Identification and synthesis of potent and selective pyridyl-isoxazole based agonists of sphingosine-1-phosphate 1 (S1P1)

Article abstract of DOI:10.1016/j.bmcl.2016.03.105

The synthesis and structure-activity relationship (SAR) of a series of pyridyl-isoxazole based agonists of S1P₁ are discussed. Compound 5b provided potent in vitro activity with selectivity, had an acceptable pharmacokinetic profile, and demonstrated efficacy in a dose dependent manner when administered orally in a rodent model of arthritis.

Full text of DOI:10.1016/j.bmcl.2016.03.105

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and synthesis of potent and selective pyridyl-isoxazole
based agonists of sphingosine-1-phosphate 1 (S1P1)
⇑
Junqing Guo , Scott H. Watterson, Steven H. Spergel, James Kempson, Charles M. Langevine,
Ding Ren Shen, Melissa Yarde, Mary Ellen Cvijic, Dana Banas, Richard Liu, Suzanne J. Suchard,
Kathleen Gillooly, Tracy Taylor, Sandra Rex-Rabe, David J. Shuster, Kim W. McIntyre, Georgia Cornelius,
Celia D’Arienzo, Anthony Marino, Praveen Balimane, Luisa Salter-Cid, Murray McKinnon, Joel C. Barrish,
Percy H. Carter, William J. Pitts, Jenny Xie, Alaric J. Dyckman
Bristol-Myers Squibb Research and Development, Princeton, NJ 08543-4000, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationship (SAR) of a series of pyridyl-isoxazole based agonists of
S1P1 are discussed. Compound 5b provided potent in vitro activity with selectivity, had an acceptable
pharmacokinetic profile, and demonstrated efficacy in a dose dependent manner when administered
orally in a rodent model of arthritis.
Received 24 February 2016
Revised 28 March 2016
Accepted 29 March 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Sphingosine-1-phosphate
Sphingosine-1-phosphate 1
S1P1
Isoxazole
FTY720
Fingolimod
Sphingosine-1-phosphate (S1P) is the endogenous ligand for the
sphingosine-1-phophate receptors (S1P1–5).1 The interaction
between S1P and the S1P receptors plays a fundamental physiolog-
ical role in a number of processes including vascular stabilization,
heart development, lymphocyte homing, and tumor related angio-
genesis.2 Agonism of S1P1, in particular, has been shown to play a
significant role in lymphocyte trafficking from the thymus and sec-
ondary lymphoid organs, resulting in immunosuppression.3 In sup-
port of this, S1P Kinase knock-out (KO) mice have been shown to
significantly reduce lymphocyte egress from the thymus and the
lymph nodes.4 Additionally, lymphocyte egress was significantly
impaired in S1P1 conditional KO mice,3,5 as well as with the treat-
ment of a selective S1P1 agonist.6 Ultimately, entrapped lympho-
cytes are unable to reach targeted tissues (Fig. 1).
Fingolimod (1, FTY720) (Fig. 2) has been validated as an
immunological therapeutic agent in a variety of preclinical models
of autoimmune disease, demonstrating immunosuppression with a
reduction in circulating lymphocytes.2b Additionally, fingolimod
(1) was shown to be rapidly phosphorylated by sphingosine-1
phosphate kinase 2 (S1PK2) to form the phosphorylated species
(2, FTY720-P) which acts as a potent agonist on the S1P1,3,4,5
subtypes (Fig. 2).7 In 2010, 1 was approved as a first line therapy
for the treatment of multiple sclerosis. In spite of its clinical
success, several concerns emerged during clinical trials including
cardiovascular effects (bradycardia and blood pressure elevation),
macular edema, and a prolonged half-life in human.8
Consistent with other research groups,9,10 our efforts have been
directed toward identifying an orally active, direct-acting small
molecule S1P1 full agonists for use in autoimmune diseases with
selectivity for S1P1 over S1P3 and improved PK properties relative
to fingolimod (1) to minimize potential host defense issues.
