affinity of pKi=6.1 and
functional activity in the reporter gene assay of pIC50=5.6 and 5.7
respectively. This was just on the borderline to be significantly
different compared to the linear compound 1 considering the
minimum discriminatory difference (MDD) of 0.24 for a compound
tested on three different occasions for this assay.
An alternative macrocyclization, based on compound 25b, of
the ether linked phenyl was also explored. In this case
macrocyclization from the meta position of the ether linked phenyl
to the sulfonamide sulfur atom using a three atom linker led to
compound 44. In this case, the binding affinity (pKi=7.3) and
functional activity (pIC50=6.6) was very similar to that of the
closest non-cyclized analogue (25b, pKi=7.8, pIC50=6.7). The
structure of MR LBD in complex with 44 revealed that despite the
conformation constraints imposed by macrocyclization, the
interactions to the protein remained intact (Figure 4). A simple
conformational search of compound 1 using a molecular
mechanics force field and an implicit solvent model suggests that
the global minimum and the low energy conformation ensemble
are in close proximity to the bound X-ray conformation of 1. We
speculate that the lack of gain in affinity upon macrocyclization is
partly due to a limited entropic contribution to binding affinity as a
consequence of locking the molecules in their bioactive
conformations.
Figure 4. Refined 2mFo-DFc electron density of MR LBD (blue) in complex
with compound 44. Selected amino acid side chains are shown in orange
and putative hydrogen bonds are shown as dotted lines. Ser811 is observed
in two distinct conformations.
replaced by the saturated 1-methyl cyclopropyl substituent, both
solubility (420 µM) and metabolic stability in human hepatocytes
(CLint: 1.5 µL/min/106 cells) were greatly improved. However, the
metabolic stability in rat, both in vitro (CLint: >300 µL/min/106 cells)
and in vivo (clearance: 56 mL/min/kg), was still low.
DMPK properties
For 25i, a more potent analogue of 2b in which the isoxazole
5-methyl was replaced by a 3-methoxy-phenoxymethyl moiety,
both solubility (0.5 µM) and metabolic stability in human
hepatocytes (CLint: 33 µL/min/106 cells) were impaired.
Furthermore, significant amounts of GSH conjugates were
trapped. The major site of metabolism was identified as the 3-
methoxy-phenoxymethyl substituent (data not shown). Also,
compound 25i was unstable in vitro (rat hepatocyte CLint
>300 µL/min/106 cells) and in vivo in rat (clearance 52 mL/min/kg).
For the macrocyclic compound 44, the issues of poor solubility
(1.5 µM) and low in vitro and in vivo metabolic stability (human
hepatocyte CLint 17 µL/min/106 cells, rat hepatocyte CLint
>300 µL/min/106 cells, rat clearance: 55 mL/min/kg) were retained.
The HTS hit (1) was moderately stable in human hepatocytes
(CLint: 7.0 µL/min/106 cells), but unstable both in rat hepatocytes
(CLint: >300 µL/min/106 cells) and in vivo in rats (clearance:
56 mL/min/kg) (Table 5). Compound 1 had moderate kinetic
solubility (62 µM) and no glutathione (GSH) conjugates were
detected when incubated with human liver microsomes (HLM). In
2b, where the phenyl sulfonamide substituent of compound 1 was
Table 5. DMPK data for selected compounds.
1
2b
25i
44
For all four compounds the Caco-2 permeability was high (Papp
53–96 110-6 cm/s).
:
Solubility (µM)
62
420
0.5
1.5
CLint human/
rat hepatocytes
(µL/min/106 cells)
7.0 /
>300
1.5 /
>300
33 /
>300
17 /
>300
Biophysical characterization
Nuclear hormone receptor ligands that lack functional
agonism at the receptor can be divided into two groups:
antagonists, which induce a conformation and an action of the
receptor distinct from that of an agonist, or inverse agonists which
promote corepressor binding.[37] To further understand the mode
of action of the three biaryl sulfonamides (1, 25d and 44) where
the MR-complex structure were solved (Table 3), we studied
ligand-dependent MR translocation into the nucleus using
EA.hy926 cells, which express MR endogenously. The
experiments were performed in the absence or presence of
0.1 nM aldosterone either to study the intrinsic ability of the
ligands to translocate MR into the nucleus or their ability to block
Clearance rat
(mL/min/kg)
51
No
70
56
No
75
52
55
No
96
GSH conjugates
detected in
HLM
Yes
53
Caco-2 Papp
(1x10-6 cm/s)
CYP inhibition[a]
(µM)
6.2
(CYP2C19)
3.2
(CYP2C9)
1.4
(CYP2C9)
5.7
(CYP3A4)
[a] Value for the most active CYP given
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10.1002/cmdc.201600529
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Table 6. Residence time and translocation data for hMR.
General Purity of all test compounds was determined by LCMS. All
screening compounds had a purity >95%. A complete list of the synthetic
procedures and compound characterization is available in the supporting
information.
Translocation
pEC50 (µM)
Translocation
Compound
koff (s-1)
0.010
block[a] pIC50 (µM)
1
<4.8
6.6 (94%)
N-[4-(3,5-Dimethyl-1,2-oxazol-4-yl)benzyl]benzenesulfonamide
(1)
25d
44
0.0048
0.0089
<5
6.4 (90%)
7.0 (72%)
1H NMR (500 MHz, CDCl3): δ = 2.24 (s, 3H), 2.38 (s, 3H), 4.21 (d, J =
6.3 Hz, 2H), 4.77–4.87 (m, 1H), 7.17 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz,
2H), 7.50–7.56 (m, 2H), 7.57–7.63 (m, 1H), 7.86–7.93 ppm (m, 2H); HRMS
(ESI) m/z [M + H]+ calcd for C18H19N2O3S: 343.1111 found: 343.1118.
