Discovery of novel 1,2,4-thiadiazole derivatives as potent, orally active agonists of sphingosine 1-phosphate receptor subtype 1 (S1P1)

Article abstract of DOI:10.1021/jm2016107

A novel series of 1,2,4-thiadiazole compounds was discovered as selective S1P₁ agonists. The extensive structure-activity relationship studies for these analogues were reported. Among them, 17g was identified to show high in vitro potency with reasonable free unbound fraction in plasma (F_u > 0.5%), good brain penetration (BBR > 0.5), and desirable pharmacokinetic properties in mouse and rat. Oral administration of 1 mg/kg 17g resulted in significant peripheral lymphocytes reduction at 4 h after dose and rapid lymphocytes recovery at 24 h. 17g showed a transient lymphopenia profile in the repeated dose study in mouse. In addition, 17g also demonstrated efficacy comparable to that of FTY720 (1) in the mouse EAE model of MS.

Full text of DOI:10.1021/jm2016107

Article
pubs.acs.org/jmc
Discovery of Novel 1,2,4-Thiadiazole Derivatives as Potent, Orally
Active Agonists of Sphingosine 1-Phosphate Receptor Subtype 1
(S1P1)
Feng Ren, Guanghui Deng, Hailong Wang, Linbo Luan, Qinghua Meng, Qiongfeng Xu, Heng Xu,
Xuesong Xu, Haibo Zhang, Baowei Zhao, Chengyong Li, Taylor B. Guo, Jiansong Yang, Wei Zhang,
Yonggang Zhao, Qiantao Jia, Hongtao Lu, Jia-Ning Xiang, John D. Elliott, and Xichen Lin*
GlaxoSmithKline, R&D China, No. 3 Building, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, PRC
S
* Supporting Information
ABSTRACT: A novel series of 1,2,4-thiadiazole compounds was discovered as
selective S1P1 agonists. The extensive structure−activity relationship studies for
these analogues were reported. Among them, 17g was identified to show high in
vitro potency with reasonable free unbound fraction in plasma (Fu > 0.5%), good
brain penetration (BBR > 0.5), and desirable pharmacokinetic properties in
mouse and rat. Oral administration of 1 mg/kg 17g resulted in significant
peripheral lymphocytes reduction at 4 h after dose and rapid lymphocytes
recovery at 24 h. 17g showed a transient lymphopenia profile in the repeated dose study in mouse. In addition, 17g also
demonstrated efficacy comparable to that of FTY720 (1) in the mouse EAE model of MS.
affects neural cell survival.10 S1P1 receptor has then emerged as
an attractive molecular target for the discovery of new
therapeutic agents for autoimmune diseases.
INTRODUCTION
Multiple sclerosis (MS) is a debilitating, progressive auto-
■
immune disorder in the CNS as a result of inflammation
mediated neural cell damage.1 As the leading cause of
2 is a nonselective S1P1 agonist over the other S1P receptors
(S1P3, S1P4, and S1P5),8b and agonism of S1P3 is thought to be
neurological disability and impairment in young adults (aged
related to hemodynamic and pulmonary side effects seen in
20−40 years), MS is believed to affect around 2.5 million
clinical trials.11 Thus, clinical compounds were optimized
individuals worldwide.2,3 Most MS patients (∼85%) are
diagnosed with the relapsing−remitting form of the disease.4
toward S1P1 agonists with increased selectivity over S1P3,
including prodrug 2-amino-2-[2-[2-chloro-4-[[3-
Currently, all available drugs on the market are administered by
(phenylmethoxy)phenyl]thio]phenyl]ethyl]-1,3-propanediol
injection except for fingolimod (FTY720, 1, Figure 1), which
hydrochloride (KRP-203),12 as well as direct S1P1 agonists (E)-
1-(4-(1-(((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)-
ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (BAF312, pro-
posed structure)13 and (Z,Z)-5-[3-chloro-4-((2R)-2,3-
dihydroxypropoxy)benzylidene]-2-propylimino-3-o-tolylthiazo-
lidin-4-one (ACT128800).14
was recently approved as the first oral drug for RRMS.3
There have been extensive SAR studies on structurally
diverse S1P1 receptor agonists reported to date,15 among which
oxadiazole derivatives were demonstrated to be potent and
selective S1P1 receptor agonists with good pharmacokinetic
properties.16 Most recently, a novel class of oxadiazole
analogues were discovered from this laboratory as selective
S1P1 receptor agonists that showed efficacy comparable to that
of 1 in the mouse EAE model, an animal model for multiple
sclerosis, by oral dosing (3 as a representative, Figure 2).17
However, there were several issues associated with this
compound that potentially can delay its clinical development.
Even though compound 3 showed reasonable CNS penetration
(BBR > 0.5), its high protein binding (Fu = 0.04%)18 indicates a
Figure 1. Structures of FTY720 (1) and its phosphate (2).
The therapeutic activity of 1 could be mainly due to its ability
to reduce lymphocyte infiltration into CNS by sequestration of
peripheral T-cells and B-cells in secondary lymphoid organs.3,5
In addition, 1 may act directly on neural cells to enhance
remyelination in cerebellar slices with lysolecithin-induced
demyelination6 and to attenuate astrogliosis in animal models
of MS.7 The pharmacological efficacy of 1 is believed to result
from the agonism activity of its monophosphate metabolite
(FTY720-p, 2, Figure 1) to S1P1,8 a G-protein-coupled
receptor that regulates peripheral lymphocyte trafficking9 and
Received: January 18, 2012
© XXXX American Chemical Society
A
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discovery of a novel 1,2,4-thiadiazole series as potent, orally
active S1P1 agonists with improved plasma protein binding (Fu
> 0.5%) and high CNS penetration (BBR > 0.5) for the
treatment of MS.
RESULTS AND DISCUSSION
■
We first turned our attention to the replacement of the
oxadiazole core by other aromatics/heteroaromatics to improve
its stability. The opening potential of the oxadiazole ring could
be represented by its low aromatic index (AI, 39 for oxadiazole
and 100 for benzene), an indication of aromaticity of five- or
six-membered ring heterocycles.20 Thus, analogues containing
different heterocyclic aromatic cores with higher aromaticity
(Table 1, AI > 45) were synthesized and evaluated in the in
vitro S1P1 assay. Even though all 21 cores synthesized (4a−u)
showed reduced S1P1 receptor agonism activities in the cell
Figure 2. Lead compound 3.
low free drug concentration in the brain. In addition, the
literature-reported opening of the oxadiazole ring under certain
circumstances was also a concern to us with regard to further
development of compound 3.19 Thus, a more stable compound
with high CNS penetration balanced with reasonable plasma
protein binding should be developed. Herein, we report the
Table 1. S1P1 Potencies with Different Aromatic/Heteroaromatic Cores
a
b
Data are the average of at least two determinations. AI data are from ref 20.
