Discovery of Highly Potent Liver X Receptor β Agonists

Article abstract of DOI:10.1021/acsmedchemlett.6b00234

Introducing a uniquely substituted phenyl sulfone into a series of biphenyl imidazole liver X receptor (LXR) agonists afforded a dramatic potency improvement for induction of ATP binding cassette transporters, ABCA1 and ABCG1, in human whole blood. The ag

Full text of DOI:10.1021/acsmedchemlett.6b00234

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Letter
pubs.acs.org/acsmedchemlett
Discovery of Highly Potent Liver X Receptor β Agonists
Ellen K. Kick,*,†,§ Brett B. Busch,‡,§ Richard Martin,‡ William C. Stevens,‡ Venkataiah Bollu,‡
Yinong Xie,‡ Brant C. Boren,‡ Michael C. Nyman,‡ Max H. Nanao,‡ Lam Nguyen,‡ Artur Plonowski,‡
Ira G. Schulman,‡ Grace Yan,‡ Huiping Zhang,† Xiaoping Hou,† Meriah N. Valente,†
Rangaraj Narayanan,# Kamelia Behnia,# A. David Rodrigues,# Barry Brock,# James Smalley,#
Glenn H. Cantor,# John Lupisella,∥ Paul Sleph,∥ Denise Grimm,∥ Jacek Ostrowski,∥ Ruth R. Wexler,†
Todd Kirchgessner,∥ and Raju Mohan‡
†Department of Discovery Chemistry, ∥Department of Cardiovascular Biology, #Pharmaceutical Candidate Optimization, Research &
Development, Bristol-Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543-5400, United States
‡Exelixis Inc., 210 East Grand Avenue, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Introducing a uniquely substituted phenyl
sulfone into a series of biphenyl imidazole liver X receptor
(LXR) agonists afforded a dramatic potency improvement for
induction of ATP binding cassette transporters, ABCA1 and
ABCG1, in human whole blood. The agonist series
demonstrated robust LXRβ activity (>70%) with low partial
LXRα agonist activity (<25%) in cell assays, providing a
window between desired blood cell ABCG1 gene induction in
cynomolgus monkeys and modest elevation of plasma
triglycerides for agonist 15. The addition of polarity to the
phenyl sulfone also reduced binding to the plasma protein, human α-1-acid glycoprotein. Agonist 15 was selected for clinical
development based on the favorable combination of in vitro properties, excellent pharmacokinetic parameters, and a favorable
lipid profile.
KEYWORDS: Liver X receptor, LXRα, LXRβ, ABCA1, ABCG1, α-1-acid glycoprotein
positive effects described above while not causing increases in
low-density lipoprotein cholesterol (LDL-C) and triglycerides
(TG). The TG increases are primarily caused by LXR induction
of the transcription factor sterol regulatory element binding
protein 1c (SREBP1c) and the enzyme fatty acid synthase
(FAS) in liver, leading to increased very low density lipoprotein
(VLDL) production and secretion from liver,14 as well as
upregulation of hepatic angiopoietin-like protein 3 and down-
regulation of apoA-V expression resulting in decreased lipolysis
of circulating TG-rich lipoproteins.16,17 Increased LDL-C has
been reported after repeat dose treatment in hamsters and
cynomolgus monkeys with LXR agonists.18 The LDL-C
increases may be caused by a combination of multiple
mechanisms, including increased VLDL production, induction
of cholesteryl ester transfer protein (CETP),19 and induction of
inducible degrader of LDL receptor (IDOL).20 The lipid effects
have been proposed to be driven by hepatic LXRα based on
knockout mice,21,22 influencing the field to optimize for LXRβ
selectivity.8
dentification of drugs to treat coronary heart disease (CHD)
Ipatients continues to be an important research area. Despite
significant advances in treatment including statin therapy, CHD
caused by atherosclerosis remains a major cause of morbidity
and mortality in the United States.1 Owing to the powerful
effect of liver X receptor (LXR) agonists on reverse cholesterol
transport (RCT)2 and immune system modulation,3,4 which
culminate in reduced atherosclerosis lesions in animals,5−7
there have been many LXR agonist medicinal chemistry
campaigns to treat atherosclerosis driven cardiovascular
disease.8 LXRα and LXRβ agonists increase RCT by induction
of ATP binding cassette transporters ABCA1 and ABCG1,9,10
which efflux cholesterol from cells to HDL particles, and
transporters ABCG5 and ABCG8, which traffic cholesterol
from liver to the feces and promote its excretion. The immune
system effects of LXR receptors continue to be elucidated, and
LXRs are involved in innate and acquired immunity processes.3
LXRs regulate many additional pathways in lipid homeostasis
and energy utilization, and as such LXR agonists have been
suggested as therapeutic treatments for other diseases including
several types of cancers,11 skin conditions,12 and heart failure.13
While many LXR agonists have been reported including the
well-studied TO-091317 (1)14 and GW3965 (2),15 a challenge
in the field has been to develop agonists that maintain the
Received: June 10, 2016
Accepted: September 27, 2016
© XXXX American Chemical Society
A
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Several reports of LXR agonists with improved therapeutic
windows compared to full pan-agonists have been described
(Figure 1). For instance, the agonist LXR-623 (3) has been
Table 1. SAR Optimization of Lead 5
LXRβ
/LXRα
Binding Ki,
nM
LXRβ
EC50 nM
(% eff)
LXRα
EC50 nM
(% eff)
ABCA1 HeLa
EC50 nM (%
eff)
hWBA
EC50 nM
(% eff)
#
5
14/68
11/16
5/31
250
220
33
1200
(55%)
380
(72%)
170
(38%)
99
(50%)
18
Figure 1. Examples of LXR agonists reported in the literature.