<5
[a] Efficacy given in parenthesis
tert-Butyl N-[[4-(3,5-dimethylisoxazol-4-yl)phenyl]methyl]-carbamate
(4) Compound 4 was synthesized from 4-bromo-3,5-dimethylisoxazole
(4.0 g, 23 mmol) to afford the title compound (4.6 g, 67%).
aldosterone-mediated nuclear translocation. All three compounds
prevent aldosterone-mediated translocation of MR to the nucleus,
which is a potential explanation for their antagonistic behavior in
reporter gene assays (Table 6). The three compounds also lack
the ability to induce nuclear translocation of MR in the absence of
aldosterone.
[4-(3,5-Dimethylisoxazol-4-yl)phenyl]methanamine hydrochloride (5)
Compound 5 was synthesized from tert-butyl N-[[4-(3,5-dimethylisoxazol-
4-yl)phenyl]methyl]-carbamate (4, 4.59 g, 15.2 mmol) to afford the title
compound (3.4 g, crude yield 93%).
Compounds 1, 25d and 44 were also kinetically characterized
using a time-resolved competition SPR assay (Table 6). The 2-
fold difference in affinity between 25d and 44 was also reflected
by a 2-fold difference in residence time. However, compound 1
had a similar dissociation rate to 44 despite a 10-fold difference
in Ki. This indicates that compound 1 has a slower on rate.
General procedure A for synthesis of N-[4-(3,5-dimethyl-1,2-oxazol-4-
yl)benzyl]-sulfonamides
(2a–2c)
[4-(3,5-Dimethylisoxazol-4-
yl)phenyl]methanamine hydrochloride (5, 70 mg, 0.29 mmol) was
dissolved in CH2Cl2 (2.5 mL). The appropriate sulfonylchloride
(0.32 mmol) and NEt3 (0.16 mL, 1.2 mmol) were added and the reaction
was stirred at rt overnight. The solvent was removed in vacuo and the
product was purified by preparative HPLC.
Conclusions
5-Chloro-N-[4-(3,5-dimethyl-1,2-oxazol-4-yl)benzyl]-1,3-dimethyl-1H-
pyrazole-4-sulfonamide (2a)[14] Compound 2a was synthesized
according to general procedure A to afford the title compound (62 mg,
58%).
We have identified a series of biaryl sulfonamide MR
antagonists that act by blocking aldosterone-induced
translocation of MR into the nucleus. Guided by the X-ray
structure of the HTS hit (1), the compounds were expanded into
a cryptic pocket formed by induced fit at the receptor. Targeting
this cryptic pocket also gave a general increase in selectivity over
GR and PR. We also report the first potent macrocyclic MR
antagonist, with a binding affinity pKi=7.3. We have demonstrated
that good solubility, good metabolic stability in human
hepatocytes, high permeability and low reactive metabolite
formation can be achieved within the compound series. However,
the low metabolic stability in rats still needs to be addressed in
order to develop a compound suitable for testing in a rat in vivo
efficacy model.
N-[4-(3,5-Dimethyl-1,2-oxazol-4-yl)benzyl]-1-methylcyclopropane-
sulfonamide (2b). Compound 2b was synthesized according to general
procedure A to afford the title compound (60 mg, 64%).
N-[4-(3,5-Dimethyl-1,2-oxazol-4-yl)benzyl]-3,5-dimethyl-1,2-oxazole-
4-sulfonamide (2c)[13] Compound 2c was synthesized according to
general procedure A to afford the title compound (63 mg, 59%).
5-Chloro-N-[(4-iodophenyl)methyl]-1,3-dimethyl-pyrazole-4-
sulfonamide (7) Compound 7 was synthesized from (4-iodophenyl)-
methanamine (6, 3.9 g, 17 mmol) to afford the title compound (6.0 g, 92%)
as an off-white solid.
General procedure B for Suzuki cross-coupling (8a–8e) Into a 40 mL
vial, was placed 5-chloro-N-[(4-iodophenyl)methyl]-1,3-dimethyl-pyrazole-
4-sulfonamide (7, 42.6 mg, 0.10 mmol), boronic acid (0.15 mmol), K3PO4
(0.20 mmol) and Pd(dppf)Cl2 (5– 10Mol%) in THF:water 4:1 (2 mL). The
mixture was stirred at 80°C for 16 h. After cooling to the ambient
temperature the crude product was purified by preparative HPLC and
lyophilized.
Abbreviations
AR, androgen receptor; CIR, confidence interval ratio; CKD,
chronic kidney disease; DBD, DNA binding domain; ER, estrogen
receptor; GR, glucocorticoid receptor; GSH, glutathione; HLM,
human liver microsomes; HTS, high throughput screening; LBD,
ligand binding domain; LLE, lipophilic ligand efficiency; MDD,
minimum discriminatory difference; MR, mineralocorticoid
receptor; PR, progesterone receptor; RCM, ring-closing
metathesis; rt, room temperature; SAR, structure–activity
relationship; SPA, scintillation proximity assay.
5-Chloro-1,3-dimethyl-N-[4-(1,2-oxazol-4-yl)benzyl]-1H-pyrazole-4-
sulfonamide (8a) Compound 8a was synthesized according to general
procedure B to afford the title compound (14 mg, 39%).
5-Chloro-N-[4-(1,4-dimethyl-1H-pyrazol-5-yl)benzyl]-1,3-dimethyl-1H-
pyrazole-4-sulfonamide (8b) Compound 8b was synthesized according
to general procedure B to afford the title compound (29 mg, 74%).