B
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a
Table 2. Physicochemical and Biological Properties of Compounds 3, 4a, 4b, 4d, 4e, 4f, 4l, and 4m
pEC50
b
d
d
d
compd
AI
S1P1
S1P3
cLogP
permeability (nm/s)
solubility (μg/mL)
pIC50(P450)
Fu (%) (human)
BBR (1 h)
BBR (4 h)
c
3
39
63
72
50
64
66
9.7
8.9
8.6
8.5
8.5
8.4
8.8
9
<5
<5
<5
<5
<5
<5
<5
<5
5.1
5
345
230
320
240
220
300
310
190
46
all <5
0.04*
0.51
0.53
0.21
0.36
0.11
0.38
1.0
0.68
0.19
0.52
0.09
0.35
1.5
c
4a
4b
4d
4e
4f
256
160
187
125
115
154
all <5
5.3
4.2
6.1
7.3
7.3
5.1
5.3 (2C9)
5.1 (2C9)
5.4 (2C9)
5.8 (2C9)
5.4 (2C9)
0.21
0.21*
0.10
0.02*
0.01*
0.19
4l
100
84
0.93
0.47
0.95
0.48
4m
161
5.2 (2D6)
a
b
c
S1P1, S1P3 assay data are the average of at least two determinations. AI data are from ref 20. All >5 except for the five CYP enzymes that were
d
included in the assay panel: 1A2, 2D6, 2C9, 2C19, and 3A4. Fu in human plasma unless otherwise indicated by an asterisk; (∗) Fu in rat plasma.
based FRET assay,21 quite a few of them demonstrated
reasonable potency (4a−g, 4l, and 4m) with pEC50 > 8.0. We
then further evaluated these compounds for their physicochem-
ical and biological properties in order to prioritize the chemical
series for further SAR studies (4g was excluded because of its
low AI).
mL). Thus, all compounds were suitable for further evaluation
in in vivo pharmacological studies. It is well established that
S1P1 agonism causes lymphocyte retention in the secondary
lymphoid organs, leading to rapid onset of lymphopenia. In the
acute mouse lymphopenia model where the number of
peripheral blood lymphocytes was measured at 4 and 24 h
after a single oral administration (Experimental Section), these
compounds (1 mg/kg) caused a pharmacologically relevant
lymphocyte reduction to 15−38% of baseline levels at 4 h,
along with rapid recovery in lymphocytes counts (88%, 63%,
and 103% of baseline lymphocyte levels by 24 h for 4b, 4e, and
4m, respectively; 4a showed sustained lymphopenia effect with
<44% lymphocyte recovery). These compounds were further
evaluated in the mouse EAE model.25 At a daily oral dose of 1
mg/kg starting from disease onset (indicated by a nonzero
mean clinical score), compounds 4b and 4m demonstrated
efficacy comparable to that of 1 (1 mg/kg) (Figure 3b).
Compounds 4a and 4e, however, showed no/marginal
beneficial effect in the EAE model (Figure 3a and Figure 3b).
Thus, the 1,2,4-thiadiazole series (4b) and the pyrimidine series
(4m), which demonstrated efficacy in both lymphopenia and
EAE models, were selected for further structure−activity
relationship studies. In this paper, we will focus on the 1,2,4-
thiadiazole series because of space limitation.
We first examined the effects of substitutions on the left-hand
side aromatic group of 4b on the S1P1 receptor potency (4b−
5h, Table 4). Replacements of the cyano group of 4b with
either chlorine (5a) or trifluoromethyl (5b) substitutions
retained potency. The isopropoxyl group could also be replaced
by the isobutyl (5c) functionality with similar or increased
potency. A slight decrease of S1P1 agonist activity was observed
once an N atom was introduced to the phenyl ring (5d). The
replacement of the isopropoxyl functionality was tolerated with
the phenyl ring (5h) or an electron-donating piperidine group
(5g), compared to 5b. However, when more heteroatoms (O
or N) were introduced to the piperidine ring, a significant
decrease of S1P1 potency was observed (5e and 5f). SAR for
substitutions on the left-hand aromatic ring suggested that
isopropoxyl and isobutyl groups are preferred at the para
position and cyano, chloro, and trifluoromethyl functionalities
are preferred at the meta position. As mentioned previously,
one problem of the 1,2,4-thiadiazole series is their high plasma
protein binding (Fu of 0−0.32% for 4b−5h), which may
contribute to the disconnection between the in vitro potency
and the in vivo efficacy. A well-established solution to improve
the protein binding is to introduce polarity to the molecule. We
first tried to introduce an N atom to the right-hand aromatic
ring (5i−l, Table 4). However, this led to a significant decrease
As shown in Table 2, these compounds should have cores
more stable than the 1,2,4-oxadiazole core, as indicated by their
AI (all >50, compared to 39 of 1,2,4-oxadiazole). They are all
selective S1P1 receptor agonists over S1P3 (pEC50 < 5)22 with
good permeability and solubility properties. None of them
raised drug−drug interaction concerns, as evidenced by their
lack of to low inhibitory activities against CYP enzymes (2C9,
2C19, 2D6, and 3A4). However, the compound with the 1,3,4-
oxadiazole core (4d) showed low CNS penetration property
(mouse, ip dosing) with BBR of 0.11 and 0.09 at 1 and 4 h,
respectively. Since the brain exposure is critical for a CNS drug,
4d is thus deprioritized from further progress. On the other
hand, analogues with the thiophene and phenyl cores (4f and
4l) are highly brain penetrable (BBR > 0.9 at both 1 and 4 h),
whereas their developability was predicted to be poor (cLogP =
7.3) according to Lipinski’s rule.23 For this reason, 4f and 4l
were also deprioritized. It is noteworthy that most of the
compounds (except for 4a) showed high protein binding
property. We decided to resolve this issue at a later stage,
knowing several strategies are available in literature to improve
protein binding.24 For further comparison, the four compounds
with the 1,3,4-thiadiazole core (4a), the 1,2,4-thiadiazole core
(4b), the thiazole core (4e), and the pyrimidine core (4m)
were progressed to in vivo studies.
Mouse pharmacokinetic studies for the four compounds
revealed a desirable PK profile (Table 3). They all had good
oral bioavailability (>75%), moderate to long half-lives (1.5−
8.0 h), and moderate to high blood exposure (2.60−11.5 μg·h/
Table 3. Mouse Pharmacokinetic and Lymphopenia Studies
for Compounds 4a, 4b, 4e, and 4m
lymphocyte counts (relative to
compd
mouse PK
baseline)
4a
T1/2 = 3.8 h; F ≈ 100%
AUC0−24h = 5.94 μg·h/mL
T1/2 = 1.5 h; F = 87%
37.9% (4 h),
43.6% (24 h)
22.7% (4 h),
87.9% (24 h)
15.3% (4 h),
62.6% (24 h)
34.9% (4 h),
103.2% (24 h)
4b
4e
AUC0−24h = 2.60 μg·h/mL
T1/2 = 3.6 h; F ≈ 100%
AUC0−24h = 8.34 μg·h/mL
T1/2 = 8.0 h; F = 78%
4m
AUC0−24h = 11.5 μg·h/mL
C
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Table 4. SAR of the Left-Hand and Right-Hand Aromatic
Rings and the Human Plasma Protein Binding Data
Measured as Fu
Figure 3. (a) Treatment efficacy of compound 4a in mouse EAE. (b)
Treatment efficacy of compounds 4b, 4e, and 4m in mouse EAE.
in the S1P1 receptor potency (S1P1 pEC50 of 6.4−7.6) without
any significant improvement in plasma protein binding (Fu of
0.12−0.19% for 5i−l). We then turned our attention to
introduce polarity (such as N) to the carboxylic side chain for
better potency and plasma protein binding.
