Optimization of a potent, orally active S1P1 agonist containing a quinolinone core

Article abstract of DOI:10.1021/ml200252b

The optimization of a series of S1P₁ agonists with limited activity against S1P₃ is reported. A polar headgroup was used to improve the physicochemical and pharmacokinetic parameters of lead quinolinone 6. When dosed orally at 1 and 3 mg/kg, the azahydroxymethyl analogue 22 achieved statistically significant lowering of circulating blood lymphocytes 24 h postdose. In rats, a dose-proportional increase in exposure was measured when 22 was dosed orally at 2 and 100 mg/kg.

Full text of DOI:10.1021/ml200252b

Letter
pubs.acs.org/acsmedchemlett
Optimization of a Potent, Orally Active S1P1 Agonist Containing a
Quinolinone Core
Paul E. Harrington,*,† Michael D. Croghan,† Christopher Fotsch,† Mike Frohn,† Brian A. Lanman,†
Lewis D. Pennington,† Alexander J. Pickrell,† Anthony B. Reed,† Kelvin K. C. Sham,† Andrew Tasker,†
Heather A. Arnett,‡ Michael Fiorino,‡ Matthew R. Lee,§ Michele McElvain,∥ Henry G. Morrison,⊥
Han Xu,∥ Yang Xu,# Xuxia Zhang,‡ Min Wong,‡ and Victor J. Cee†
‡
§
∥
†Chemistry Research and Discovery, Inflammation Research, Molecular Structure, HTS and Molecular Pharmacology,
⊥Pharmaceutics, and Pharmacokinetics and Drug Metabolism, Amgen, One Amgen Center Drive, Thousand Oaks, California
#
91320-1799, United States
S
* Supporting Information
ABSTRACT: The optimization of a series of S1P1 agonists with limited activity against
S1P3 is reported. A polar headgroup was used to improve the physicochemical and
pharmacokinetic parameters of lead quinolinone 6. When dosed orally at 1 and 3 mg/kg, the
azahydroxymethyl analogue 22 achieved statistically significant lowering of circulating blood
lymphocytes 24 h postdose. In rats, a dose-proportional increase in exposure was measured
when 22 was dosed orally at 2 and 100 mg/kg.
KEYWORDS: Sphingosine-1-phosphate receptor, S1P1, agonist, multiple sclerosis
bind to the S1P1 receptor, resulting in receptor internalization,
which prevents lymphocyte egress from lymphoid organs.10,11
The sequestration of lymphocytes in the lymphoid organ and
resulting decrease in circulating lymphocytes has been
postulated to explain the efficacy of S1P1 agonists in preclinical
models of autoimmune diseases including multiple sclerosis
(MS),12,13 where these cells participate in the pathogenesis of
the disease.14
he lysophospholipid sphingosine-1-phosphate 1 has been
Timplicated in an array of cellular signaling pathways such
as apoptosis, proliferation, migration, and differentiation
(Figure 1).1 S1P signaling is mediated by five G-protein
Until recently, agonism of S1P3 by 2 was believed to be the
exclusive cause of the transient dose-dependent bradycardia
that was observed in clinical trials of FTY720.15 This
conclusion was supported by rodent data wherein dosing of 2
to normal mice resulted in bradycardia, whereas administration
of 2 to S1P3 knockout mice caused no change in heart rate.16,17
More recent literature18,19 suggests that S1P1 rather than S1P3
is responsible for the observed heart rate changes in humans.
Despite this side effect, FTY720 was approved by the United
States Food and Drug Administration for the treatment of
relapsing forms of MS.20,21 Herein, we report the discovery of a
S1P1 agonist with minimal S1P3 activity and excellent
pharmacokinetic parameters.
Figure 1. Agonists of S1P1.
In prior work,22 we reported a series of conformationally
restricted S1P1 agonists derived from 5.9 Quinolinone 6
demonstrated excellent potency in an assay that measured
receptor internalization (RI) of hS1P1-GFP fusion protein in
U2OS cells, in addition to limited hS1P3 activity as determined
coupled receptor (GPCR) subtypes known as S1P1−5.2 The
role of S1P1 in lymphocyte trafficking in the lymph system,
where it is highly expressed, has been the focus of intense
research.3 These efforts were aided by the disclosure that 2
(FTY720P), which is derived from 3 (FTY720) in vivo by
selective phosphorylation by sphingosine kinase, is a non-
selective S1P1 agonist.4,5 In addition, FTY720P is also an
agonist of S1P3−5. Agonists6,7 such as 2, 4 (AMG 369),8 and 59
Received: October 21, 2011
Accepted: November 23, 2011
Published: November 23, 2011
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
by Ca2+ mobilization in hS1P3/Gq/i5-transfected Chinese
hamster ovary (CHO)-K1 cells. In vivo, 6 lowered circulating
lymphocytes in rats after a single oral dose of 1 mg/kg when
measured at 24 h (vide infra). However, when dosed orally in
rats at 100 mg/kg, the exposure of 6 failed to increase
proportionally relative to the 3 mg/kg dose despite efforts to
optimize the formulation (Figure 1). The lack of dose-
proportional exposure limited further development of 6.
