Discovery of AMG 369, a thiazolo[5,4-b]pyridine agonist of S1P1 and S1P5

Article abstract of DOI:10.1021/ml100306h

The optimization of a series of thiazolopyridine S1P₁ agonists with limited activity at the S1P₃ receptor is reported. These efforts resulted in the discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5, 4-b]pyridin-2-yl)ben

Full text of DOI:10.1021/ml100306h

pubs.acs.org/acsmedchemlett
Discovery of AMG 369, a Thiazolo[5,4-b]pyridine
Agonist of S1P1 and S1P5
Victor J. Cee,*,† Mike Frohn,† Brian A. Lanman,† Jennifer Golden,† Kristine Muller,†
Susana Neira,† Alex Pickrell,† Heather Arnett,‡ Janet Buys,† Anu Gore,† Mike Fiorino,†
Michelle Horner,† Andrea Itano,† Matt R. Lee,† Michele McElvain,† Scot Middleton,†
Michael Schrag,† Dalia Rivenzon-Segal,§ Hugo M. Vargas,† Han Xu,† Yang Xu,† Xuxia Zhang,†
†
†
†
€
Jerry Siu, Min Wong, and Roland W. Burli
†Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States, ‡Amgen Inc., 1201 Amgen Court West,
Seattle, Washington 98119, United States, and §EPIX Pharmaceuticals Inc., 167 Worcester Street, Suite 201, Wellesley Hills,
Massachusetts 02481, United States
ABSTRACT The optimization of a series of thiazolopyridine S1P1 agonists with
limited activity at the S1P3 receptor is reported. These efforts resulted in the
discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)-
azetidine-3-carboxylic acid (5d, AMG 369), a potent dual S1P1/S1P5 agonist with
limited activity at S1P3 and no activity at S1P2/S1P4. Dosed orally at 0.1 mg/kg, 5d is
shown to reduce blood lymphocyte counts 24 h postdose and delay the onset and
reduce the severity of experimental autoimmune encephalomyelitis in rat.
KEYWORDS Sphingosine-1-phosphate receptor, S1P1, lymphocyte, inflammation,
multiple sclerosis
helysophospholipidsphingosine-1-phosphate(1,S1P)
has emerged as a versatile signaling molecule,1,2
T
capable of impacting a wide range of cellular functions,
including proliferation and apoptosis, differentiation, and
migration. The diverse activities of S1P are believed to be
mediated through its interaction with the five S1P GPCRs
(S1P1-5) encoded by the EDG (endothelial differentiation
gene) family as well as possible additional intracellular
targets.1-6 The discovery that the potent immunomodulator
FTY720 (fingolimod, 2) is phosphorylated in vivo to the
monophosphate ester FTY720P,7,8 an agonist of all S1P GPCRs
except S1P2, sparked intense research into the functions of the
individual S1P receptors (see Figure 1 for structures). From
these studies, a consensus emerged that the immunomodu-
latory effects of FTY720 are primarily due to agonism and
subsequent downregulation of the S1P1 receptor by FTY720P,
which interrupts the normal pattern of lymphocyte migration
and leads to a state of peripheral lymphocyte depletion.3-8 It
was also established that, in rodents, agonism of the S1P3
receptor by FTY720P causes a reduction in heart rate9,10
similar to that observed in human subjects.11 These findings,
combined with the impressive efficacy of FTY720 in clinical
trials for the treatment of transplant rejection12 and multiple
sclerosis,13,14 led to a wide-ranging search for S1P1 agonists
with limited activity at the S1P3 receptor.15,16 Herein we report
the discovery of a dual S1P1/S1P5 thiazolo[5,4-b]pyridine
agonist (5d) and describe its in vivo pharmacological and
pharmacokinetic characteristics.