In order to efficiently explore the SAR of the carboxylic acid
side chain, convergent syntheses of the general structures 12
and 17 were developed, where the diversity (amino acid side
chains) was introduced at the last step (Scheme 1). Suzuki
coupling of the commercially available starting material 5-
chloro-3-bromo-1,2,4-thiadiazole (6) with boronic ester 7
provided diaryl bromide 8. The second Suzuki coupling of 8
with boronic ester 9 afforded aldehyde 10. The final step of the
reductive amination with different amino acids 11 gave benzylic
amino acid analogues 12 with generally acceptable yield. For
the synthesis of homobenzylic amino acid derivatives, the
boronic ester for the second Suzuki coupling is methyl enoether
13. After acid catalyzed hydrolysis of the methyl enoether 14,
aldehyde 15 was obtained with a moderate to good yield.
Reductive amination then afforded homobenzylic amino acid
derivatives 17.
Having developed a robust synthetic procedure for various
amino acid analogues, we started to investigate the right side of
SAR. Trifluoromethyl substitution on the left-hand phenyl ring
was our first focus (12a−d and 17a−d) because of the higher
potency. As shown in Table 5, straight chain benzylic amino
acid derivatives (12a and 12b) showed moderate S1P1 agonist
activities. Extending the side chain by one carbon to
homobenzylic amino acid analogues provided much improved
potency (17a and 17b). Cyclic amino acids resulted in either
similar or higher potency (12c and 12d, respectively). Further
extending the side chain to their homobenzylic analogues led to
a
b
Data are the average of at least two determinations. Fu in human
plasma unless otherwise indicated by an asterisk; (∗) Fu in rat plasma.
a substantial increase of activity (piperidine analogue 17c) or a
similar activity (azetidine analogue 17d). However, these
compounds (12a−d and 17a−d) still showed high protein
binding except for 12a. Apparently, a further lipophilicity
reduction of these compounds is needed. We then turned our
attention to replace the trifluoromethyl group with less
lipophilic Cl and CN. As the straight chain amino acid
derivatives provided limited exposure probably because of high
clearance, the cyclic amino acid analogues were chosen to be
explored. As expected, replacement of the trifluoromethyl
group with Cl (12e, 17e, and 17f) or CN (12f, 17g, and 17h)
resulted in much improved protein binding property with Fu at
0.45−1.28%. In addition, all of them showed excellent S1P1
potency (pEC50 > 9.0) except for the two benzylic azetidine
derivatives (12e and 12f). The carboxylic acid functionality
proved to be critical for S1P1 activity. Replacement or removal
of the carboxylic acid resulted in dramatic decrease in potency
(17i and 17j). All compounds (12a−f and 17a−h) proved to
be selective S1P1 receptor agonists versus S1P3 (>1000 fold).
D
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a
Scheme 1. General Synthetic Procedure for the 1,2,4-
Thiadiazole Amino Acid Derivatives 12 and 17
Table 5. SAR for Amino Acid Side Chains
a
a
(a) 7, PdCl2(dppf), K3PO4, 53−97%. (b) 9, Pd(PPh3)4, K3PO4, 77−
94%. (c) 11, NaBH(OAc)3, HOAc, 10−60%. (d) 13, Pd(PPh3)4,
K3PO4, 65−98%. (e) HCl, 80−100%. (f) 16, NaBH(OAc)3, HOAc,
10−60%.
Among these compounds, 17e and 17g demonstrated good
overall balanced profile (S1P1 pEC50 > 9.0; Fu > 0.4%; BBR >
0.5, Table 6) and were then selected for further in vitro and in
vivo evaluation.
In a mouse CNS penetration study, both 17e and 17g
showed good BBR (1.49 and 0.68, respectively, at 4 h after
dosing; Table 6). Mouse PK studies revealed that both
compounds had decent oral bioavailability (∼40%) and
moderate to long half-lives (15.6 and 5.3 h for 17e and 17g,
respectively). In rat, 17e also showed a much longer half-life
(27.7 h) than 17g (6.2 h). Again, both compounds
demonstrated good bioavailability (>50%) in rat. Encouraged
by their mouse and rat PK profile, we evaluated both
compounds in in vivo efficacy studies. In the acute mouse
lymphopenia model, both compounds turned out to be
efficacious at 1 mg/kg (62−83% lymphocyte reduction at 4
h). On the basis of the lymphocyte counts measured by 24 h,
compound 17g (72% of baseline at 24 h) had a transient effect
whereas compound 17e resulted in a sustained lymphopenia
profile (only 40% of baseline at 24 h). Thus, duration of the PD
effect produced by these compounds seemed to correlate with
their half-lives. As expected, both compounds demonstrated
good stability and no glutathione (GSH) adducts were
observed in the GSH conjugation study. Neither compound
showed inhibitory activity in the human CYP enzymes (1A2,
2D6, 2C9, 2C19, and 3A4), indicating that there is no drug−
drug interaction concern. They were both progressed to the
chronic mouse EAE evaluation. To our delight, both
compounds were efficacious at a daily oral dose of 1 mg/kg
(Figure 4a and 4b).
Compound 17g was further progressed in both single and
repeat dosing lymphopenia studies to assess its PK−PD
relationship to facilitate human dose selection. The single
dose rat PK−PD study demonstrated a dose-dependent
lymphocyte reduction and later on reversal over a 60 h period
(Figure 5a), with a calculated IC50 of 12 ng/mL. In addition,
the repeat-dose study showed that at the EAE-efficacious dose
of 1 mg/kg, compound 17g rapidly produced a daily
a
S1P1, S1P3 assay data are the average of at least two determinations.
Fu in human plasma unless otherwise indicated by an asterisk; (∗) Fu
b
in rat plasma.
lymphocyte reduction at the range 20−60% of baseline over
the treatment period (Figure 5b). Perhaps more important,
E
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Table 6. In Vitro and in Vivo Data for 17e and 17g
17e
17g
a
BBR
0.52 (1 h)
0.23 (1 h)
0.65 (4 h)
Fpo = 45%
1.48 (4 h)
mouse PK
Fpo = 40%
T1/2 = 15.6 h
Fpo = 70%
T1/2 = 5.3 h
Fpo = 50%
b
rat PK
T1/2 = 27.7 h
16% (4 h),
40% (24 h)
all pIC50 < 5
not observed
T1/2 = 6.2 h
23% (4 h),
72% (24 h)
all pIC50 < 5
not observed
a
lymphopenia
CYP (1A2, 2D6, 2C9, 2C19, 3A4)
GSH conjugates
a
b
See Experimental Section. Male Sprague−Dawley (for iv, 1 mg/kg,
1%DMSO in 10% HP-β-CD (w/v), N = 3; for po, 2 mg/kg, 1%
DMSO in 1% MC (w/v), N = 3).
Figure 5. (a) Dose-dependent lymphopenia produced by single
ascending dose of compound 17g. (b) Chronic lymphopenia elicited
by repeat dosing of compound 17g.
EXPERIMENTAL SECTION
■
Chemistry. Compounds not described below were purchased from
commercial vendors or as previously reported. Compound purity was
determined using LC/MS analysis. Purification of the compounds was
carried out by conventional methods such as chromatography and/or
recrystallization using suitable solvents. Chromatographic methods
include column chromatography, flash chromatography, and MDAP
(mass directed autopurification system).