Furthermore, 6 suffered from poor solubility (5 μg/mL) in
simulated intestinal fluid (SIF) and relatively high lipophilicity
(cLog P = 3.5).23 Efforts aimed at introducing additional
nitrogen atoms to the carbon skeleton were only moderately
successful. Thus, more significant structural modifications of 6
were directed at decreasing the cLog P and improving the
aqueous solubility while maintaining or improving the
pharmacological profile.
6 (EC50 = 0.024 μM) was slightly more potent than the parent
7 (EC50 = 0.070 μM) and 22 was equipotent with 9.
Alcohol 22 was prepared as described in Scheme 1.
Condensation of 23 with 2-chloropyridin-4-amine provided
a
Scheme 1. Synthesis of 22
Many of the S1P1 agonists that have been disclosed contain a
polar headgroup (Figure 1).6,7 This group is usually comprised
of an amino acid, amino alcohol, or a diol that is attached to a
lipophilic carbon skeleton. Quinolinone 6 lacked a polar
headgroup, which could provide a handle to modulate the
physicochemical properties without detrimentally affecting the
pharmacological profile. To investigate whether attachment of a
polar moiety to 6 would result in improved properties, a
hydroxymethyl group was attached at several positions about a
fused bicyclic core of the scaffold. Compounds derived from
the carbon analogue 7 were initially targeted to simplify the
challenge of synthesizing azaquinolinones containing a polar
headgroup. Gratifyingly, as shown in Table 1, alcohols 8 and 9
demonstrated that a hydroxymethyl group could be incorpo-
rated into the carbon skeleton at either the 8- or the 7-position,
respectively, without compromising potency (8, EC50 = 0.039
μM; 9, EC50 = 0.018 μM vs 7, EC50 = 0.070 μM). However, the
6-substituted analogue 10 was substantially less potent than 7.
Additional efforts aimed at improving S1P1 potency by
incorporating different groups (11, 12, and 13) at the 8-
position resulted in diminished activity relative to 8.
Furthermore, the significant activity of 12 and 13 in our S1P3
assay suggested that maintaining minimal activity against S1P3
may be a challenge with 8-substituted analogues. Therefore, we
focused on optimizing the polar headgroup at the 7-position.
The chain length of the primary alcohol was extended by two
(14, EC50 = 0.008 μM) and three (15, EC50 = 0.015 μM)
carbon atoms without significantly impacting S1P1 potency or
S1P3 activity. Carboxylic acids were also examined at the 7-
position. The propionic acid analogue 18 provided excellent
S1P1 potency (EC50 = 0.004 μM) and limited activity against
S1P3 (EC50 = 0.94 μM). Two amino acids (19 and 20) were
also examined. Although 19 had only moderate S1P1 activity
(EC50 = 0.15 μM), incorporating the azetidine carboxylic acid
found in AMG 369 led to an analogue, 20 (EC50 = 0.025 μM),
with little difference in potency relative to 7 (EC50 = 0.070
μM). With several polar head groups identified, the
corresponding 6-aza analogs of two analogues (18 and 9)
that provided an acceptable combination of potency, limited
activity against S1P3, solubility, and structural diversity were
prepared with the expectation that the combination would
provide improved physicochemical properties. Analogue 22 was
equipotent (EC50 = 0.018 μM) with 9, while maintaining
limited activity against S1P3 (EC50 >25 μM). However, 21 had
diminished potency relative to the parent compound 18 in
addition to increased potency against S1P3 (EC50 = 1.3 μM).
This result was somewhat unexpected since the 6-aza analogue
a
Reagents and conditions: (a) 2-Chloropyridin-4-amine, 120 °C. (b)
Ph2O, 220 °C. (c) K2CO3, MeI, 7% yield (3 steps). (d) KOH, 89%
yield. (e) Compound 28, HBTU, Et3N, 92% yield. (f) Tributyl-
(vinyl)stannane, Pd[P(tert-Bu)3]2, CsF, 80 °C, 76% yield. (g) OsO4,
NaIO4, 97% yield. (h) LiAlH(Otert-Bu)3, 68% yield.