Figure 1. S1P, FTY720, benzothiazole 3a, and optimized thiazolo-
[5,4-b]pyridine 5d (AMG 369).
was representative of this series and exhibited double-digit
nanomolar potency in an assay measuring receptor inter-
nalization (RI) of an hS1P1-GFP fusion protein in U2OS cells,
and it exhibited limited activity at hS1P3 as determined by
Ca2þ mobilization in hS1P3- and Gq/i5-transfected CHO-K1
cells (Table 1).18 Compound 3a also demonstrated on-me-
chanism activity in rat, with a single oral dose of 1 mg/kg
producing a significant reduction of circulating blood lym-
phocytes 24 h postdose. However, 3a is characterized by
both relatively high lipophilicity (CLogP=3.9) and low topo-
logical polar surface area (tPSA = 53 Å2),19 and therefore, it
Received Date: September 27, 2010
Accepted Date: December 21, 2010
Published on Web Date: December 29, 2010
Previously, our research team produced a series of potent
and selective benzothiazole S1P1 agonists.17 Compound 3a
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Table 1. SAR of Thiazolopyridine Analoguesa
compd
X
Y
Z
hS1P1 RI EC50, μM (% efficacy)
hS1P3 Ca2þ EC50, μM (% efficacy)
3a
3b
3c
3d
4a
4b
4c
4d
CH
N
CH
CH
N
CH
CH
CH
N
0.041 (102)
0.144 (106)
0.429 (115)
0.138 (125)
0.221 (84)
1.60 (75)
1.21 (24)
10.1 (63)
>25
CH
CH
CH
N
CH
CH
CH
N
2.89 (36)
3.47 (50)
>25
CH
CH
CH
N
CH
CH
3.98 (77)
5.46 (64)
0.478 (94)
CH
0.199 (92)
a Data represents an average of at least two determinations. % efficacy is reported relative to 1.00 μM 1-(4-(6-benzylbenzofuran-2-yl)-3-
fluorobenzyl)azetidine-3-carboxylic acid and 0.200 μM S1P for hS1P1 RI and hS1P3 Ca2þ assays, respectively; >[highest concentration tested] is
reported for compounds that do not achieve >10% of control activity. For statistical analysis, see the Supporting Information.
resides in a region of physicochemical space with a higher
probability of off-target toxicity (CLogP>3, tPSA<75 Å2).20
Therefore, modifications of 3a were undertaken with the
goal of identifying S1P1 agonists with lower CLogP and
higher tPSA. Single nitrogen for CH substitutions were
identified as a means of lowering CLogP (-1.1 units) and
raising tPSA (þ12 Å2). The three possible aza-analogues
of 3a (3b-3d) arising from nitrogen substitution at each
benzothiazole C-H position were investigated (Table 1)
and found to exhibit reduced potency (3-10-fold loss vs 3a)
in the S1P1 receptor internalization assay. Aza-derivatives
(4b-4d) of the isomeric 6-benzyl-benzothiazole 4a were
also investigated, and in one case, the substitution of nitro-
tPSA = 65 Å2) were also favorable relative to 3a (ClogP=
3.9, tPSA = 53 Å2). Further testing of 5d in a Ca2þ mobiliza-
tion format revealed potent agonism of the hS1P5 receptor
(EC50 = 0.029 μM, 50% efficacy) and a lack of activity at
hS1P2 and hS1P4 receptors at concentrations up to 2.5 μM.