1H NMR spectral data were recorded on a Bruker 400 NMR
spectrometer operating at 400 MHz. 13C NMR spectra were recorded
at 100 MHz. CDCl3 is deuteriochloroform, and DMSO-d6 is
hexadeuteriodimethylsulfoxide. Chemical shifts are given in parts per
million (δ) downfield from the internal standard tetramethylsilane
(TMS) or the NMR solvent. Abbreviations for NMR data are as
follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
dd = doublet of doublets, dt = doublet of triplets, app = apparent, br =
broad. J indicates the NMR coupling constant measured in hertz. High
resolution mass spectrometry (HRMS) was operated in positive mode
of electrospray ionization (ESI) at an orthogonal acceleration time-of-
flight (oa-TOF) SYNAPT G2 HDMSTM (Waters, Manchester, U.K.).
Solutions (500 ng/mL in ACN/H2O (1:1, v/v)) were introduced via
infusion at a flow rate of 5 μL/min to acquire accurate mass. LC/MS
(Agilent 1200SL-6110) analysis was conducted for all assayed
compounds in either acidic or basic conditions. (1) Acidic conditions
refer to water containing 0.05% TFA/acetonitrile as mobile phase on
an Agilent SB-C18 column (1.8 μm, 4.6 mm × 30 mm) with MS
instrument and photodiode array detector. The following conditions
were used: a gradient from 5% to 95% in 4 min (or 6 min) and held at
95% for 1 min; UV detection at 214 and 254 nm; a flow rate of 1.5
mL/min; full scan; mass range from 100 to 1000 amu. (2) Basic
conditions refer to water containing 10 mM aqueous NH4HCO3/
acetonitrile as mobile phase on a Waters XBridge C18 column (3.5
μm, 4.6 mm × 50 mm) with MS instrument and photodiode array
detector. The following conditions were used: a gradient from 5% to
Figure 4. (a) Treatment efficacy of compound 17e in mouse EAE. (b)
Treatment efficacy of compound 17g in mouse EAE.
lymphocyte counts recovered to normal range (∼70% of
baseline) 3 days after treatment cessation.
CONCLUSION
■
We have discovered a novel 1,2,4-thiadiazole series of
compounds as selective S1P1 agonists. Extensive SAR studies
led to the discovery of compound 17g, a potent S1P1 agonist
(pEC50 > 9) with reasonable Fu in plasma (0.64%), good brain
penetration (BBR > 0.5), and desirable PK properties in rodent
species. Oral administration of 1 mg/kg of 17g resulted in
significant peripheral lymphocyte reduction at 4 h after dose
and rapid lymphocyte recovery at 24 h. A transient
lymphopenia profile for 17g was observed in the repeat dosing
lymphopenia study. In addition, 17g demonstrated comparable
efficacy to 1 in the mouse EAE model of MS.
F
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1
95% in 5 min and held at 95% for 1 min; UV detection at 214 and 254
nm; a flow rate of 2 mL/min; full scan; mass range from 100 to 1000
amu. All the assayed compounds possess ≥95% purity determined
using LC/MS analysis. Column chromatography was performed on
Isco or Biotage instrument using a prepacked silica gel column, a
detector with UV wavelength at 254 nm, and mixed solvents. MDAP
equipped with 2489 UV detector, 2767 sample manager, 2545 pump,
and 3100 single quadrupole mass spectrometer was performed on
Sunfire Prep C18 column (5 μm, 19 mm × 50 mm) using water
containing 0.05% TFA/acetonitrile as mobile phase. The following
conditions were used: a gradient from 5% to 95% in 8 min and held in
95% for 2 min; a flow rate of 30 mL/min.
3-Bromo-5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-1,2,4-
thiadiazole (8a). To a solution of 2-(3-chloro-4-isopropoxyphenyl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane 7a26 (500 mg, 1.51 mmol), 3-
bromo-5-chloro-1,2,4-thiadiazole 6 (604 mg, 3.03 mmol), and
tripotassium phosphate (964 mg, 4.54 mmol) in DMF (3 mL) and
water (0.6 mL) was added PdCl2(dppf)−CH2Cl2 adduct (618 mg,
0.76 mmol). The vessel was sealed and heated in a microwave reactor
at 80 °C for 1 h. After cooling, the mixture was concentrated in vacuo,
and the residue was purified by chromatography on silica gel with
petroleum/ethyl acetate (4:1) to give 294 mg (53%) of 8a as a white
solid. ESMS m/z: 366.9 (M + H)+.
3-Bromo-5-(3-chloro-4-isopropoxyphenyl)-1,2,4-thiadiazole
(8b). Compound 8b (6.2 g, 66%) was prepared from 2-(3-chloro-4-
isopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 7b26 (6 g,
28 mmol) and 6 (11.2 g, 56 mmol) in the same manner as described
for 8a. 1H NMR (600 MHz, CDCl3) δ ppm 1.44 (d, J = 6.40 Hz, 6 H),
4.69 (dt, J = 12.05, 6.40 Hz, 1 H), 7.01 (d, J = 8.66 Hz, 1 H), 7.78 (dd,
J = 8.28, 2.26 Hz, 1 H), 7.99 (d, J = 2.26 Hz, 1 H). ESMS m/z: 333.0
(M + H)+.
5-(3-Bromo-1,2,4-thiadiazol-5-yl)-2-isobutylbenzonitrile
(8c). Compound 8c (1.14 g, 88%) was prepared from 2-(2-
methylpropyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzonitrile 7c26 (1 g, 3.51 mmol) and 6 (1.399 g, 7.01 mmol) in the
same manner as described for 8a. 1H NMR (600 MHz, CDCl3) δ ppm
1.00 (d, J = 6.78 Hz, 6 H), 2.01−2.10 (m, 1 H), 2.81 (d, J = 7.53 Hz, 2
H), 7.47 (d, J = 8.28 Hz, 1 H), 8.06 (dd, J = 8.28, 1.88 Hz, 1 H), 8.23
(d, J = 1.88 Hz, 1 H). ESMS m/z: 321.9 (M + H)+.
2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-1,2,4-
thiadiazol-3-yl)benzaldehyde (10a). To a solution of 8a (0.93 g,
2.53 mmol) in DMF (6 mL) and water (1.5 mL) were added 2-ethyl-
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde 917
(0.659 g, 2.53 mmol), tripotassium phosphate (1.344 g, 6.33 mmol),
and Pd(Ph3P)4 (0.585 g, 0.507 mmol). The reaction vessel was sealed
and heated with a microwave reactor at 120 °C for 15 min. After
cooling, the reaction mixture was filtered and the solid was washed
with ethyl acetate (2 × 15 mL). The filtrate and the washing solution
were combined. The combined solution was washed with water (2 ×
20 mL), then dried over anhydrous sodium sulfate. The dried solution
was concentrated in vacuo, and the residue was purified by
chromatography on silica gel with petroleum/ethyl acetate (9:1) to
give 1 g (94%) of 10a as a brown oil. 1H NMR (400 MHz, CDCl3) δ
ppm 1.37 (t, J = 7.40 Hz, 3 H), 1.44 (d, J = 6.02 Hz, 6 H), 3.39 (q, J =
7.40 Hz, 2 H), 4.78 (dt, J = 12.11, 6.02 Hz, 1 H), 7.14 (d, J = 8.78 Hz,
1 H), 7.50 (t, J = 7.78 Hz, 1 H), 8.02 (dd, J = 7.78, 1.51 Hz, 1 H),
8.12−8.17 (m, 2 H), 8.24 (d, J = 2.01 Hz, 1 H), 10.48 (s, 1 H). ESMS
m/z: 421.1 (M + H)+.