24, which was cyclized to 25. Methylation of 25 gave 26 in 7%
yield over three steps from 23. Ester 26 was hydrolyzed to 27
and coupled with 289 to provide 29. The hydroxymethyl was
introduced using a three step sequence.
With several polar head groups identified, the most potent
and S1P3 sparing analogues were further profiled. As
anticipated, the addition of the polar headgroup increased the
solubility in SIF of most of the analogs shown in Table 2
relative to 6 and 7. None of the compounds distinguished
themselves in terms of cell permeability, and only one, 16, was
an efflux substrate in the parental porcine proximal tubule cell
line (LLC-PK1). All of the compounds in Table 2 exhibited
excellent stability in rat liver microsomes (CLint < 14 μL/min/
mg). The rat plasma protein binding was similar (91.7−92.7%
bound) for the analogues containing a polar headgroup derived
from an alcohol (9, 14, and 22), whereas acids 16 (98.0%
bound) and 18 (97.8% bound) were more extensively protein-
bound. All five analogues that contained a polar headgroup had
acceptable characteristics for advancement into an in vivo assay
that measures circulating lymphocytes.
Compounds were dosed orally at 1 mg/kg in female Lewis
rats, and after 24 h, blood lymphocyte counts and plasma
concentration of total drug were measured. The 24 h time point
was chosen to minimize the impact that dosing has on
lymphocyte counts. Both 16 and 18 had low plasma
concentrations 24 h postdose and did not lower blood
lymphocytes at 24 h. However, 9, 14, and 22 achieved
statistically significant lowering of blood lymphocytes. Of the
compounds in Table 2, 22 appeared the most promising with
high lymphocyte reduction and excellent SIF solubility.
Therefore, 22 was investigated in a dose−response experiment.
When dosed orally, 22 achieved statistically significant (P <
0.05) reduction of circulating blood lymphocytes after 24 h
when dosed at 1 and 3 mg/kg (Figure 2). The 3 mg/kg dose
corresponds to a 88% reduction in lymphocytes relative to
vehicle at 24 h.
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ACS Medicinal Chemistry Letters
Letter
a
Table 1. SAR of Analogues Containing a Polar Head Group
a
Data represent an average of at least two determinations. The percent efficacy is reported relative to 1.00 μM 1-(4-(6-benzylbenzofuran-2-yl)-3-
fluorobenzyl)azetidine-3-carboxylic acid (see ref 8) and 0.200 μM S1P for hS1P1 RI and hS1P3 Ca2+ assays, respectively; > [highest concentration
tested] is reported for compounds that do not achieve >10% of control activity.
Table 2. Solubility, Permeability, Protein Binding, and in
Vivo Characterization of Select Compounds
Table 3. Preclinical Pharmacokinetic Parameters for 22
IV/oral
dose
(mg/kg)
oral
AUC0‑inf
(μM·h)
CL
Vss, iv MRT,
F
Papp
rat
species
(L/h/kg) (L/kg) iv (h)
(%)
(LLC-
PK1) ×
LLC-
PK1
plasma
protein
binding
(%)
PLC
Cplasma
at 24 h
(ng/
b
a
SIF
solubility
compd (mg/mL)
POC 1
mouse
1/3
0.31
0.31
−
6.0
5.8
−
20
19
−
16
19
75
132
>93
67
10−6
a
parental
mg/kg,
24 h (%)
b
a
rat
1/2
cm/s
(ER)
mL)
bc
,
rat
−/100
4/10
1630
141
c
6
9
0.005
0.027
0.006
0.048
0.20
5.7
2.8
2.2
3.0
1.6
2.5
0.8
1.0
1.0
6.7
1.0
1.0
91.7
92.4
92.7
98.0
97.8
91.7
−64
350
65
d
NHP
0.11
4.3
40
c
−44
a
c
Studies were conducted in female C57BL/6 mice; iv doses were
14
16
18
22
−48
27
administered in 40% hydroxypropyl β-cyclodextrin/60% water; oral
d
+4
BQL
doses in 1% HPMC/1% pluronic F68/20% HPBCD/78% water, MSA,
−25
−60
4
b
pH 2. Studies were conducted in male Sprague−Dawley rats; iv doses
c
>0.20
84
were administered in DMSO; oral doses in 20% Captisol/1% Tween
a
c
d
Apparent permeability (Papp) through porcine proximal tubule cells
80, pH 2, MSA. Oral data only. PK studies were conducted in male
nonhuman primates (NHP); iv doses were administered in 40%
hydroxypropyl β-cyclodextrin/60% water, MSA, pH 3; oral doses in
1% HPMC/1% pluronic F68/20% HPBCD/78% water, MSA, pH 2.