The S1P5 activity of 5d was not considered a liability, as it has
been suggested that agonism of oligodendrocyte S1P5 re-
ceptors by FTY720P may contribute to the efficacy of
FTY720 (2) in the treatment of multiple sclerosis.21
The greater than 10-fold difference in S1P1 potency
between 5d and both the gem-dimethyl analogue 5c and
des-aza cyclopropane analogue 6 (Table 2) suggests that the
presence of both cyclopropane and pyridine endow 5d with
either a unique conformation or unique interactions with the
S1P1 receptor, or both. To further understand the conforma-
tional preferences of 5c, 5d, and 6, quantum mechanical
calculations22 were performed on fragments corresponding
to 5c, 5d, and 6 (Figure 2, A, B, and C, respectively) to
characterize the rotational energy profile about the indicated
NCCC or CCCC bonds. The profiles for the gem-dimethyl
fragment A and cyclopropyl fragment B show striking differ-
ences. Fragment Aexhibits a fairly shallow profile and global
minima at 120 and 240° angles, whereas B exhibits a steep
profile greatly favoring a 180° dihedral angle. The des-aza
fragment C does not share the profile of B; rather, a slight
preference for a 0° dihedral angle is observed. It was further
established that, like B, the simple fragment 2-(1-phenylcy-
clopropyl)pyridine also shows a strong preference for a 180°
NCCC dihedral angle (data not shown, see Supporting In-
formation). The unique conformational profile of 2-(1-phe-
nylcyclopropyl)pyridines has, to the best of our knowledge,
not been previously described. It may be that the 5d-S1P1
complex that is conducive to efficient receptor internali-
zation contains 5d with a 180° NCCC dihedral angle
and, therefore, benefits entropically from ligand preorgani-
zation.
gen for CH resulted in no loss in potency (4d, S1P1 RI EC50
0.199 μM) relative to the parent (4a, hS1P1 RI EC50
=
=
0.221 μM). Of the six aza-analogues, 3b, 3d, and 4d exhib-
ited reasonable S1P1 potency (EC50 = 0.138-0.199 μM)
and were selected for further optimization.
Given the high lipophilicity of the endogenous S1P1 ligand,
S1P (1, CLogP = 5.5), we hypothesized that the reduced
potency of the aza-analogues could be remedied by addition
of small lipophilic groups, with the goal of not exceeding the
lipophilicity of 3a (CLogP = 3.9). After extensive exploration
of more than seventy analogues of 3b, 3d, and 4d bearing
small lipophilic groups, it was found that the R-methylbenzyl
analogues of 4d, the thiazolo[5,4-b]pyridines 5a-b (Table 2),
showed a 5-fold improvement in S1P1 potency and a 2-fold
loss of S1P3 activity relative to the parent 4d. Geminal
dimethyl substitution (5c) resulted in comparable S1P1
potency and reduced S1P3 activity. Encouraged by these
results, benzylic 1,1-disubstituted carbocycles were investi-
gated (5d-g). Of this series, cyclopropane-containing 5d
was found to uniquely exhibit single-digit nanomolar po-
tency at the S1P1 receptor (EC50 = 0.002 μM), with much
weaker activity at the S1P3 receptor (EC50 = 0.888 μM). The
calculated physicochemical properties of 5d (ClogP = 3.2,
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Table 2. Optimization of Thiazolo[5,4-b]pyridine Agonistsa
compd
Z
R1
R2
hS1P1 RI EC50, μM (% efficacy)
hS1P3 Ca2þ EC50, μM (% efficacy)
4d
5a
5b
5c
5d
5e
5f
N
H
H
0.199 (92)
0.035 (99)
0.038 (87)
0.033 (103)
0.002 (98)
0.119 (124)
0.026 (76)
0.033 (108)
0.038 (93)
0.478 (94)
0.877 (44)
0.996 (45)
2.51 (64)
N
Me
H
N
H
Me
Me
N
Me
N
-(CH2)2-
-(CH2)3-
-(CH2)4-
-(CH2)5-
-(CH2)2-
0.888 (26)
1.31 (68)
N
N
12% @ 2.5 μM
0.836 (41)
4.67 (56)
5g
6
N
CH
a Data represents an average of at least two determinations. % efficacy is reported as in Table 1. For statistical analysis, see the Supporting
Information
Figure 2. Rotational energy profile of fragments A (blue),
B (black), and C (red) corresponding to 5c, 5d, and 6, respectively
(B3LYP/6-31G* with Polarizable Continuum Model to simulate
aqueous media). Minimization of fragment A at a 0° NCCC dihedral
was not possible.