Experimental Section
5-Chloro-N-[4-(3,5-dimethyl-1H-pyrazol-4-yl)benzyl]-1,3-dimethyl-1H-
pyrazole-4-sulfonamide (8c) Compound 8c was synthesized according
to general procedure B to afford the title compound (4.1 mg, 10%).
Chemistry
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10.1002/cmdc.201600529
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5-Chloro-N-[(3'-cyanobiphenyl-4-yl)methyl]-1,3-dimethyl-1H-
pyrazole-4-sulfonamide (8d) Compound 8d was synthesized according
to general procedure B to afford the title compound (17 mg, 43%).
carbonitrile (18, 150 mg, 0.70 mmol) to afford the title compound (56 mg,
17% over two steps) as a white solid.
(3-Methylisoxazol-5-yl)methanol (21) Compound 21 was synthesized
from 3-methylisoxazole-5-carboxylic acid (20, 1.0 g, 7.9 mmol) to give the
title compound as a colorless syrup (0.89 g, crude yield 100%).
5-Chloro-N-[(3'-cyano-2'-fluorobiphenyl-4-yl)methyl]-1,3-dimethyl-
1H-pyrazole-4-sulfonamide (8e) Compound 8e was synthesized
according to general procedure B to afford the title compound (19 mg,
46%).
(4-Iodo-3-methylisoxazol-5-yl)methanol (22) Compound 22 was
synthesized from (3-Methylisoxazol-5-yl)methanol (21, 20.0 g, 159 mmol,
90%) to afford the title compound as a light yellow solid (23 g, 57%).
Ethyl 3-methylisoxazole-5-carboxylate (10) Ethyl (4E)-4-hydroxyimino-
2-oxo-pentanoate was prepared as described by Sumimoto[25] (20 g, 91%).
Compound 10 was prepared from ethyl (4E)-4-hydroxyimino-2-oxo-
pentanoate as described by Dannhardt et al.[26] to afford the title compound
(4.0 g, 74%).
[4-[[(1-Methylcyclopropyl)sulfonylamino]methyl]phenyl]-boronic
acid (23) Compound 23 was synthesized from [4-([[(tert-
butoxy)carbonyl]amino]methyl)phenyl]boronic acid (3, 14 g, 53 mmol,
95%) to afford the title compound as a light yellow solid (14 g, crude yield
80%).
Ethyl
4-(4-cyanophenyl)-3-methylisoxazole-5-carboxylate
(11)
Compound 11 was synthesized from ethyl 3-methylisoxazole-5-
carboxylate (10, 12.5 g, 80.6 mmol) to afford the title compound (6.2 g,
37%) as a pale yellow solid.
[3-Methyl-4-[4-[[(1-methylcyclopropyl)sulfonylamino]-methyl]-
phenyl]isoxazol-5-yl]methyl methanesulfonate (24) Compound 24 was
synthesized from (4-iodo-3-methylisoxazol-5-yl)methanol (22, 10.4 g,
41.3 mmol) to afford the title compound as a yellow oil (8.0 g, crude yield
47% over two steps).
4-[4-(Aminomethyl)phenyl]-3-methylisoxazole-5-carboxamide
(12)
Compound 12 was synthesized from ethyl 4-(4-cyanophenyl)-3-
methylisoxazole-5-carboxylate (11, 6.5 g, 25 mmol) to afford the title
compound (2.0 g, 35% over two steps) as a pink solid.
General procedure C for synthesis of ether linked compounds (25a–
25o) Into a 40 mL vial, was placed the appropriate alcohol (0.11 mmol)
and NaH (0.11 mmol) in DMF (3 mL). The mixture was stirred at 60°C for
2 h. After cooling to the ambient temperature [3-methyl-4-[4-[[(1-methyl-
cyclopropyl)sulfonylamino]-methyl]phenyl]isoxazol-5-yl]methyl methane-
sulfonate (24, 29 mg, 0.07 mmol) was added into the vial and the mixture
was stirred for 16 h at rt and then concentrated in vacuo. The crude
product was purified by preparative HPLC.
5-Chloro-N-[4-(5-cyano-3-methyl-1,2-oxazol-4-yl)benzyl]-1,3-
dimethyl-1H-pyrazole-4-sulfonamide (13a) Compound 13a was
synthesized
from
4-(4-(aminomethyl)phenyl)-3-methylisoxazole-5-
carboxamide (12, 300 mg, 1.30 mmol) to afford the title compound
(100 mg, 19% over two steps) as a white solid.
N-[4-(5-Cyano-3-methyl-1,2-oxazol-4-yl)benzyl]-1-(difluoromethyl)-
3,5-dimethyl-1H-pyrazole-4-sulfonamide (13b) Compound 13b was
synthesized 4-(4-(aminomethyl)phenyl)-3-methylisoxazole-5-carboxamide
(12, 300 mg, 1.30 mmol) to afford the title compound (15 mg, 3% over
three steps) as a white solid.
N-(4-(5-((3-Chlorophenoxy)methyl)-3-methylisoxazol-4-yl)benzyl)-1-
methylcyclopropane-1-sulfonamide (25a). Compound 25a was
synthesized according to general procedure C to afford the title compound
(67 mg, 44%).
Ethyl 5-methylisoxazole-3-carboxylate (14) was prepared as described
1-Methyl-N-(4-(3-methyl-5-(phenoxymethyl)isoxazol-4-
yl)benzyl)cyclopropane-1-sulfonamide (25b). Compound 25b was
synthesized according to general procedure C to afford the title compound
(11 mg, 8%).
by Rao et al.[27] to afford the title compound (25 g, 85%).