Agonism of S1P3 was originally thought to be the source of the car-
diovascular effects observed with 1, based on rodent studies,11 but
more recent clinical studies with S1P3 sparing agonists have shown
that the effects seen in humans are dependent, at least in part, on
S1P1 activation.12 Since S1P3 agonism does not appear to con-
tribute to the efficacy, selectivity remains a desirable attribute.
The key structural features of fingolimod (1) include an amino-
diol polar head group which is phosphorylated by S1PK2, a 1,4-dis-
ubstituted phenyl ring which acts as a rigid linker, and a lipophilic
tail which is important for interacting with the hydrophobic bind-
ing pocket of the S1P receptors.13,14 We recently disclosed the
identification of BMS-520 (3) as a potent and selective S1P1 agonist
⇑
Corresponding author. Tel.: +1 609 252 5071.
E-mail address: junqing.guo@bms.com (J. Guo).
http://dx.doi.org/10.1016/j.bmcl.2016.03.105
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Guo, J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.105

2
J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
O
P
ester afforded carboxylic acids 9. Pyridyl-isoxazole carboxylates
OH OH
NH2
OH
O-
Sphingosine
kinases
OH
O
12, used in the preparation of isoxazoles agonist 5, were prepared
by coupling bromoenotes 11 with the dipole generated in situ from
N-hydroxypicolinimidoyl chloride 10 in the presence of triethy-
lamine to form pyridyl-isoxazole carboxylates as a mixture of
two regioisomers. Chromatographic separation provided the
desired regioisomers 12 in low yield (ꢀ10%) which were then
hydrolyzed to afford pyridyl-isoxazole carboxylic acids 13.
Pyridyl-isoxazoles 4 and 5 were prepared by coupling 17 with isox-
azoles 9 or 13, followed by cyclodehydration and deprotection of
the azetidine carboxylic acid, as depicted in Scheme 2.
Natural ligand:
Sphingosine-1
phosphate(S1P)
12
12
S1P
+
NH3
Sphingosine
Figure 1. Structures of sphingosine and sphingosine 1-phosphate (S1P).
NH2
OH
R
O
1
2
R = H
R = PO(OH)2
As access to intermediate 12 (R = CF3) was limited due to poor
yields, an improved synthetic route was developed to prepare
compound 5b, as described in Scheme 3. This approach involved
a [3+2]-dipolar cycloaddition reaction between the dipole gener-
ated from N-hydroxypicolinimidoyl chloride 19, in the presence
of trimethylamine, and ethyl propiolate to afford pyridyl isoxazole
carboxylate 20. Subsequent condensation with N0-hydroxy-4-
methylbenzimidamide under basic conditions provided intermedi-
ate 21. A selective palladium catalyzed bromination of 21 provided
22 in good yield, which was then treated with methyl 2,2-difluoro-
2-(fluorosulfonyl)acetate to afford trifluoromethyl pyridyl-isoxa-
zole 23 in 45% yield. Compound 23 was brominated with NBS to
give 24, and the resulting bromide was displaced with 15 followed
by the deprotection of the azetidine carboxylic acid to provide 5b.
Figure 2. Structures of fingolimod (1) and fingolimod-P (2).
N
O
N
N
N
N
F3C
O
N
CO2H
3 (BMS-520)
O
R
N
O
N
N
N
O
O
N
Pyridyl-isoxazoles 4 and 5 were evaluated in a functional GTPcS
assay16 looking at both human S1P1 and S1P3 receptor agonism, as
summarized in Table 1. In general, all compounds were potent S1P1
full agonists and highly selective over S1P3. With desirable intrinsic
potency, each compound was evaluated in vivo in a rat blood lym-
phocyte reduction (BLR) pharmacodynamic/pharmacokinetic
(PD/PK) assay17 in which healthy rats were dosed orally with a
1.0 mg/kg dose. Blood samples were drawn at 4 h and 24 h time
points to assess the reduction in circulating lymphocytes relative
to control and to evaluate plasma concentration for each com-
pound. In this assay, all pyridyl-isoxazoles (4a–c and 5a–b)
demonstrated >50% lymphocyte reduction in plasma at 24 h with
C5-linked pyridyl-isoxazoles 5a and 5b providing a near maximal
response relative to the C3-linked analogs 4a–c (e.g. 4b vs 5a).