6
(41%)
60
(20%)
100
(42%)
9
(32%)
300
7
reported to decrease LDL-C in cynomolgus monkeys, while
inducing transporters such as ABCA1; however, the mechanism
for the LDL-C decrease has not been reported.23,24
Unfortunately neurological effects were observed in humans
after 1 day of dosing 3.25 Further supporting that efficacy can
be achieved without lipid effects, AZ876 (4) was reported to
reduce atherosclerosis plaques and improve heart failure
outcomes in mice at doses that did not cause increased
TGs.26 We have previously reported that an improved
therapeutic window can be achieved with agonist BMS-
779788 (5).27,28
Agonist 5 shows a preference for LXRβ in binding and
functional assays and induces LXR target genes ABCA1 and
ABCG1 in human whole blood with an EC50 value of 1.2 μM
and 55% efficacy (Table 1).27 An improved lipid profile was
observed in mouse and cynomolgus monkeys with 5 compared
to TO-091317 (1), and agonist 5 had a good pharmacokinetic
and safety profile so it was taken into human trials.28 Key goals
for optimization were to identify a molecule with lower LXRα
agonist activity to minimize the TG effects, while improving the
potency for on-target ABCA1 and ABCG1 induction in human
whole blood.
The imidazole agonists were prepared as reported
previously27 and as described in Supporting Information.
Agonists were profiled in a suite of five LXR assays. The
binding affinity was determined with full-length LXRα−RXRα
and LXRβ−RXRα heterodimers.29 Functional isoform activity
was assessed using LXRα and LXRβ transactivation assays in
CV-1 cells,30 and in HeLa cells with endogenous LXRα and
LXRβ receptors and an ABCA1 LXREx3 reporter. Compounds
were tested for ABCA1 and ABCG1 induction in a human
whole blood assay (hWBA). The hWBA potency was a key
driver because we anticipated it would predict clinical efficacy.
The hWBA EC50 value for ABCA1 gene induction is reported
because we used that data to evaluate SAR; in general, ABCG1
EC50 values were within 2-fold and are reported for 5 and 15 in
the Supporting Information.
(50%)
170
(39%)
110
(54%)
12
(72%)
300
8
13/74
40/180
10/53
(74%)
220
(46%)
120
(56%)
21
(46%)
870
9
(93%)
72
(47%)
76
(68%)
8
(47%)
57
10
(83%)
(29%)
(43%)
(47%)
a
Standard deviations are reported in the Supporting Information when
n > 2.
In pursuit of improving properties, several areas were
explored simultaneously, including substitutions on the A and
D rings (Table 1). While many compounds were prepared with
different R1 substitution patterns that had similar activities to 5
(structures and data not shown), the 2,6-dichloro substitution
(6) achieved a 3-fold boost in hWBA potency with lower LXRα
efficacy (20%) compared to 5 (38% LXRα efficacy).
Substituted phenyl sulfones were prepared based on the
LXRβ crystal structure obtained with agonist 5 that had a
water channel where R4 is positioned.27 Investigations to
exploit this position led to the synthesis of the hydroxymethyl
sulfones 7 and 8. Small gains in potency were observed in
LXRα and LXRβ agonist assays, which translated to 300 nM
EC50 values for hWBA ABCA1 induction. Since 7 was prepared
first it was dosed to mice at 10 mg/kg, and good plasma
exposure was observed (Supporting Information); however,
when dosed to cynomolgus monkeys, the observed clearance
was higher than hepatic blood flow at 61 mL/min/kg (Table
2). Cynomolgus monkeys were critical to compound
progression because they were a key model to study the lipid
effects. High clearance was also observed with other analogues
containing this D-ring substitution (data not shown), so
compounds were prepared to address the high clearance.