5-Methylisoxazole-3-carboxamide (15) was prepared as described by
Collins et al.[28] to afford the title compound (1.7 g, 70%).
N-(4-(5-((3,5-Difluorophenoxy)methyl)-3-methylisoxazol-4-yl)benzyl)-
1-methylcyclopropane-1-sulfonamide (25c). Compound 25c was
synthesized according to general procedure C to afford the title compound
(2 mg, 4%).
5-Methylisoxazole-3-carbonitrile (16) was prepared as described by
Collins et al.[28] to afford the title compound (9.4 g, quantitative yield).
tert-Butyl
N-[[4-(3-cyano-5-methylisoxazol-4-yl)phenyl]methyl]-
1-Methyl-N-[[4-[3-methyl-5-(phenoxymethyl)isoxazol-4-
yl]phenyl]methyl]cyclopropanesulfonamide (25d). Compound 25d
was synthesized according to general procedure C to afford the title
compound (8.0 mg, 26%).
carbamate (17) Compound 17 was synthesized from 5-methylisoxazole-
3-carbonitrile (16, 10.0 g, 92.5 mmol) to afford the title compound (1.2 g,
21% over two steps) as a brown oil which solidified on standing.
4-[4-(Aminomethyl)phenyl]-5-methylisoxazole-3-carbonitrile
(18)
1-Methyl-N-[[4-[3-methyl-5-[(4-methylphenoxy)methyl]isoxazol-4-
yl]phenyl]methyl]cyclopropanesulfonamide (25e). Compound 25e was
synthesized according to general procedure C to afford the title compound
(5.6 mg, 19%).
Compound 18 was synthesized from tert-butyl N-[[4-(3-cyano-5-
methylisoxazol-4-yl)phenyl]methyl]carbamate (17, 1.2 g, 3.8 mmol) to
afford the title compound (0.70 g, 73%) as a pale yellow oil which solidified
on standing.
N-[[4-[5-[(4-Chloro-3-fluoro-phenoxy)methyl]-3-methylisoxazol-4-
5-Chloro-N-[4-(3-cyano-5-methyl-1,2-oxazol-4-yl)benzyl]-1,3-
yl]phenyl]methyl]-1-methyl-cyclopropanesulfonamide
(25f).
dimethyl-1H-pyrazole-4-sulfonamide (19a) Compound 19a was
Compound 25f was synthesized according to general procedure C to
synthesized
from
4-(4-(aminomethyl)phenyl)-5-methylisoxazole-3-
afford the title compound (0.9 mg, 28%).
carbonitrile (18, 90 mg, 0.42 mmol) to give the desired product (40 mg,
23%) as a white solid.
N-[[4-[5-[(3-Fluoro-5-methyl-phenoxy)methyl]-3-methylisoxazol-4-
yl]phenyl]methyl]-1-methyl-cyclopropanesulfonamide
Compound 25g was synthesized according to general procedure C to
(25g).
N-[4-(3-Cyano-5-methyl-1,2-oxazol-4-yl)benzyl]-1-(difluoromethyl)-
3,5-dimethyl-1H-pyrazole-4-sulfonamide (19b) Compound 19b was
afford the title compound (3.2 mg, 10%).
synthesized
from
4-(4-(aminomethyl)phenyl)-5-methylisoxazole-3-
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N-[[4-[5-[(2-Fluoro-5-methyl-phenoxy)methyl]-3-methylisoxazol-4-
[4-[4-[(tert-Butoxycarbonylamino)methyl]phenyl]-3-methylisoxazol-
5-yl]methyl acetate (32) Compound 32 was synthesized from (4-bromo-
3-methylisoxazol-5-yl)methyl acetate (31, 10.0 g, 42.7 mmol) to afford the
title compound as a yellow oil (7.0 g, 46% over two steps).
yl]phenyl]methyl]-1-methyl-cyclopropanesulfonamide
Compound 25h was synthesized according to general procedure C to
afford the title compound (2.8 mg, 9.0%).
(25h).
N-(4-[5-[(3-Methoxyphenoxy)methyl]-3-methyl-1,2-oxazol-4-
yl]benzyl)-1-methyl-cyclopropane-sulfonamide (25i) Compound 25i
was synthesized according to general procedure C to afford the title
compound (9.2 mg, 34%).
[4-[4-(Aminomethyl)phenyl]-3-methylisoxazol-5-yl]methyl
(33) Compound 33 was synthesized from
butoxycarbonylamino)methyl)phenyl)-3-methylisoxazol-5-yl)methyl
acetate (32, 10.0 g, 27.8 mmol) to afford the title compound as a brown oil
(6.0 g, crude yield 83%).
acetate
(4-(4-((tert-
1-Methyl-N-[[4-[3-methyl-5-(2-naphthyloxymethyl)isoxazol-4-
yl]phenyl]methyl]cyclopropanesulfonamide (25j). Compound 25j was
synthesized according to general procedure C to afford the title compound
(5.1 mg, 16%).
3-Bromo-N-[[4-[5-(hydroxymethyl)-3-methylisoxazol-4-yl]phenyl]-
methyl]benzenesulfonamide (34a) Compound 34a was synthesized
from [4-[4-(aminomethyl)phenyl]-3-methylisoxazol-5-yl]methyl acetate (33,
1.5 g, 5.8 mmol) to afford the title compound as a yellow oil (1.3 g, 51%
over two steps).
N-[3-({3-Methyl-4-[4-({[(1-methylcyclopropyl)sulfonyl]-
amino}methyl)phenyl]-1,2-oxazol-5-yl}methoxy)phenyl]acetamide
(25k) Compound 25k was synthesized according to general procedure C
to afford the title compound (3.4 mg, 7%).