Additionally, when 5b was dosed at a 0.3 mg/kg, ꢀ50% lymphope-
nia was still maintained at 24 h. When compared to isoxazole 3, 5b
was equipotent and selective, provided a similar plasma concen-
tration at 4 h, but had reduced exposure at 24 h (54 nM vs
703 nM).15 This attenuated exposure at the later time point could
potentially be advantageous since a potent compound with a
shorter half-life compared with 1 could mitigate potential host–
defense issues upon drug discontinuation.
CO2H
CO2H
4
5
N
R
N
N
Figure 3. Discovery of pyridyl-isoxazole based agonists of S1P1.
with a phenyl-isoxazole as lipophilic tail fragment.15 In this Letter,
we describe our efforts toward exploring pyridyl-isoxazoles as
novel lipophilic tail fragments. This effort resulted in highly potent
and selective S1P1 full agonists derived from both C3-linked iso-
mers (4) and C5-linked isomers (5) (Fig. 3).
The general synthetic approaches for C-4 alkyl pyridyl-isoxa-
zoles carboxylic acids 9 and 13, used in the preparation of com-
pounds 4 and 5, are outlined in Scheme 1. Pyridyl-isoxazole
carboxylates 8, used in the preparation of isoxazoles agonists 4,
were accessed via a regioselective [3+2] dipolar cycloaddition reac-
tion between acetylene pyridines 6 and the dipole generated in situ
from dimethyl 2-nitromalonate 7 under microwave conditions at
170 °C in the presence of an ionic liquid, 1-butyl-3-methylimida-
zoliumhexafluorophosphate. Subsequent hydrolysis of the t-butyl
With a desirable potency and selectivity profile and a clean
liability profile (Table 2), 5b was further evaluated in vivo. In
N
N
O
O
CO2Me
CO2H
a
b or c
MeO2C
CO2Me
NO2
+
R
R
R
N
N
N
R = alkyl
Regioselective
6
8
7
9
O
O
N
N
Cl
CO2H
CO2Me
Br
b or c
d
OH
+
R
N
CO2Et
R
R
N
N
N
R = alkyl
12
13
10
11
Scheme 1. Reagents and conditions: (a) 1-butyl-3-methyl-1H-imidazol-3-ium hexafluorophosphate, toluene, Microwave, 170 °C, 10%; (b) LiOH–H2O, MeOH/H2O; (c) 1 N
aqueous NaOH, MeOH, microwave, 100 °C; (d) Et3N, CH2Cl2, ꢀ10%.
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J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Cbz
N
N
HN
a,b
c
OH
O
O
NC
O
O
O
14
16
18
15
O
R
N
N
d
e,f
O
N
H2N
HO
N
N
O
O
N
N
OH
17
O
Scheme 2. Reagents and conditions: (a) t-BuOH, DMAP, EDCI, CH2Cl2, 81%; (b) 10% Pd/C, EtOAc, 92%; (c) 4-cyanobenzaldehyde, Na(CN)BH3; MeOH, 89%; (d) HONH2–HCl;
NaHCO3, t-BuOH, 55%; (e) 9 or 13, EDCI, HOBt, DMF, 53–86%; (f) TFA.