The remarkable in vitro potency of 5d translated to in vivo
activity at doses that were more than an order of magnitude
lower than had previously been achieved in our program
(Figure 3a). Dosed orally at 0.01, 0.03, and 0.1 mg/kg to
Lewis rats, 5d produced a dose-dependent reduction in
circulating blood lymphocyte counts consistent with S1P1
agonism2 24 h postdose.23 Statistical significance (P < 0.05)
was achieved at the 0.03 mg/kg (45% reduction in lympho-
cytes vs vehicle) and 0.1 mg/kg doses (65% reduction in
lymphocytes vs vehicle). Since S1P1 agonists typically pro-
duce a maximal 70% reduction in lymphocyte counts,24 the
0.1 mg/kg dose of 5d in the rat provides a nearly maximal
Figure 3. In vivo characterization of 5d. (a) 5d dosed orally
reduces blood lymphocyte counts in female Lewis rats 24 h
postdose (N = 5/group; bars represent average blood lymphocyte
counts þ SE; circles represent average plasma concentration ( SE;
*P < 0.05 vs vehicle by ANOVA/Dunnett's Multiple Comparison
Test). (b) Efficacy of 5d dosed orally at 2 and 20 μg/rat/day
(approximately 0.01 and 0.1 mg/kg, respectively) in delaying onset
and reducing disease score in an experimental autoimmune
encephalomyelitis (EAE) model in female Lewis rats (N = 15/
group; *P < 0.05 vs vehicle by Wilcoxon exact 2-sided test).
Vehicle = 20% captisol, pH 2.
pharmacologic effect.25 Compound 5d was further profiled
in a rat experimental autoimmune encephalomyelitis (EAE)
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Table 3. Pharmacokinetic Parameters for 5d
Scheme 1. Synthesis of 5da
species
Cl (L/(h kg))
V
ss (L/kg)
T1/2 (h)
MRT (h)
% F
rata
canineb
NHPc
0.18
0.071
0.50
1.8
1.2
2.3
5.9
38
24
10
17
64
41
34
4.7
a Male Sprague-Dawley (iv: 1 mg/kg, DMSO, N = 3; po: 2 mg/kg,
20% captisol pH 4.0, N = 2). b Male beagle (iv: 1 mg/kg, 20% captisol pH
4.0, N = 3; po: 1 mg/kg, 20% captisol/1% pluronic F68/1% HPMC
pH 2.1, N = 3). c NHP = nonhuman primate, male cynomolgus (iv:
1 mg/kg, 20% captisol pH 4.0, N = 3; po: 1 mg/kg, 20% captisol/1%
pluronic F68/1% HPMC pH 2.1, N = 3).
model which has characteristics of the human disease multi-
ple sclerosis (Figure 3b).26 In this EAE model, Lewis rats are
immunized with guinea pig myelin basic protein and scored
daily on a 0-6 scale reflecting the degree of disability
observed (0 = no sign of disability), with the investigator
assessing the clinical score blinded to the study group of the
individual rats. The onset of disease typically occurs 10 days
postimmunization, with complete resolution of disease at
20 days postimmunization. 5d dosed orally once daily at
20 μg per day (approximately 0.1 mg/kg) at the time of
immunization resulted in delayed onset and significant
reduction of disease score (Figure 3). A 10-fold lower dose
of 5d (2 μg, approximately 0.01 mg/kg) did not significantly
impact disease score in this model.
Additional profiling established that 5d possesses accep-
table characteristics for further development. In rat, non-
human primate, and dog (Table 3), the compound had low
clearance (4-21% of liver blood flow), a moderate steady
state volume of distribution of total drug (Vss = 1.2-2.3
L/kg), a moderate to long mean residence time (5-17 h),
and acceptable oral bioavailability (34-64%). Cardiovascu-
lar safety studies in telemetered rats established a no-effect
level for heart rate and mean arterial pressure changes of 10
mg/kg (po), indicating a wide margin for S1P3-associated
cardiovascular toxicity. In vitro studies established that 5d
was not an inhibitor or inducer of human cytochrome P450
enzymes, was nonmutagenic (Ames and micronucleus
negative), and did not significantly inhibit the hERG channel
in an electrophysiology assay at the highest concentration
tested (2.3 μM).27
a (a) n-BuLi, 2,6-dichloro-3-isothiocyanatopyridine, THF, -78 °C;
(b) Na2CO3, DMF, 90 °C, 83% (two steps); (c) bis(4-(di-tert-butylpho-
sphino)-N,N-dimethylbenzenamine) palladium dichloride (2.5 mol
%), 1-phenylvinylboronic acid, K2CO3, dioxane/water, 80 °C, 74%;
(d) Me3SOI, tBuOK, DMSO/THF, 75%; (e) 5 N HCl, THF, 65 °C, 93%;
(f) methyl azetidine-3-carboxylate hydrochloride, AcOH, DIPEA,
NaBH3CN, MeOH/CH2Cl2, 75%; (g) NaOH/THF, then HCl and pH 6
sodium phosphate buffer, 86%.