3-Bromo-N-[[4-(5-formyl-3-methylisoxazol-4-yl)phenyl]methyl]-
benzenesulfonamide (35a) Compound 35a was synthesized from 3-
bromo-N-[[4-[5-(hydroxymethyl)-3-methylisoxazol-4-yl]phenyl]methyl]-
benzenesulfonamide (34a, 1.3 g, 3.0 mmol) to afford the title compound
as a yellow oil (1.1 g, 85%).
4-({3-Methyl-4-[4-({[(1-methylcyclopropyl)sulfonyl]-
amino}methyl)phenyl]-1,2-oxazol-5-yl}methoxy)benzenesulfonamide
(25l) Compound 25l was synthesized according to general procedure C to
afford the title compound (2.9 mg, 5%).
3-Bromo-N-[[4-(3-methyl-5-vinyl-isoxazol-4-
yl)phenyl]methyl]benzenesulfonamide (36a) Compound 36a was
synthesized
from
3-bromo-N-[[4-(5-formyl-3-methylisoxazol-4-
1-Methyl-N-[4-(3-methyl-5-{[(1-methyl-1H-imidazol-2-
yl)phenyl]methyl]benzene-sulfonamide (35a, 1.1 g, 2.5 mmol) to afford the
title compound as a yellow solid (0.45 g, 41%).
yl)methoxy]methyl}-1,2-oxazol-4-yl)benzyl]cyclopropane-
sulfonamide (25m) Compound 25m was synthesized according to
general procedure C to afford the title compound (8.9 mg, 19%).
3-Allyl-N-[[4-(3-methyl-5-vinyl-isoxazol-4-yl)phenyl]methyl]benzene-
sulfonamide (37a) Compound 37a was synthesized from 3-bromo-N-[[4-
(3-methyl-5-vinyl-isoxazol-4-yl)phenyl]-methyl]benzenesulfonamide (36a,
0.45 g, 1.0 mmol) to afford the title compound as a white solid (0.19 g,
46%).
1-Methyl-N-(4-{3-methyl-5-[(oxetan-3-yloxy)methyl]-1,2-oxazol-4-
yl}benzyl)cyclopropanesulfonamide (25n) Compound 25n was
synthesized according to general procedure C to afford the title compound
(9.8 mg, 23%).
(17Z)-3-Methyl-8,16-dihydro-9H-4,7-etheno-11,15-
N-[4-(5-{[(1-Fluorocyclobutyl)methoxy]methyl}-3-methyl-1,2-oxazol-
4-yl)benzyl]-1-methylcyclopropanesulfonamide (25o) Compound 25o
was synthesized according to general procedure C to afford the title
compound (9.1 mg, 20%).
(metheno)[1,2]oxazolo[4,5-h][1,2]thiazacycloheptadecine
dioxide (38a) Compound 38a was synthesized from 3-allyl-N-[[4-(3-
methyl-5-vinyl-isoxazol-4-yl)phenyl]methyl]-benzenesulfonamide (37a,
10,10-
1.1 g, 2.8 mmol) to afford the title compound as a white solid (0.03 g, 3%).
N-Methoxy-N-methyl-2-phenyl-acetamide (27) was prepared as
described by Rudzinski et al.[29] to afford the title compound (664 mg,
quantitative yield).
3-Methyl-8,16,17,18-tetrahydro-9H-4,7-etheno-11,15-(metheno)[1,2]-
oxazolo[4,5-h][1,2]thiazacycloheptadecine
10,10-dioxide
(39a)
Compound 39a was synthesized from (17Z)-3-methyl-8,16-dihydro-9H-
4,7-etheno-11,15-(metheno)[1,2]oxazolo[4,5-
h][1,2]thiazacycloheptadecine 10,10-dioxide (38a, 19 mg, 0.05 mmol) to
afford the title compound as a white solid (11 mg, 58%).
5-Benzyl-3-methylisoxazole (28) was prepared as described by Nitz et
al.[30] to afford the title compound (105 mg, quantitative yield).
5-Benzyl-4-bromo-3-methylisoxazole (29) Compound 29 was
synthesized from 5-Benzyl-3-methylisoxazole (28, 0.105 g, 0.61 mmol) to
afford crude product (131 mg, crude yield 86%) which was directly used in
next synthesis step.
4-Bromo-N-[[4-[5-(hydroxymethyl)-3-methylisoxazol-4-yl]phenyl]-
methyl]benzenesulfonamide (34b) Compound 34b was synthesized
from [4-[4-(aminomethyl)phenyl]-3-methylisoxazol-5-yl]methyl acetate (33,
6.0 g, 23 mmol) to afford the title compound as a red oil (6.0 g, 60%).
N-[4-(5-Benzyl-3-methyl-1,2-oxazol-4-yl)benzyl]-1-methylcyclo-
propanesulfonamide (30) Compound 30 was synthesized from 5-Benzyl-
4-bromo-3-methylisoxazole (29, 0.131 g, 0.52 mmol) to afford the title
compound (4.9 mg, 2% over three steps).
4-Bromo-N-(4-(5-formyl-3-methylisoxazol-4-yl)benzyl)benzene-
sulfonamide (35b) Compound 35b was synthesized from 4-bromo-N-[[4-
[5-(hydroxymethyl)-3-methylisoxazol-4-
yl]phenyl]methyl]benzenesulfonamide (34b, 2.6 g, 6.0 mmol) to give the
title compound as a yellow oil in (1.5 g, 58%).
tert-Butyl N-[(4-bromophenyl)methyl]carbamate (45) was prepared as
described by Tashima et al.[38] to afford the title compound (25 g, 81%).