Cl
N
N
O
O
O
OH
N
a
OEt
b
N
Me
Me
N
N
N
N
O
O
N
19
20
21
N
N
O
d
c
N
N
Me
N
Br
O
F3C
O
N
N
22
23
N
O
N
O
N
f,g
e
N
N
N
N
F3C
O
N
Br
F3C
O
N
OH
5b
24
O
Scheme 3. Reagents and conditions: (a) ethyl propiolate, Et3N, CH2Cl2, 90%; (b) N0-hydroxy-4-methyl benzenecarboximidamide, NaH or Cs2CO3, DMF, 86%; (c) NBS, Pd(OAc)2,
MeCN, 87%; (d) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, CuI, HMPA, DMF, 45%; (e) NBS, AIBN, 90%; (f) 15, Et3N, DMF, 75%; (g) TFA.
Table 1
SAR of the C3-linked pyridyl-isoxazoles (4) and C5-linked pyridyl-isoxazoles (5)
O
R
N
R
N
O
N
N
N
N
N
N
O
O
N
N
CO2H
CO2H
4
5
Rat Blood Lymphocycte Reduction Assay – 1.0 mg/kg
#
R
hS1P1 GTPgS EC50 hS1P3 GTPgS EC50 S1P3/S1P1
Lymphocyte reduction
4 h (%)
Concn
(nM)
Lymphocyte reduction
24 h (%)
Concn
(nM)
4 h/24 h concn
ratio
nM
nM
Sel.
4a Et
4b Pr
4c iPr
5a Pr
3.4
1900
2100
8200
630
2900
1600
550ꢁ
84
81
84
82
85
78
972
292
686
318
2510
2134
58
53
64
85
80
91
64
9.9
31
26
54
15
30
22
12
47
3.0
0.82
1.00
0.21
2500ꢁ
8200ꢁ
3000ꢁ
4100ꢁ
3400ꢁ
5b CF3 0.71
CF3 0.47
3
703
Concn: plasma concentrations at 4 and 24 h timepoints.
multi-species pharmacokinetic (PK) studies (Table 3), 5b exhibited
good oral absorption and low clearance in mouse, rat, and dog with
low volume of distribution and acceptable half-lives. Cyno, on the
other hand, was a clear outlier relative to the other species display-
ing low bioavailability, high clearance, and a short half-life. With a
reasonable PK profile, 5b was evaluated in a rat adjuvant induced
arthritis model of human rheumatoid arthritis.18 In this model,
arthritis was induced by the subcutaneous injection of Complete
Freund’s Adjuvant containing heat-killed Mycobacterium butyricum
into the base of the tail of male Lewis rats. Pyridyl-isoxazole 5b
was administered at doses of 0.03. 0.1, 0.5, and 3 mg/kg po/QD,
along with fingolimod (1) at 1 mg/kg po/QD, for 21 days starting
at day 0 (Fig. 4). Baseline measurements of hind paw volume were
obtained by water displacement plethysmometry. Additional paw
volume measurements were performed over the next three weeks,
and the increases in paw volume above baseline were calculated.
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4
J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
arthritis at a dose of 3.0 mg/kg QD. Overall, isoxazoles 5b and 3
have demonstrated that an isoxazole based lipophilic fragment
can provide highly desirable agonists of S1P1.
Partial in vitro profiling data for isoxazole 5b
N
O
N
N
N
F3C
References and notes
O
N
CO2H
5b
Result
1. (a) Hla, T. Semin. Cell Dev. Biol. 2004, 15, 513; (b) McVerry, B. J.; Garcia, J. G. Cell.
Signal. 2004, 17, 131; (c) Lee, J.-F.; Zeng, Q.; Ozaki, H.; Wang, L.; Hand, A. R.; Hla,
T., et al. J. Biol. Chem. 2006, 281, 29190.
Parameter
2. (a) Chun, J.; Hartung, H. Clin. Neuropharmacol. 2010, 33, 91; (b) Mandala, S.;
Hajdu, R.; Bergstorm, J.; Quackenbush, E.; Xie, J.; Milligan, J.; Thornton, R.; Shei,
G.-J.; Card, D.; Keohane, C. A.; Rosenbach, M.; Hale, J.; Lynch, C. L.; Rupprecht,
K.; Parsons, W.; Rosen, H. Science 2002, 296, 346; (c) Brinkmann, V.; Davis, M.