3-(5-(3-Chloro-4-isopropoxyphenyl)-1,2,4-thiadiazol-3-yl)-2-
ethylbenzaldehyde (10b). Compound 10b (450 mg, 78%) was
prepared from 8b (500 mg, 1.51 mmol) and 9 (390 mg, 1.51 mmol) in
the same manner as described for 10a. 1H NMR (400 MHz, CDCl3) δ
ppm 1.37 (t, J = 7.40 Hz, 3 H), 1.45 (d, J = 6.02 Hz, 6 H), 3.38 (q, J =
7.40 Hz, 2 H), 4.71 (dt, J = 12.11, 6.02 Hz, 1 H), 7.05 (d, J = 8.78 Hz,
1 H), 7.49 (t, J = 7.65 Hz, 1 H), 7.88 (dd, J = 8.53, 2.26 Hz, 1 H), 8.01
(dd, J = 7.65, 1.38 Hz, 1 H), 8.08 (d, J = 2.01 Hz, 1 H), 8.14 (dd, J =
7.78, 1.51 Hz, 1 H), 10.48 (s, 1 H). ESMS m/z: 387.1 (M + H)+.
5-(3-(2-Ethyl-3-formylphenyl)-1,2,4-thiadiazol-5-yl)-2-isobu-
tylbenzonitrile (10c). Compound 10c (520 mg, 89%) was prepared
from 8c (500 mg, 1.55 mmol) and 9 (424 mg, 1.63 mmol) in the same
manner as described for 10a. H NMR (600 MHz, CDCl3) δ ppm
1.01 (d, J = 6.78 Hz, 6 H), 1.38 (t, J = 7.34 Hz, 3 H), 2.03−2.10 (m, 1
H), 2.82 (d, J = 7.53 Hz, 2 H), 3.40 (q, J = 7.34 Hz, 2 H), 7.47−7.54
(m, 2 H), 8.04 (d, J = 7.53 Hz, 1 H), 8.12−8.19 (m, 2 H), 8.31 (d, J =
1.88 Hz, 1 H), 10.48 (s, 1 H). ESMS m/z: 376.1 (M + H)+.
2-((2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)benzyl)(methyl)amino)acetic Acid (12a).
To a solution of 10a (80 mg, 0.19 mmol) in DCM (6 mL) were
added N-methylglycine (51 mg, 0.57 mmol) and acetic acid (0.022
mL, 0.38 mmol). The reaction solution was stirred at room
temperature overnight. Sodium triacetoxyborohydride (81 mg, 0.38
mmol) was added, and stirring continued for another 2 h. The reaction
mixture was concentrated in vacuo, and the residue was purified by
1
MDAP to give 35 mg (30%) of 12a as a white solid. H NMR (400
MHz, DMSO-d6) δ ppm 1.09 (t, J = 7.28 Hz, 3 H), 1.34 (d, J = 6.02
Hz, 6 H), 2.74 (s, 3 H), 3.08 (q, J = 7.28 Hz, 2 H), 4.11 (s, 2 H), 4.42
(s, 2 H), 4.96 (dt, J = 11.98, 6.02 Hz, 1 H), 7.47 (t, J = 7.65 Hz, 1 H),
7.54 (d, J = 8.78 Hz, 1 H), 7.68 (d, J = 7.28 Hz, 1 H), 7.90 (d, J = 7.28
Hz, 1 H), 8.26 (d, J = 2.26 Hz, 1 H), 8.33 (dd, J = 8.66, 2.13 Hz, 1 H).
HRMS C24H27N3O3F3S (M + H)+ calcd 494.1725, found 494.1728.
LC/MS: tR = 4.23 min, >95%, m/z 494.2 (M + H)+.
2-((2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)benzyl)(methyl)amino)-2-methylpropa-
noic Acid (12b). To a solution of 10a (650 mg, 1.55 mmol) in
MeOH (10 mL) at 0 °C was added sodium borohydride (117 mg, 3.09
mmol). The reaction mixture was stirred at room temperature
overnight. After removal of the solvent, the residue was dissolved in
ethyl acetate (50 mL). The solution was washed with water and dried
over anhydrous sodium sulfate. The dried solution was concentrated in
vacuo to give the crude alcohol. The alcohol was dissolved in DCM
(10 mL), and perbromomethane (615 mg, 1.86 mmol) was added at 0
°C, followed by a solution of triphenylphosphine (608 mg, 2.32
mmol) in DCM (10 mL). The reaction solution was stirred at room
temperature for 1 h. The reaction mixture was concentrated in vacuo,
and the residue was purified by chromatography on silica gel with
petroleum/ethyl acetate (4:1) to give 400 mg (53%) of 3-(3-
(bromomethyl)-2-ethylphenyl)-5-(4-isopropoxy-3-(trifluoromethyl)-
phenyl)-1,2,4-thiadiazole. 1H NMR (400 MHz, CDCl3) δ ppm 1.29 (t,
J = 7.40 Hz, 3 H), 1.44 (d, J = 6.02 Hz, 6 H), 3.14 (q, J = 7.40 Hz, 2
H), 4.67 (s, 2 H), 4.77 (dt, J = 12.11, 6.02 Hz, 1 H), 7.13 (d, J = 8.78
Hz, 1 H), 7.30−7.36 (m, 1 H), 7.50 (dd, J = 7.53, 1.25 Hz, 1 H), 7.90
(dd, J = 7.78, 1.25 Hz, 1 H), 8.14 (dd, J = 8.66, 2.13 Hz, 1 H), 8.24 (d,
J = 2.01 Hz, 1 H). ESMS m/z: 485.0 (M + H)+.
To a solution of the above bromide (100 mg, 0.21 mmol) in DMF
(5 mL) were added potassium carbonate (85 mg, 0.62 mmol) and 2-
methyl-2-(methylamino)propanoic acid (29.0 mg, 0.25 mmol). The
reaction mixture was heated at 60 °C overnight. After cooling, the
reaction mixture was diluted with ethyl acetate (20 mL). The organic
fraction was separated, washed with water, concentrated, and purified
by MDAP to give 27 mg (21%) of 12b as a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 1.16 (t, J = 7.28 Hz, 3 H), 1.34 (d, J = 6.02
Hz, 6 H), 1.51 (s, 6 H), 2.58 (s, 3 H), 2.99 (q, J = 7.28 Hz, 2 H), 4.96
(dt, J = 11.98, 6.02 Hz, 1 H), 5.42 (s, 2 H), 7.42 (t, J = 7.65 Hz, 1 H),
7.53 (d, J = 9.03 Hz, 1 H), 7.60 (d, J = 7.53 Hz, 1 H), 7.89 (d, J = 7.53
Hz, 1 H), 8.26 (s, 1 H), 8.29−8.37 (m, 1 H), 9.31 (br s, 1 H). 13C
NMR (100 MHz, DMSO-d6) δ 185.4, 173.5, 170.7, 158.5, 158.1,
157.8, 142.2, 133.8, 133.7, 132.8, 131.6, 125.9, 121.7, 118.8, 118.5,
115.8, 71.7, 65.8, 60.9, 27.6, 22.2, 21.5, 21.1, 15.5. HRMS
C26H31N3O3F3S (M + H)+ calcd 522.2038, found 522.2048. LC/
MS: tR = 4.67 min, >95%, m/z 522.2 (M + H)+.