4-Bromo-N-[[4-(3-methyl-5-vinylisoxazol-4-yl)phenyl]methyl]-
benzenesulfonamide (36b) Compound 36b was synthesized from 4-
bromo-N-(4-(5-formyl-3-methylisoxazol-4-yl)-benzyl)benzene-
sulfonamide (35b, 4.0 g, 9.2 mmol) to afford the title compound as a
colorless oil (1.5 g, 38%).
(3-Methylisoxazol-5-yl)methyl acetate (46) was prepared as described
by Gehling et al.[31] to afford the title compound as a yellow oil (8.0 g, 51%).
(4-Bromo-3-methylisoxazol-5-yl)methyl acetate (31) was prepared as
described by Gehling et al.[31] to afford the title compound as a yellow oil
(15.0 g, 66%).
4-Allyl-N-[[4-(3-methyl-5-vinyl-isoxazol-4-yl)phenyl]methyl]benzene-
sulfonamide (37b) Compound 37b was synthesized from 4-bromo-N-[[4-
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10.1002/cmdc.201600529
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(3-methyl-5-vinylisoxazol-4-yl)phenyl]methyl]benzenesulfonamide (36b,
1.5 g, 3.5 mmol) to afford the title compound as a brown solid (0.30 g,
22%).
Elmer) and 2 µg/mL anti-MBP antibody (Abcam ltd.) were mixed in the
assay buffer and 20 µL of the mix was added to the assay plates. Plates
were incubated for at least 8 h in darkness at rt before read on
LEADseeker. Compounds were tested in ten-point concentration response
(½ log serial dilution) with 37.5 µM starting concentration. Raw data output
was analyzed in Screener software (Genedata AG) and IC50 values
calculated using a four parameter logistic fit. pIC50 was reported as mean
with the number of experiments n≥3. Confidence interval ratio (CIR) was
1.5 for the MR binding assay with MDD of 0.26 for compounds tested on
three occasions. The 95% confidence interval for the true IC50 of the
compound is the single calculated value of IC50 ×/÷ CIR or on a log scale
pIC50 ± log10(CIR). Two compounds are considered different if their pIC50
differ more than ± MDD (p = 0.05).
3-Methyl-8,15-dihydro-9H-4,7:11,14-dietheno[1,2]oxazolo[4,5-h][1,2]-
thiazacyclohexadecine 10,10-dioxide (38b) Compound 38b was
synthesized
from
4-allyl-N-[[4-(3-methyl-5-vinyl-isoxazol-4-
yl)phenyl]methyl]benzenesulfonamide (37b, 450 mg, 1.14 mmol) to afford
the title compound as a white solid (50 mg, 12%).
3-Methyl-8,15,16,17-tetrahydro-9H-4,7:11,14-dietheno[1,2]oxazolo-
[4,5-h][1,2]thiazacyclohexadecine 10,10-dioxide (39b) Compound 39b
was
synthesized
from
3-methyl-8,15-dihydro-9H-4,7:11,14-
10,10-dioxide
dietheno[1,2]oxazolo[4,5-h][1,2]thiazacyclo-hexadecine
Mineralocorticoid receptor reporter gene assay A 384-well UAS-MR-
bla HEK293 cell based reporter assay (GeneBLAzer®MR DA,
ThermoFisher Scientific) was used to identify compounds showing
antagonist activity at the human mineralocorticoid receptor LBD. This
nuclear receptor assay uses a beta-lactamase reporter (bla) under
transcriptional control of an Upstream Activator Sequence (UAS). The
UAS is activated by the GAL4 transcription factor DNA Binding Domain
(DBD), which is expressed as a fusion protein with the MR LBD (residues
682–984). Upon ligand binding of an agonist (aldosterone), the
GAL4(DBD)-MR(LBD) binds to the UAS, which controls transcription of
beta-lactamase. Cryo preserved, stably transfected HEK293 cells were
thawed and diluted in assay medium (DMEM (Gibco), 2% charcoal
stripped fetal bovine serum (Gibco), 100 µg/mL streptomycin,10 units/mL
penicillin, 1 mM sodium pyruvate and 1X non-essential amino acid). The
cells were seeded, 25000/well in 30 µL assay medium, in a 384 well
black/clear TC-treated plate (Corning) and incubated for 4 h at 37°C, 5%
CO2 and 95% humidity. Compound plate with 0.6 µL test or control
compound was prediluted 100 times with assay medium and 4 µL of the
diluted compound was transferred to the cell plate. The cell plate was
incubated for 30 min prior to addition of 4 µL of aldosterone in a final
concentration of 1 nM, followed by incubation (as described above) for 16–
24 h. The cells were loaded with 8 µL CCF4, a fluorescent substrate buffer
containing two connected fluorophores, coumarin and fluorescein, to
assess the beta-lactamase activity. The plate was incubated in darkness
for 2 h at rt before fluorescence was measured on PHERAstar FS (BMG
Labtech) with optic module (FI 405/530/560). The result was presented as
the ratio of coumarin/fluorescein fluorescence intensities. Raw data output
was analyzed in Screener software (Genedata AG) and IC50 values
calculated using a four parameter logistic fit. CIR for the MR reporter gene
assay was 1.48, for a compound tested on three occasions and the MDD
was 0.24.