b
(LLC-PK1 cell line) and efflux ratio (ER). Percent-of-control (POC)
reduction vs vehicle in peripheral lymphocyte count (PLC) 24 h after
a single oral dose (1 mg/kg; vehicle = 20% captisol, 1% HPMC, and
1% pluronic F68, pH 2.1, with MSA) administered to female Lewis
c
rats (N = 5/group). The measured POC reduction in PLC is
The area under the plasma concentration−time curve
(AUC0‑inf) increased from 19 to 1630 μM·h for the 2 and
100 mg/kg doses, respectively, thereby confirming that the
exposure was dose porportional in rats up to at least 100 mg/
kg. In addition, the clearance (CL = 0.31 L/h/kg) and long
mean residence time (19 h) were favorable. In nonhuman
primates, 22 displayed excellent pharmacokinetic parameters
with low clearance (CL = 0.11 L/h/kg), long mean residence
time (40 h), and good oral bioavailability (F = 67%).
statistically significant (P < 0.05 vs vehicle by ANOVA/Dunnett's
d
multiple comparison test). BQL, below the quantifiable limit.
Although 22 achieved dose-proportional exposure over a
narrow range of 0.3−3 mg/kg, the exposure at higher doses was
critical for advancement of 22. The exposure of 22 in rats was
measured after single oral doses of 2 and 100 mg/kg (Table 3).
Its oral absorption was complete, with % F of 132 at 2 mg/kg.24
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ACS Medicinal Chemistry Letters
Letter
clearance; cLog P, calculated logarithm of octanol/water
partition coefficient; Cplasma, total compound concentration in
plasma; DMSO, dimethyl sulfoxide; EC50, molar concentration
that produces half maximal response; F, bioavailability; HBTU,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluor-
ophosphate; HPBCD, hyroxypropyl β-cyclodextrin; HPMC,
hydroxypropyl methylcellulose; iv, intravenous; LLC-PK1,
porcine proximal tubule cell line; MRT, mean residence time;
MSA, methanesulfonic acid; Papp, apparent permeability; Vss,
volume of distribution in steady state
Figure 2. After a single oral dose, 22 reduced blood lymphocyte
counts in female Lewis rats 24 h postdose (N = 3/group; bars
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represent average blood lymphocyte counts
SE; circles represent
average plasma concentration SE; *P < 0.05 vs vehicle by ANOVA/
Dunnett's multiple comparsion test). Compound was administered in
20% captisol/1% HPMC/1% pluronic F68, pH 2, with MSA.
In summary, a polar headgroup was used to improve the
physicochemical and pharmacokinetic parameters of a series of
S1P1 agonists without detrimentally affecting the pharmaco-
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examined for appending the polar headgroup. The 7-position
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ASSOCIATED CONTENT
* Supporting Information
■
S
Statistical analysis for in vitro data in Table 1, experimental
procedures and characterization data for 8−22, PK data for 22,
and experimental details for in vitro and in vivo assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
X.; Siu, J.; Wong, M.; Burli, R. W. Discovery of AMG 369, a
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thiazolo[5,4-b]pyridine agonist of S1P1 and S1P5. ACS Med. Chem.
Lett. 2011, 2, 107−112.
AUTHOR INFORMATION
Corresponding Author
*Tel: 805-313-5564. Fax: 805-480-1337. E-mail: pharring@
amgen.com.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Ronya Shatila and Elizabeth Tominey (Amgen) for
in vivo pharmacokinetic studies and Laura Scott (Amgen) for
executing the HTS campaign that identified the quinolinone
series. All live animal studies were conducted in an AAALAC
accredited facility, and husbandry procedures met all of the
recommendations of the Guide for the Care and Use of
Laboratory Animals. All work using research animals was
conducted under Institutional Animal Care and Use
Committee (IACUC) approved protocols.
ABBREVIATIONS
AUC, area under the plasma concentration−time curve; CHO,
Chinese hamster ovary; CL, clearance; CLint, intrinsic
■
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(23) cLog P values were calculated using software from Daylight
Chemical Information Systems.
(24) The % F was not accurately estimated for the 100 mg/kg oral
dose due to the long half-life of 57 h with up to 48 h of plasma
collection. In contrast, the half-lives after either iv or oral dose (1−2
mg/kg) were only 13−16 h, and thus, the AUC for these two doses
was sufficiently characterized by collecting plasma samples up to 48 h.
However, the % F for the 100 mg/kg oral dose would be at least 93%
based on estimation using AUC0−48 h for the 100 mg/kg oral dose.
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