1-(2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)benzyl)piperidine-4-carboxylic Acid (12c).
To a solution of 10a (80 mg, 0.19 mmol) in DCM (5 mL) and MeOH
(5 mL) were added ethyl 4-piperidinecarboxylate (90 mg, 0.57 mmol)
and acetic acid (0.022 mL, 0.38 mmol). The reaction solution was
stirred at room temperature overnight. Sodium triacetoxyborohydride
(81 mg, 0.381 mmol) was added, and stirring continued for another 2
h. After removal of the solvent, the residue was dissolved in ethyl
acetate (50 mL). The organic fraction was separated, washed with
water (2 × 10 mL), and concentrated to afford the ester. The ester was
G
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dissolved in isopropanol (5 mL) and water (5 mL), and NaOH (0.5 M
in water) (1.90 mL, 0.95 mmol) was added. The reaction solution was
heated at 90 °C for 2 h. After removal of the solvent, the residue was
acidified to pH 3−4, extracted with ethyl acetate (2 × 20 mL). The
combined organic phases were dried over sodium sulfate and
concentrated, and the residue was purified by MDAP to give 44 mg
(335 mg, 1.16 mmol) in the same manner as described for 10a as a
colorless oil except that the reaction time was 10 min. ESMS m/z:
415.1 (M + H)+.
5-(3-(2-Ethyl-3-(2-methoxyvinyl)phenyl)-1,2,4-thiadiazol-5-
yl)-2-isobutylbenzonitrile (14c). Compound 14c (928 mg, 66%)
was prepared from 8c (1.13 g, 3.51 mmol) and 13 (1.11 g, 3.86 mmol)
in the same manner as described for 10a as an oil except that the
reaction temperature was 110 °C. ESMS m/z: 404.2 (M + H)+.
2-(2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)phenyl)acetaldehyde (15a). To a solution
of 14a (140 mg, 0.31 mmol) in THF (10 mL) under nitrogen was
added HCl (0.2 mL, 0.40 mmol). The reaction mixture was heated at
70 °C for 5 h. After the mixture was cooled, the solvent was
concentrated under reduced pressure. The remaining trace hydro-
chloric acid was removed under high vacuum for 2 h to give 108 mg
(80%) of 15a, which was used for the following step without further
purification. ESMS m/z: 435.2 (M + H)+.
1
(36%) 12c as a white solid. H NMR (400 MHz, DMSO-d6) δ ppm
1.08 (t, J = 7.40 Hz, 3 H), 1.34 (d, J = 6.02 Hz, 6 H), 1.77 (q, J = 12.30
Hz, 2 H), 2.08 (d, J = 13.30 Hz, 2 H), 3.04 (q, J = 6.78 Hz, 2 H),
3.12−3.26 (m, 2 H), 3.41−3.50 (m, 3 H), 4.38−4.54 (m, 2 H), 4.96
(dt, J = 12.05, 6.02 Hz, 1 H), 7.44−7.57 (m, 2 H), 7.71 (d, J = 7.53
Hz, 1 H), 7.91 (d, J = 7.53 Hz, 1 H), 8.26 (d, J = 2.01 Hz, 1 H), 8.33
(dd, J = 8.78, 2.01 Hz, 1 H), 9.24 (br s, 1 H), 12.58 (br s, 1 H). 13C
NMR (100 MHz, DMSO-d6) δ 185.5, 174.6, 173.4, 158.5, 158.1,
143.7, 133.7, 133.3, 132.4, 126.4, 125.8, 124.5, 121.7, 118.9, 118.5,
115.8, 71.8, 56.1, 51.2, 38.0, 25.3, 22.7, 21.5, 16.0. HRMS
C27H31N3O3F3S (M + H)+ calcd 534.2038, found 534.2041. LC/
MS: tR = 4.26 min, >95%, m/z 534.3 (M + H)+.
1-(2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)benzyl)azetidine-3-carboxylic Acid (12d).
Compound 12d (44 mg, 37%) was prepared from 10a (100 mg, 0.24
mmol) and methyl azetidine-3-carboxylate, HCl salt (72 mg, 0.48
mmol) in the same manner as described for 12c. 1H NMR (400 MHz,
DMSO-d6) δ ppm 1.12 (t, J = 7.40 Hz, 3 H), 1.34 (d, J = 6.02 Hz, 6
H), 2.93 (q, J = 7.40 Hz, 2 H), 3.18−3.28 (m, 3 H), 3.42 (t, J = 6.65
Hz, 2 H), 3.66 (s, 2 H), 4.95 (dt, J = 12.05, 6.02 Hz, 1 H), 7.30 (t, J =
7.65 Hz, 1 H), 7.45 (d, J = 6.53 Hz, 1 H), 7.52 (d, J = 8.78 Hz, 1 H),
7.67−7.73 (m, 1 H), 8.24 (d, J = 2.01 Hz, 1 H), 8.31 (dd, J = 8.78,
2.01 Hz, 1 H). LC/MS: tR = 4.27 min, >95%, m/z 506.1 (M + H)+.
1-(3-(5-(3-Chloro-4-isopropoxyphenyl)-1,2,4-thiadiazol-3-
yl)-2-ethylbenzyl)azetidine-3-carboxylic Acid (12e). Compound
12e (40 mg, 33%) was prepared from 10b (100 mg, 0.26 mmol) and
methyl azetidine-3-carboxylate, HCl salt (59 mg, 0.39 mmol) in the
same manner as described for 12c. 1H NMR (400 MHz, DMSO-d6) δ
ppm 1.11 (t, J = 7.28 Hz, 3 H), 1.34 (d, J = 6.02 Hz, 6 H), 2.93 (q, J =
7.28 Hz, 2 H), 3.19−3.28 (m, 3 H), 3.42 (t, J = 6.53 Hz, 2 H), 3.67 (s,
2 H), 4.85 (dt, J = 12.05, 6.02 Hz, 1 H), 7.26−7.32 (m, 1 H), 7.37 (d, J
= 8.78 Hz, 1 H), 7.44 (d, J = 7.28 Hz, 1 H), 7.71 (d, J = 7.03 Hz, 1 H),
8.00 (dd, J = 8.66, 2.13 Hz, 1 H), 8.13 (d, J = 2.01 Hz, 1 H). 13C NMR
(100 MHz, DMSO-d6) δ 185.2, 174.3, 174.1, 156.0, 141.7, 136.8,
132.6, 130.7, 129.7, 128.8, 127.9, 125.6, 123.1, 123.0, 115.5, 71.6, 60.4,
56.6, 33.6, 22.0, 21.7, 15.3. HRMS C24H27N3O3SCl (M + H)+ calcd
472.1462, found 472.1462. LC/MS: tR = 4.04 min, >95%, m/z 472.2
(M + H)+.