(38b, 28 mg, 0.08 mmol) to afford the title compound as a white solid
(20.0 mg, 71.0%).
tert-Butyl
N-[[4-[5-(hydroxymethyl)-3-methylisoxazol-4-yl]phenyl]-
methyl]carbamate (40) Compound 40 was synthesized from [4-[4-[(tert-
butoxycarbonylamino)methyl]phenyl]-3-methylisoxazol-5-yl]methyl
acetate (32, 8.0 g, 22 mmol) to afford the title compound as a yellow solid
(6.5 g, 92%).
tert-Butyl N-[[4-[3-methyl-5-[(3-vinylphenoxy)methyl]-isoxazol-4-yl]-
phenyl]methyl]carbamate (41) Compound 41 was synthesized from tert-
butyl
N-[[4-[5-(hydroxymethyl)-3-methylisoxazol-4-yl]phenyl]methyl]-
carbamate (40, 6.50 g, 20.4 mmol) to afford the title compound as a grey
solid (4.5 g, 52%).
[4-[3-Methyl-5-[(3-vinylphenoxy)methyl]isoxazol-4-yl]phenyl]methan-
amine (42) Compound 42 was synthesized from tert-butyl N-[[4-[3-methyl-
5-[(3-vinylphenoxy)methyl]isoxazol-4-yl]phenyl]methyl]carbamate
(41,
4.50 g, 10.7 mmol) to afford the title compound as a yellow solid (2.5 g,
crude yield 73%).
N-[[4-[3-Methyl-5-[(3-vinylphenoxy)methyl]isoxazol-4-yl]phenyl]-
methyl]prop-2-ene-1-sulfonamide (43) Compound 43 was synthesized
from
[4-[3-methyl-5-[(3-vinylphenoxy)-methyl]isoxazol-4-yl]phenyl]-
methanamine (42, 2.5 g, 7.8 mmol) to afford the title compound as a yellow
solid (2.0 g, 60%).
3-Methyl-8,12,13,20-tetrahydro-9H,11H-4,7-etheno-14,18-
(metheno)[1,2]oxazolo[4,5-q][1,10,11]oxathiazacyclononadecine
10,10-dioxide (44) Compound 44 was synthesized from N-[[4-[3-methyl-
5-[(3-vinylphenoxy)methyl]isoxazol-4-yl]phenyl]methyl]prop-2-ene-1-
sulfonamide (43, 0.50 g, 1.2 mmol) to afford the title compound as a white
solid (60 mg, 12% over two steps).
Fluorescence polarization (FP) ligand binding assays for AR, GR and
PR Competition binding studies were performed using Polarscreen™ AR,
GR and PR Competitor Assay kits (Life Technologies). Compounds were
tested in 10-point concentration response, ½ log serial dilution, final assay
start concentration of 50 µM, in black 384-well low volume Corning plates
(NBS surface, U bottom). To assay plates with 50 nL test or control
compound, 5 µL of either AR Fluormone RED, GR Fluormone RED or PR
Fluormone RED were added, followed by 5 µL AR (Rat LBD), GR (Human
full length protein) or PR (Human LBD). Final concentrations in the three
different assays were 2 nM AR Fluormone RED/ 120 nM AR, 1 nM GR
Fluormone RED/ 1500 nM GR or 2 nM PR Fluormone RED/ 150 nM PR
respectively. After a 4 h (AR and GR assays) or 2 h (PR assay) incubation
time in darkness at rt, the plates were read on a PHERAstar Plus (BMG
Protein purification, crystallography, and biological assays
Mineralocorticoid receptor binding assay A 384-well format scintillation
proximity assay (SPA) was used to identify compounds that show binding
to the human mineralocorticoid receptor LBD. The immobilization of the
fusion protein to the scintillation beads was done via rabbit MBP (maltose-
binding protein) antibodies that were captured by the anti-rabbit SPA PVT
(Polyvinyltoluene) beads. The inhibition of the scintillation signal by
displacement of 3H-aldosterone by test compounds was measured on
LEADseeker (GE Healthcare). Assay was run in 384-well, white, medium
binding plates (Greiner). Assay buffer contained 10 mM Tris pH 7.5
(Sigma-Aldrich), 0.5 mM EDTA (Sigma-Aldrich), 20 mM sodium molybdate
dihydrate (Sigma-Aldrich), 10% Glycerol (KEBO Lab) and 0.1 mM DTT
(Sigma-Aldrich). A MR/3H-aldosterone solution was prepared by mixing 7
µg/mL MBP-tagged human mineralocorticoid receptor ligand binding
domain (residues 729–984) co-infected with p23 lysate (MBP-MR
LBD/p23) with 5 nM 3H-aldosterone (Perkin Elmer). 20 µL of MR/3H-
aldosterone solution was added to assay plates with 150 nL test or control
compound and incubated for 1 h. 2.5 mg/mL SPA imaging beads (Perkin
Labtech) using
a
FP optic module (540/590/590). Results, in
millipolarization (mP), were analyzed in Screener (Genedata AG) and the
IC50 values were calculated using a 4 parameter logistic fit. pIC50 was
reported as mean with the number of experiments n≥3. CIR for AR was
1.44, GR 1.61 and PR 1.58 respectively for compounds tested on three
occasions and the MDD for each assay was 0.23 for AR, 0.29 for GR and
0.28 for PR.