Anticipating that a secondary alcohol would have reduced
clearance, 9 was prepared and found to show partial 47%
activity at LXRα with an hWBA EC50 value of 870 nM.
B
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Table 2. PK in Cynomolgus Monkeys after i.v. Dosing
Table 3. Optimization of R1, R2, and R3 with (2-Fluoro-6-
(methylsulfonyl)phenyl)methanol D-Ring
example
5
5
7
9
10
15
dose (mg/kg)
Cl (mL/min/kg)
t1/2 (h)
1.0
1.9
7.4
0.2
2.9
8.9
0.25
0.2
5.6
5.5
0.2
8.4
5.6
3.0
8.0
61
0.8
12
a
n = 2 for 0.2 and 0.25 mg/kg doses, and n = 3 for 1 mg/kg doses.
Standard deviations for 5 and 15 are in the Supporting Information.
Analogue 10 was prepared with an adjacent electron with-
drawing fluorine at R5 to try to slow metabolism of the
hydroxymethyl R4 substituent. Compound 10 had LXRβ
agonist potency of 72 nM with a dramatic improvement in
hWBA potency to 57 nM (47% efficacy), which was 20-fold
better than 5. While limited selectivity was observed in binding
assays, 10 showed differential agonist activity with 29% LXRα
efficacy and robust LXRβ 83% efficacy. Both 9 and 10 had
improved clearance rates of 5.6 and 8.4 mL/min/kg in
cynomolgus monkeys (Table 2). Due to the significant potency
improvement with 10 we progressed this series and did not test
the closest comparator 8 in cynomolgus monkey PK to confirm
the improved clearance was entirely due to the fluorine.
With optimized D-ring substituents, we focused SAR
exploration on the A and C aryl rings (Table 3). Our goal
was to identify the most potent analogues with LXRα efficacy
≤25% and robust LXRβ efficacy. The 2-fluoro substitution (11)
at R1 had a similar profile to 10, although the % efficacy was
modestly reduced across all agonist assays. Mirroring the
observation with 6, the 2,6-dichloro analogue 12 had a 3−4-
fold improvement in hWBA potency to 15 nM with limited
25% LXRα efficacy. Interestingly, the 2-Cl,3-F analogue 13 had
very limited LXRα activity at 12% while maintaining a hWBA
EC50 value of 76 nM. The R3 fluorine substitution (14)
maintained similar activity assays compared to 10. When the
fluorine R3 substitution was combined with the 2,6-diCl A-ring
(15) very potent hWBA activity was observed with an EC50
value of 9 nM (26% efficacy). Although 15 has similar LXRα
and LXRβ binding Ki values (19 and 12 nM, respectively), in
agonist assays the compound achieved 88% efficacy toward
LXRβ and only 20% efficacy toward LXRα compared to a full
pan-agonist. When tested in antagonist mode, 15 was a potent
LXRα antagonist with an IC50 value of 69 nM (83% inhibition);
whereas no antagonism was observed in LXRβ assays up to
10,000 nM (Supporting Information). Further SAR inves-
tigation with a 2-Cl,6-F R1 substitution pattern provided 16
with a hWBA potency of 41 nM (33%). Introduction of an R3
chlorine atom in 17 caused a decrease in efficacy in all four
agonist assays with a potent hWBA EC50 value of 5 nM just
above the limit of assay detection (16%). Analogue 18 with
hydrogen at R2 had in vitro activity consistent with the gem-
dimethyl analogue 12. The monomethyl analogue 19 was
identified as a metabolite of 15, and upon synthesis the profile
showed it to be a potent, partial LXR agonist as well.