D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof, R.; Bruns, C.; Prieschl, E.; Baumruker,
T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K. R. J. Biol. Chem. 2002, 277,
21453; (d) Takuwa, N.; Okamoto, Y.; Yoshioka, K.; Takuwa, Y. Biomed. Rev.
2011, 22, 15.
Human S1P4/S1P1 selectivity
Human S1P5/S1P1 selectivity
Protein binding (bound)
3.2ꢁ
3.2ꢁ
99.5% human
99.4% mouse
97.6% rat
72.6% dog
93.5% monkey
3. Matloubian, M. M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.; Xu, Y.; Brinkmann, V.;
Allende, M. L.; Prola, R. L.; Cyster, J. G. Nature 2004, 427, 355.
Mutagenicity
Ames negative
4. Pappu, R.; Schwab, S. R.; Cornelissen; Pereira, J. P.; Regard, J. B.; Xu, Y.; Camerer,
E.; Zheng, Y.-W.; Huang, Y.; Cyster, J. G.; Coughlin, S. R. Science 2007, 316, 295.
5. Allende, M. L.; Dreier, J. L.; Mandala, S.; Proia, R. L. J. Biol. Chem. 2004, 279,
15396.
6. (a) Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.; Thangada, S.; Tigyi, G.; Osborne,
D. A.; Hla, T.; Parrill, A. L.; Rosen, H. Chem. Biol. 2005, 12, 703; (b) Roberts, E.;
Guerro, M.; Urbano, M.; Rosen, H. Expert Opin. Ther. Pat. 2013, 23, 817.
7. Sanchez, T.; Estrada-Hernandez, T.; Paik, J.-H.; Wu, M.-T.; Venkataraman, K.;
Brinkmann, V.; Claffey, K.; Hla, T. J. Biol. Chem. 2003, 278, 47281.
hERG (patch clamp)
13% @ 10
34% @ 30
l
l
M
M
Na+ (patch clamp)
Ca+ (patch clamp)
<5% @ 10
12% @ 10
l
l
M (1 and 4 Hz)
M
CYPa inhibition (IC50
)
>40
3A4
lM 1A2, 2B6, 2C8, 2C9, 2C19, 2D6,
Liver microsomal stability (%
remaining)
h 88, r 100, m 100
8. (a) Pelletier, D.; Hafler, D. A. N. Eng. J. Med. 2012, 366, 339; (b) Kappos, L.; Antel,
J.; Comi, G.; Montalban, X.; O’Connor, P.; Polman, C. H.; Haas, T.; Korn, A. A.;
Karlsson, G.; Radue, E. W. N. Eng. J. Med. 2006, 355, 1124.
a
CYP = cytochrome P450.