2-(3-(5-(3-Chloro-4-isopropoxyphenyl)-1,2,4-thiadiazol-3-
yl)-2-ethylphenyl)acetaldehyde (15b). Compound 15b (300 mg,
99%) was prepared from 14b (313 mg, 0.76 mmol) in the same
manner as described for 15a. ESMS m/z: 401.1 (M + H)+.
5-(3-(2-Ethyl-3-(2-oxoethyl)phenyl)-1,2,4-thiadiazol-5-yl)-2-
isobutylbenzonitrile (15c). Compound 15c (990 mg, 100%) was
prepared from 14c (1.03 g, 2.55 mmol) in the same manner as
1
described for 15a. H NMR (600 MHz, CDCl3) δ ppm 1.00 (d, J =
6.40 Hz, 6 H), 1.19 (t, J = 7.34 Hz, 3 H), 2.03−2.09 (m, 1 H), 2.81 (d,
J = 7.15 Hz, 2 H), 2.97 (q, J = 7.34 Hz, 2 H), 3.86 (s, 2 H), 7.32−7.38
(m, 2 H), 7.45−7.49 (m, 1 H), 7.89 (d, J = 7.15 Hz, 1 H), 8.14 (d, J =
7.91 Hz, 1 H), 8.30 (s, 1 H), 9.79 (s, 1 H). ESMS m/z: 390.2 (M +
H)+.
2-((2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)phenethyl)amino)acetic Acid (17a). Com-
pound 17a (5.6 mg, 11%) was prepared from 15a (45 mg, 0.10 mmol)
and glycinate (21 mg, 0.21 mmol) in the same manner as described for
12c, giving a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.13
(t, J = 7.40 Hz, 3 H), 1.34 (d, J = 6.02 Hz, 6 H), 2.91 (q, J = 7.40 Hz, 2
H), 3.02 (s, 4 H), 3.22 (s, 2 H), 4.95 (dt, J = 12.05, 6.02 Hz, 1 H),
7.27−7.40 (m, 2 H), 7.52 (d, J = 9.03 Hz, 1 H), 7.68−7.72 (m, 1 H),
8.24 (d, J = 2.01 Hz, 1 H), 8.31 (dd, J = 8.78, 2.01 Hz, 1 H). 13C NMR
(100 MHz, DMSO-d6) δ 185.2, 174.0, 158.4, 141.4, 136.8, 133.6,
132.8, 131.4, 129.5, 126.1, 125.9, 121.8, 118.5, 115.7, 71.7, 50.0, 47.8,
29.1, 22.2, 21.5, 15.7. HRMS C24H27N3O3F3S (M + H)+ calcd
494.1725, found 494.1726. LC/MS: tR = 3.37 min, >95%, m/z 494.2
(M + H)+.
2-((2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)phenethyl)(methyl)amino)acetic Acid
(17b). Compound 17b (9.2 mg, 17%) was prepared from 15a (45
mg, 0.10 mmol) and ethyl 2-(methylamino)acetate (24.27 mg, 0.21
mmol) in the same manner as described for 12c, giving a white solid.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.13 (t, J = 7.28 Hz, 3 H),
1.34 (d, J = 6.02 Hz, 6 H), 2.58 (s, 3 H), 2.88−2.97 (m, 4 H), 3.32 (s,
4 H), 4.95 (dt, J = 12.05, 6.02 Hz, 1 H), 7.30 (t, J = 7.65 Hz, 1 H),
7.38 (d, J = 6.78 Hz, 1 H), 7.53 (d, J = 8.78 Hz, 1 H), 7.67 (d, J = 7.78
Hz, 1 H), 8.25 (d, J = 1.76 Hz, 1 H), 8.32 (dd, J = 8.78, 2.01 Hz, 1 H).
HRMS C25H29N3O3F3S (M + H)+ calcd 508.1882, found 508.1888.
LC/MS: tR = 3.36 min, >95%, m/z 508.2 (M + H)+.
1-(3-(5-(3-Cyano-4-isobutylphenyl)-1,2,4-thiadiazol-3-yl)-2-
ethylbenzyl)azetidine-3-carboxylic Acid (12f). Compound 12f
(51 mg, 33%) was prepared from 10c (100 mg, 0.27 mmol) and
methyl azetidine-3-carboxylate, HCl salt (42 mg, 0.28 mmol) in the
same manner as described for 12c. 1H NMR (400 MHz, DMSO-d6) δ
ppm 0.94 (d, J = 6.78 Hz, 6 H), 1.10 (t, J = 7.40 Hz, 3 H), 1.99 (dt, J =
13.49, 6.78 Hz, 1 H), 2.78 (d, J = 7.28 Hz, 2 H), 3.00 (q, J = 7.03 Hz, 2
H), 3.66 (quin, J = 8.28 Hz, 1 H), 4.19−4.38 (m, 4 H), 4.58 (s, 2 H),
7.46 (t, J = 7.65 Hz, 1 H), 7.58 (d, J = 7.53 Hz, 1 H), 7.70 (d, J = 8.03
Hz, 1 H), 7.90 (d, J = 7.28 Hz, 1 H), 8.33 (dd, J = 8.16, 1.88 Hz, 1 H),
8.54 (d, J = 1.76 Hz, 1 H), 10.31 (br s, 1 H), 13.19 (br s, 1 H). 13C
NMR (100 MHz, DMSO-d6) δ 185.1, 173.6, 158.1, 149.0, 142.7,
133.1, 132.0, 131.8, 131.5, 128.5, 126.4, 117.2, 113.2, 55.6, 54.4, 42.7,
32.3, 29.7, 22.4, 22.0, 15.7. HRMS C26H29N4O2S (M + H)+ calcd
461.2011, found 461.2011. LC/MS: tR = 4.11 min, >95%, m/z 461.1
(M + H)+.
3-(2-Ethyl-3-(2-methoxyvinyl)phenyl)-5-(4-isopropoxy-3-
(trifluoromethyl)phenyl)-1,2,4-thiadiazole (14a). Compound 14a
(580 mg, 98%) was prepared from 8a (487 mg, 1.33 mmol) and 2-(2-
ethyl-3-(2-methoxyvinyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane 1326 (573 mg, 1.99 mmol) in the same manner as described for
10a as a colorless oil except that the reaction time was 10 min. ESMS
m/z: 449.1 (M + H)+.