LXRβ/
LXRα/
Binding Ki,
nM
LXRβ
EC50 nM
(%Eff)
LXRα
EC50 nM
(%Eff)
ABCA1 HeLa
EC50 nM (%
Eff)
hWBA
EC50 nM
(%Eff)
#
11
14/81
160
130
12
43
(68%)
42
(13%)
30
(15%)
3
(28%)
15
12
13
14
15
16
17
18
19
6/38
(72%)
72
(25%)
72
(23%)
8
(43%)
76
14/53
18/9
(68%)
50
(12%)
57
(16%)
2
(34%)
46
(79%)
24
(25%)
8
(29%)
0.6
(35%)
9
12/19
14/70
48/50
11/75
13/17
(88%)
20
(20%)
11
(29%)
1
(26%)
41
(86%)
27
(15%)
8
(30%)
2
(33%)
5
(51%)
140
(6%)
69
(12%)
5
(16%)
42
(54%)
25
(17%)
12
(43%)
2
(51%)
23
(67%)
(18%)
(9%)
(17%)
a
Standard deviations are reported in the Supporting Information when
n > 2.
distribution and slow clearance in humans was tight binding
to α-1-acid glycoprotein (α1 AGP), which was different
between human and preclinical species (manuscript in
preparation). A similar explanation has been proposed for the
human PK of UCN-01 (7-hydroxystaurosporine).31 Equili-
brium dialysis with 5 demonstrated 99.9% binding to human α1
AGP. In contrast 5 has moderate binding of 97, 92, and 98% to
human serum albumin (HSA) and rat and dog α1 AGP,
respectively. Binding to human α1 AGP was measured for
several agonists (Table 4). Whereas the biphenyl sulfones 5 and
6 had >99% binding to α1 AGP, introduction of the polar R4
hydroxymethyl group in 8, 10, 13, and 15 reduced α1 AGP
binding, consistent with the hWBA potency improvement.
Binding to HSA, a major plasma protein, did not differentiate
the analogues with 94−98% bound.
During the characterization of our lead molecules the clinical
single ascending dose PK results were available for 5. The
human plasma t1/2 was 100−200 h, which was 5−10-fold longer
than predicted by preclinical studies. Studies with clinical
plasma samples showed high binding (i.e., >99.9%) of 5 to
plasma proteins. The volume of distribution in humans was at
least 10× smaller than the projected values based on preclinical
species, suggesting limited distribution of this molecule outside
of systemic circulation. One hypothesis brought forward to
explain the unexpected long half-life, limited volume of
C
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effects. In addition, 15 was very potent in the hWBA.
Analogues 13 and 15 were not active in 16 nuclear hormone
receptor agonist assays (>10 μM), except PXR with EC50 values
of 3 μM (85% of full agonism) and 1 μM (108% of full
agonism), respectively. When dosed in mice at 10 mg/kg, the
Cmax coverage was high compared to the hWBA potency
(Supporting Information). Given that LXR agonists could have
deleterious effects in brain, as observed with LXR-623, the brain
levels were measured and found to be low with 15 having a
brain to plasma ratio of <0.05. In cynomolgus monkeys, 13 and
15 displayed good bioavailability, moderate clearance rates, and
10−12 h plasma half-lives (Supporting Information).
Table 4. Equilibrium Dialysis with Human α1 AGP and HSA
example
human α1 AGP (% bound)
HSA (% bound)
5
99.9 0.0
99.6 0.2
90.2 4.2
86.6 0.9
87.6 1.3
97.3 0.1
97.2 0.3
97.9 0.5
96.1 0.7
95.5 0.2
94.4 1.1
96.6 0.1
6
8
10
13
15
a
The average is reported with standard deviation (n = 3).
The crystal structure of 15 complexed with the ligand
binding domain of LXRβ has been determined to 2.4 Å
While 15 was considered the lead compound due to
exceptional hWBA potency coupled with low LXRα efficacy
(Table 3), both 13 and 15 were studied in cynomolgus
monkeys for 14 days to investigate the ABCG1 dose response
to the lipid effects compared to those of 1. The agonists
showed robust induction of the RCT target gene ABCG1 in
plasma at drug concentrations that were predicted by the
cynomolgus monkey WBA potency (1 cynoWBA EC50 = 310
nM (100%); 13 cynoWBA EC50 = 52 nM (29%); 15 cynoWBA
EC50 = 5 nM (32%)). ABCA1 had shown variable vehicle
effects in multiple cynomolgus monkey studies, precluding its
use as a pharmacodynamic biomarker. Both 13 and 15 had
improved TG profiles compared to 1. Fourteen days of dosing
1 at 10 mg/kg (200 nM plasma concentration at 5 h) caused a
6-fold ABCG1 induction in blood cells with TGs elevated 140%
over baseline values (p < 0.05, ANOVA). After 14 days, the 1
and 3 mg/kg doses of 13 afforded 4- and 10-fold ABCG1
induction in blood cells with 85 and 310 nM plasma exposures,
respectively. These doses yielded TGs of 2% and 58% above
baseline (not significant). Comparatively the 0.1, 0.3, and 1
mg/kg doses of 15 provided 5 h plasma exposures of 7.5, 22,
and 57 nM with 4.7-, 15-, and 11-fold ABCG1 induction on day
14. The TGs were elevated nonsignificantly 20, 8, and 10% over
baseline, respectively. As anticipated, 15 provided robust
ABCG1 induction at very low plasma drug concentrations,
with little effect on plasma TGs. A full data set from this
cynomolgus monkey study 15 is reported elsewhere.32
In summary, we have identified a potent biphenyl imidazole
series of LXR partial agonists containing a (2-fluoro-6-
(methylsulfonyl)phenyl)methanol substituent. Importantly,
agonist 15 induces ABCA1 and ABCG1 RCT targets in
human whole blood at nanomolar drug exposures with robust
LXRβ agonism and limited LXRα agonist activity (Table 3 and
Supporting Information). In cynomolgus monkeys this profile
gave robust blood cell activity with limited elevations of TGs.