9. Dyckman, A. J. Ann. Rep. Med. Chem. 2012, 195. and references cited therein.
10. (a) Buzard, D. J.; Kim, S. H.; Lopez, L.; Kawasaki, A.; Zhu, X.; Moody, J.; Thoresen,
L.; Calderon, I.; Ullman, B.; Han, S.; Lehmann, J.; Gharbaoui, T.; Sengupta, D.;
Calvano, L.; Montalban, A. G.; Ma, Y.-A.; Sage, C.; Gao, Y.; Semple, G.; Edwards,
J.; Barden, J.; Morgan, M.; Chen, W.; Usmani, K.; Chen, C.; Sadeque, A.;
Christopher, R. J.; Thatte, J.; Fu, L.; Solomon, M.; Mills, D.; Whelan, K.; Al-
Shamma, H.; Gatlin, J.; Le, M.; Gaidarov, I.; Anthony, T.; Unett, D. J.; Blackburn,
A.; Rueter, J.; Stirn, S.; Behan, D. P.; Jones, R. M. Med. Chem. Lett. 2014, 5, 1313;
(b) Buzard, D. J.; Schrader, O.; Zhu, X.; Lehmann, J.; Johnson, B.; Kasem, M.; Kim,
S.-H.; Kawasaki, A.; Lopez, L.; Moody, J.; Han, S.; Gao, Y.; Edwards, J.; Barden, J.;
Thatte, J.; Gatlin, J.; Jones, R. M. Bioorg. Med. Chem. Lett. 2015, 25, 659; (c)
Skidmore, J.; Heer, J.; Johnson, C. N.; Norton, D.; Redshaw, S.; Sweeting, J.;
Hurst, D.; Cridland, A.; Vesey, D.; Wall, I.; Ahmed, M.; Rivers, D.; Myatt, J.;
Giblin, G.; Philpott, K.; Kumar, U.; Stevens, A.; Bit, R. A.; Haynes, A.; Taylor, S.;
Watson, R.; Witherington, J.; Demont, E.; Heightman, T. D. J. Med. Chem. 2014,
57, 10424; (d) Bolli, M. H.; Abele, S.; Birker, M.; Brave, R.; Bur, D.; de Kanter, R.;
Kohl, C.; Grimont, J.; Hess, P.; Lescop, C.; Mathys, B.; Muller, C.; Nayler, O.; Rey,
M.; Scherz, M.; Schmidt, G.; Seifert, J.; Steiner, B.; Velker, J.; Weller, T. J. Med.
Chem. 2014, 57, 110.
Table 3
Multi-species PK parameters of compound 5b
Parameter
Mouse
Rat
Cynoa
Dog
po dose (mg/kg)
iv dose (mg/kg)
T1/2 (h), iv
Cl (mL/min/kg), iv
% hepatic flow
Vss (L/kg), iv
5
2
5
2
1
1
1
1
4.5
0.3
<1
0.1
70
2.8
4.8
1.0
1.4
0.4
100
26
1.1
14
32
0.5
12
>400
6.1
2.6
8.4
0.7
87
7.4
Fpo (%)
Peak/trough ratio
a
Cynomolgus monkey.
11. (a) Forrest, M.; Sun, S.-Y.; Bergstrom, J.; Card, D.; Doherty, G.; Hale, J.; Keohane,
C.; Meyers, C.; Milligan, J.; Mills, S.; Nomura, N.; Rosen, H.; Rosenbach, M.; Shei,
G.-J.; Singer, I. I.; Tian, M.; White, V.; Xie, J.; Proia, R. L.; Mandala, S. J. Pharmacol.
Exp. Ther. 2004, 309, 758; (b) Li, Z.; Chen, Z.; Hale, J. J.; Lynch, C. L.; Mills, S. G.;
Hajdu, T.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G. J.; Chrebet, G.;
Parent, S. A.; Bergstrom, J.; Card, D.; Forrest, M.; Quackenbush, E. J.; Wickham,
L. A.; Vargas, H.; Evans, R. M.; Rosen, H.; Mandala, S. J. Med. Chem. 2005, 48,
6169.
12. Gergely, P.; Nuesslein-Hildesheim, B.; Guerini, D.; Brinkmann, V.; Traebert, M.;
Bruns, C.; Pan, S.; Gray, N. S.; Hinterding, K.; Cooke, N. G.; Groenewegen, A.;
Vitaliti, A.; Sing, T.; Luttringer, O.; Yang, J.; Gardin, A.; Wang, N.; Crumb, W. J.,
Jr; Saltzman, M.; Rosenberg, M.; Wallstrom, E. Br. J. Pharmacol. 2012, 167, 1035.
13. Parrill, A. L.; Wang, D-A.; Bautista, D. L.; Van Brocklyn, J. R.; Lorincz, Z.; Fischer,
D. J.; Baker, D. L.; Liliom, K.; Spiegel, S.; Tigyi, G. J. Biol. Chem. 2000, 275, 39379.
14. Deng, Q.; Clemas, J. A.; Chrebet, G.; Fischer, P.; Hale, J. J.; Li, Z.; Mills, S. G.;
Bergstrom, J.; Mandala, S.; Mosley, R.; Parent, S. A. Mol. Pharmacol. 2007, 71,
724.