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Mineralcorticoid receptor translocation assay To study ligand activated
MR translocation into the nucleus and the blocking effect on MR
translocation, we used EA.hy926 cells (ATCC®CRL-2922). Cells were
either treated with compounds for MR translocation assay or compounds
in presence of 0.1 nM aldosterone for assay in MR translocation blocking
mode. Cryo preserved cells were thawed and washed once with assay
medium (DMEM (Gibco) with 10% charcoaled stripped FBS (Gibco))
before seeded, 6000 cells/well in 25 µL assay medium, in 384-well Optilux
black/clear plates (Corning). Plates were incubated for 24 h at the same
conditions as above. 25 nL compound solution was added to the plates by
acoustic dispensing (Echo, Labcyte) with a ½ log serial dilution to give a
ten point concentration response curve starting at 10 µM. For the MR
blocking mode we used 0.1 nM aldosterone (corresponding to EC80 value)
as an agonist and 25 nL was added to all compound wells. Plates were
incubated for 1 h in 37°C, 10% CO2 and 95% humidity. Medium was
completely removed and cells were fixed with 4% buffered
paraformaldehyde (Sigma-Aldrich), 50 µL/well, for 20 min at rt. Plates
were washed 3 times with PBS, permeabilised with 0.2% Triton-X100
(Sigma-Aldrich) in PBS, 25 µL/well for 15 min at rt and washed three times
with PBS. Blocking solution, containing 5% BSA (Sigma-Aldrich) and 5%
normal goat serum (Jackson Immunoresearch) in PBS was added,
15 µL/well for 30 min at rt. Blocking solution was removed and primary
antibody rMR 1–18 1D5 (DSHB) added, 1/1500 dilution in 5% BSA/PBS
and incubated at 4°C ON. Plates were washed twice with PBS/0.05%
TWEEN and twice with PBS, followed by addition of 15 µL of secondary
antibody, goat-anti-mouse AF488 (InVitrogen) and stained with Hoechst
33342 (InVitrogen) 10 µg/mL in 5% BSA/PBS for 1 h in rt. Plates were
washed twice with PBS/0.05% TWEEN and twice with PBS, left with 50 µL
PBS/well and sealed. The plates were imaged on ImageXpress Micro
system (MolecularDevices) with a 10X Sfluor objective, taking one site per
well. Two wavelengths were measured, DAPI and FITC, using the
standard filters. The images were quantified using MetaXpress software
(MolecularDevices). The difference in fluorescence intensity between the
cytoplasm and the nucleus was used to quantify the nuclear
translocation/blocking event. Raw data output was analyzed in Screener
software (Genedata AG) and EC50/IC50 values were calculated using a 4-
parameter logistic fit.
dialyzed with compound of interest (50 µM). Directly upon dilution and
addition of >100-fold excess of competitor (100 µM Aldosterone), the
change in receptor binding to surface-immobilized co-factor peptide
(PRGC2 146-166), caused by the ligand-induced change in affinity, was
monitored by consecutive injection cycles (typically 61 min injections) in
SPR. The dissociation rate was determined by exponential fits to the
positive change in binding rate as a function of time. 120 nM MR in
HBSP(+) buffer (10 mM HEPES, 150 mM NaCl, 0,005% P20, pH 7.4) was
used for all measurements. The peptide surface was regenerated with
0.005% SDS after each injection.
Lipophilicity[45] Lipophilicity, measured as the distribution coefficient
(logD) between octanol and 10 mM sodium phosphate buffer with pH
adjusted to 7.4. For all DMPK assays a more detailed assay description is
given in the supporting information.
Solubility[45] This method measures the thermodynamic solubility of
research compounds based on a shake-flask approach.[47]
Metabolic stability in human and male Han Wistar rat hepatocytes[48]
Hepatocyte metabolic stability was determined in accordance with the
method described by Jacobson et al.[49]
Intrinsic permeability across Caco-2 cell monolayers A monolayer of
Caco-2 cells was used to study the permeability in the apical to basolateral
direction.
In vivo rat PK was assessed as previously described.[48] The studies were
approved by the Göteborg Animal Research Ethical Board.
Glutathione trapping[50] Compounds were incubated in human liver
microsomes in the presence of the trapping agent glutathione and NADPH
at pH 7.4 and 37°C.
CYP inhibition in human liver microsomes The objective of this study
is to measure the potential of a compound to inhibit a specific cytochrome
P450 enzyme in human liver microsomes (HLM).
Protein expression, purification and crystallization Expression and
purification of MR-LBD (amino acids 735-984) with the mutations C808S,
C910S and S810L, an N-terminal, TEV cleavable 6-HN tag, and a C-
terminal thrombin cleavable co-activator peptide PQAQQKSLLQQLLTE
was performed as described previously.[39] Crystals of MR (residues 735–
984) C808S, C910S and S810L co-expressed and purified with 44 were
grown by sitting drop vapor diffusion in 0.9 M (NH4)2SO4, 0.1 M CHES pH
9.5, and 10% (v/v) of condition G5 of Morpheus® Screen (Molecular
Dimensions). The same crystallization conditions were used for MR in
complex with compounds 25d and 1 with the exception that 10% (v/v) of
Morpheus® conditions C9 and F12 were added to the well solutions
respectively instead of condition G5. The crystals were quickly dipped into
well solution supplemented with 18% ethylene glycol before being flash
frozen in liquid nitrogen.
Acknowledgements
The authors would like to acknowledge the Separation Science
Group at AstraZeneca Gothenburg for help with compound
purification and HRMS as well as and Global DMPK for support
with DMPK and physicochemical profiling.
Keywords: Drug design • Macrocycles • Nuclear hormone
receptor • SBDD • NR3C2
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Entry for the Table of Contents
A biarylsulfonamide was identified as a mineralocorticoid receptor antagonist in an HTS campaign. Structure–based drug design was
used to improve selectivity over the oxosteroid receptors by exploring an induced fit pocket and expanding the ligands towards an
area where the receptors differ from each other. X-ray crystallography confirmed the binding mode and inspired macrocyclization to
test conformationally-restrained MR antagonists.
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