In conclusion, a thiazolopyridine series of S1P1 agonists
was developed with the goal of reducing the lipophilicity and
increasing the topological polar surface area of an existing
benzothiazole series. The moderate potency of the thiazolo-
[5,4-b]pyridine series was improved by addition of small
lipophilic groups at the benzylic position. The cyclopropyl-
containing derivative 5d had improved calculated physico-
chemical properties, exhibited single-digit nanomolar S1P1
potency with selectivity against S1P2, S1P3, and S1P4, and
was capable of reducing blood lymphocyte counts and the
severity of experimental autoimmune encephalomyelitis
in rat at a dose of 0.1 mg/kg. Molecular modeling experi-
ments revealed an unanticipated preference of the 5-(1-
phenylcyclopropyl)thiazolo[5,4-b]pyridine fragment for a
rotamer with an NCCC dihedral angle of 180°, which differ-
entiates 5d from related less potent analogues. Additional in
vitro and in vivo experiments established that 5d possesses
acceptable properties for further development.
SUPPORTING INFORMATION AVAILABLE Statistics for in
vitro data presented in Tables 1 and 2. Tabulated data for quantum
mechanical calculations for fragments A-C and 2-(1-phenylcyclo-
propyl)pyridine, as well as a complete ref 22. Experimental proce-
dures and characterization data for 3a-d, 4a-d, 5a-g, and 6.
Details of in vitro and in vivo assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
The synthesis of 5d is detailed in Scheme 1. Aryl bromide 7,
synthesized in one step from the readily available 4-bromo-
3-fluorobenzaldehyde, was treated with n-BuLi to effect
lithium-halogen exchange, and the resulting anion was
allowed to react with 2,6-dichloro-3-isothiocyanatopyridine
to give an intermediate thioamide. Treatment of the thio-
amide with sodium carbonate in DMF at elevated tempera-
ture effected cyclization to the thiazolo[5,4-b]pyridine 8. A
Suzuki reaction between 8 and 1-phenylvinylboronic acid
AUTHOR INFORMATION
Corresponding Author: *E-mail: vcee@amgen.com; 805-313-
5500. Fax: 805-480-1337.
28
catalyzed by PdCl2{PtBu2-p-NMe2-Ph}2 provided 9. The
cyclopropane was installed with dimethyloxosulfonium
methylide under Corey-Chaykovsky conditions29 to give
10, and deprotection of the acetal revealed aldehyde 11.
Reductive amination of 11 with methyl azetidine-3-carboxylate
hydrochloride provided the penultimate intermediate 12.
Saponification of 12 afforded 5d, which was isolated in high
yield as its zwitterion.
ACKNOWLEDGMENT We thank Ronya Shatila and Beth Tominey
for in vivo PKDM studies, Jay Larrow, Bobby Riahi, and Filisaty
Vounatsos for large-scale synthesis (7 and methyl azetidine-3-
carboxylate hydrochloride), Zheng Hua for chiral separation
support, and Srinivasa Chekru, Mercedes Lobera, Yael Marantz,
Sharon Shacham, Anurag Sharadendu, Xiang Yu, and Zhaoda
Zhang (EPIX Pharmaceuticals).
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