1.5
1.0
0.5
0.0
Vehicle
5b 0.03mg/kg
5b 0.1mg/kg
5b 0.5mg/kg
5b 3.0mg/kg
1 0.3mg/kg
*
*
*
*
7
9
11
13
15
17
19
21
Study Day
Figure 4. Effect of once daily dosing of 5b vs 1 (FTY720) on paw volume in rat AA.
*p <0.05 vs vehicle.
15. Watterson, S. H.; Guo, J.; Spergel, S. H.; Langevine, C. L.; Moquin, R. V.; Shen, D.
R.; Yarde, M.; Cvijic, M. E.; Banas, D.; Liu, R.; Suchard, S. J.; Gillooly, K.; Taylor,
T.; Rex-Rabe, S.; Shuster, D. J.; McIntyre, K. W.; Cornelius, G.; Darienzo, C.;
Marino, A.; Balimane, P.; Warrack, B.; Saltercid, L.; McKinnon, M.; Barrish, J. C.;
Carter, P. C.; Pitts, W. J.; Xie, J.; Dyckman, D. J. J. Med. Chem. 2016, 59, 2820.
16. hS1P-1 GTPg35S binding assay: Compounds were loaded in a 384 Falcon v-
bottom plate (0.5 ml/well in a 3-fold dilution). Membranes prepared from
S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells were added to the compound
plate (40 ml/well, final protein 3 mg/well) with multidrop. [35S]GTP (1250 Ci/
mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH 7.5, 10 mM
MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 mM GDP, 0.1% fatty acid free
BSA, and 10 mg/ml Saponin to 0.4 nM. 40 ml of the [35S] GTP solution was
added to the compound plate with a final concentration of 0.2 nM. The reaction
was kept at room temperature for 45 min. At the end of incubation, all the
mixtures in the compound plate were transferred to a 384 well FB filter plates
via GPCR robot system. The filter plate was washed with water 4 times by
using the modified manifold Embla plate washer and dried at 60 °C for 45 min.
As seen in Figure 4, doses of 0.5 and 3.0 mg/kg provided dose-
dependent reduction of paw swelling relative to the vehicle treated
rats, with the 3.0 mg/kg dose providing complete suppression of
disease development.
In conclusion, a potent and selective series of pyridyl-isoxazole
based S1P1 agonists has been identified. Compound 5b was identi-
fied as a highly potent drug-like agonist of S1P1 with significant
selectivity over S1P3. With a desirable potency and selectivity pro-
file and a clean liability profile, 5b was advanced into in vivo stud-
ies, providing a differentiated and potentially more desirable PK
profile compared with fingolimod 1. Additionally, 5b demonstrated
robust efficacy in a pre-clinical, chronic model of rheumatoid
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J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
30 ml of MicroScint 20 scintillation fluid was added to each well for counting at
Packard TopCount. EC50 is defined as the agonist concentration that
corresponds to 50% of the Ymax (maximal response) obtained for each
individual compound tested.
Hematology Analyzer (Siemens Healthcare Diagnostics). The results were
measured as a reduction in the percentage of circulating lymphocytes as
compared to the vehicle treated group at the 4 h and 24 h measurement. The
results represent the average results of all animals within each treatment
group (n = 3–4).
17. Blood lymphocyte reduction assay (BLR) in rat: Lewis rats were dosed orally with
test article (as
a
solution or suspension in the vehicle) or vehicle alone
18. Bendele, A.; McComb, J.; Gould, T.; McAbee, T.; Sennello, G.; Chlipala, E.; Guy,
M. Toxicol. Pathol. 1999, 27, 134.
(polyethylene glycol 300, ‘PEG300’). Blood was drawn at 4 h and 24 h by retro-
orbital bleeding. Blood lymphocyte counts were determined on an ADVIA 120
Please cite this article in press as: Guo, J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.105