Based on coupling the in vitro properties with an excellent
pharmacokinetic profile and favorable lipid profile in
cynomolgus monkeys, 15 (BMS-852927) advanced into
clinical studies. In human trials the PK was well predicted by
preclinical data; however, TGs and LDL-C were observed to be
elevated after multiple days of dosing with a limited therapeutic
window, indicating that the low LXRα efficacy was not
sufficient to protect from deleterious lipid elevations.32
resolution (Figure 2). The complex crystallized in space group
Figure 2. LXRβ complexed with 15 to 2.4 Å resolution (PDB code:
5JY3).
C2 with four independent subunits in the asymmetric unit, with
subunits A and B forming a canonical dimer, as did C and D.
Helix 12 from subunit A was bound in the coactivator binding
pocket of subunit B. The binding mode of 15 is very similar to
that of 5, and there do not appear to be any major changes to
side chain positions. The sulfone interacts with the backbone
Leu330 as was observed with 5. The hydroxymethyl interacts
with Ser-278, Glu281, and a bound butane diol molecule from
the crystallization solvent. The fluorine gives an improved
shape complementarity to the pocket near the “D” ring of 15
compared to 5, likely providing some of the improved potency.
The benzylic phenyl (A-ring) forms a pi-stacking interaction
with Phe340. The second chlorine substituent causes the
benzylic methyl groups to rotate compared to 5, improving the
molecular shape complementarity to the LXR pocket. Whereas
many LXR agonists have a direct interaction with His435
stabilizing helix 128 that is important for LXR agonist activity,
the carbinol hydroxyl group of 17 appears to interact with a
water in the active site that looks to be positioned to H-bond
with His435. While we are not able to give a definitive
structural reason for the low LXRα agonist activity from the
LXRβ structure, it is possible that the indirect interaction
through a water molecule with His 435 provides some of the
observed differences between LXRα and LXRβ agonist activity.
Agonists 13 and 15 were nominated for further study
because both compounds had robust LXRβ efficacy with low
LXRα agonist efficacy (<20%), which was anticipated to
improve the separation of desired efficacy from TG and LDL-C
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00234.
D
DOI: 10.1021/acsmedchemlett.6b00234
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ACS Medicinal Chemistry Letters
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AUTHOR INFORMATION
Corresponding Author
*Tel: 609 466-5053. E-mail: ellen.kick@bms.com.
■
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Author Contributions
§These authors contributed equally to this work.
Funding
Bristol-Myers Squibb and Exelixis funded the described work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Akbar Nayeem for preparing Figure 2; Amy
Gallagher, Chris Bonagura, and Hangjun Zhan for LXRβ
crystallization efforts; Bang-Chi Chen for help with large scale
synthesis of 15; and Petia Shipkova, Robert Langish, Michael
Witkus, Yingru Zhang, Michael Hicks, Cindy Li, and Rich
Dalterio for analytical characterization of 13 and 15.
ABBREVIATIONS
■
α1 AGP, α-1-acid glycoprotein; ABCA1, ABCG1, ABCG5, or
ABCG8, ATP binding cassette transporter; apoA, apolipopro-
tein A; CHD, coronary heart disease; cyno, cynomolgus
monkey; HDL, high density lipoprotein; HSA, human serum
albumin; hWBA, human whole blood assay; LDL-C, low-
density lipoprotein cholesterol; LXR, liver X receptor; PAMPA,
parallel artificial membrane permeability assay; PK, pharmaco-
kinetics; PXR, pregnane X receptor; RCT, reverse cholesterol
transport; RXR, retinoid X receptor; SAR, structure−activity
relationship; TG, triglygerides; VLDL, very low density
lipoprotein
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