1-(2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)phenethyl)piperidine-4-carboxylic Acid
(17c). Compound 17c (5 mg, 9%) was prepared from 15a (45 mg,
0.10 mmol) and ethyl 4-piperidinecarboxylate (323 mg, 0.21 mmol) in
1
the same manner as described for 12c, giving a white solid. H NMR
(400 MHz, DMSO-d6) δ ppm 1.13 (t, J = 7.28 Hz, 3 H), 1.34 (d, J =
6.02 Hz, 6 H), 1.49−1.61 (m, 2 H), 1.80 (d, J = 13.80 Hz, 2 H), 2.01−
2.10 (m, 2 H), 2.12−2.18 (m, 1 H), 2.81−2.94 (m, 8 H), 4.96 (dt, J =
12.11, 6.02 Hz, 1 H), 7.27 (t, J = 7.53 Hz, 1 H), 7.36 (d, J = 7.78 Hz, 1
H), 7.53 (d, J = 9.03 Hz, 1 H), 7.64 (dd, J = 7.65, 1.38 Hz, 1 H), 8.25
(d, J = 2.01 Hz, 1 H), 8.32 (dd, J = 8.66, 2.13 Hz, 1 H). 13C NMR
(100 MHz, DMSO-d6) δ 185.6, 1774.7, 158.8, 141.5, 139.7, 134.1,
134.1, 133.0, 132.2, 129.3, 126.3, 122.3, 122.3, 116.2, 72.2, 60.5, 52.9,
30.1, 28.6, 22.8, 21.9, 16.1. HRMS C28H33N3O3F3S (M + H)+ calcd
5-(3-Chloro-4-isopropoxyphenyl)-3-(2-ethyl-3-(2-
methoxyvinyl)phenyl)-1,2,4-thiadiazole (14b). Compound 14b
(313 mg, 65%) was prepared from 8b (388 mg, 1.16 mmol) and 13
H
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548.2195, found 548.2205. LC/MS: tR = 3.48 min, >95%, m/z 548.3
(M + H)+.
8.53 (d, J = 1.76 Hz, 1 H), 10.06 (br s, 1 H), 13.14 (br s, 1 H). 13C
NMR (100 MHz, DMSO-d6) δ 184.9, 174.0, 158.3, 157.9, 148.9,
141.5, 135.6, 132.7, 131.7, 131.6, 131.5, 129.9, 128.5, 126.1, 117.2,
113.1, 55.3, 54.9, 42.7, 32.3, 29.7, 26.9, 22.3, 21.9, 15.6. HRMS
C27H31N4O2S (M + H)+ calcd 475.2168, found 475.2176. LC/MS: tR
= 4.10 min, >95%, m/z 475.1 (M + H)+.
1-(2-Ethyl-3-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1,2,4-thiadiazol-3-yl)phenethyl)azetidine-3-carboxylic Acid
(17d). Compound 17d (7 mg, 13%) was prepared from 15a (45
mg, 0.10 mmol) and azetidinecarboxylate (24 mg, 0.21 mmol) in the
same manner as described for 12c, giving a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 1.12 (t, J = 7.28 Hz, 3 H), 1.34 (d, J = 6.02
Hz, 6 H), 2.58−2.64 (m, 2 H), 2.64−2.71 (m, 2 H), 2.89 (q, J = 7.28
Hz, 2 H), 3.12−3.21 (m, 5 H), 4.95 (dt, J = 12.05, 6.02 Hz, 1 H),
7.24−7.30 (m, 1 H), 7.33−7.38 (m, 1 H), 7.53 (d, J = 8.78 Hz, 1 H),
7.65 (dd, J = 7.53, 1.25 Hz, 1 H), 8.24 (d, J = 2.26 Hz, 1 H), 8.31 (dd,
J = 8.66, 2.13 Hz, 1 H). HRMS C26H29N3O3F3S (M + H)+ calcd
520.1882, found 520.1884. LC/MS: tR = 3.43 min, >95%, m/z 520.2
(M + H)+.
1-(3-(5-(3-Chloro-4-isopropoxyphenyl)-1,2,4-thiadiazol-3-
yl)-2-ethylphenethyl)piperidine-4-carboxylic Acid (17e). Com-
pound 17e (40 mg, 24%) was prepared from 15b (100 mg, 0.25
mmol) and ethyl 4-piperidinecarboxylate (78 mg, 0.50 mmol) in the
same manner as described for 12c, giving a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 1.14 (t, J = 7.28 Hz, 3 H), 1.35 (d, J = 6.02
Hz, 6 H), 1.65−2.08 (m, 2 H), 2.10−2.18 (m, 2 H), 2.87−2.98 (m, 2
H), 2.98−3.17 (m, 4 H), 3.22−3.35 (m, 3 H), 3.70 (d, J = 11.80 Hz, 2
H), 4.86 (dt, J = 12.05, 6.02 Hz, 1 H), 7.29−7.46 (m, 3 H), 7.69−7.78
(m, 1 H), 8.02 (dd, J = 8.53, 2.26 Hz, 1 H), 8.14 (d, J = 2.26 Hz, 1 H),
9.56 (br s, 1 H), 12.66 (br s, 1 H). HRMS C27H33N3O3SCl (M + H)+
calcd 514.1931, found 514.1935. LC/MS: tR = 3.39 min, >95%, m/z
514.2 (M + H)+.
1-(3-(5-(3-Chloro-4-isopropoxyphenyl)-1,2,4-thiadiazol-3-
yl)-2-ethylphenethyl)azetidine-3-carboxylic Acid (17f). Com-
pound 17f (32 mg, 21%) was prepared from 15b (100 mg, 0.25
mmol) and methyl 3-azetidinecarboxylate (57 mg, 0.50 mmol) in the
same manner as described for 12c, giving a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 1.12 (t, J = 7.40 Hz, 3 H), 1.35 (d, J = 6.02
Hz, 6 H), 2.86−2.96 (m, 4 H), 3.38−3.44 (m, 2 H), 3.60−3.66 (m, 1
H), 4.18−4.39 (m, 4 H), 4.86 (dt, J = 12.05, 6.02 Hz, 1 H), 7.31−7.46
(m, 3 H), 7.71−7.76 (m, 1 H), 8.02 (dd, J = 8.66, 2.13 Hz, 1 H), 8.14
(d, J = 2.26 Hz, 1 H), 10.17 (br s, 1 H), 13.21 (br s, 1 H). 13C NMR
(100 MHz, DMSO-d6) δ 185.3, 173.9, 156.0, 141.5, 135.6, 132.9,
131.5, 129.8, 128.7, 128.0, 126.1, 123.1, 122.9, 115.5, 71.6, 55.4, 55.1,
32.4, 27.0, 22.3, 21.6, 15.6. HRMS C25H29N3O3SCl (M + H)+ calcd
486.1618, found 486.1620. LC/MS: tR = 3.32 min, >95%, m/z 486.2
(M + H)+.
1-(3-(5-(3-Cyano-4-isobutylphenyl)-1,2,4-thiadiazol-3-yl)-2-
ethylphenethyl)piperidine-4-carboxylic Acid (17g). Compound
17g (86 mg, 49%) was prepared from 15c (110 mg, 0.28 mmol) and
ethyl 4-piperidinecarboxylate (58 mg, 0.37 mmol) in the same manner
as described for 12c, giving a white solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 0.94 (d, J = 6.53 Hz, 6 H), 1.15 (t, J = 7.28 Hz, 3
H), 1.69−2.03 (m, 3 H), 2.13 (d, J = 13.05 Hz, 2 H), 2.78 (d, J = 7.28
Hz, 2 H), 2.94 (q, J = 7.28 Hz, 2 H), 3.00−3.16 (m, 4 H), 3.25−3.32
(m, 2 H), 3.53 (d, J = 12.55 Hz, 1 H), 3.70 (d, J = 11.80 Hz, 2 H),
7.33−7.40 (m, 1 H), 7.40−7.46 (m, 1 H), 7.70 (d, J = 8.28 Hz, 1 H),
7.73−7.79 (m, 1 H), 8.33 (dd, J = 8.16, 1.88 Hz, 1 H), 8.53 (d, J =
1.76 Hz, 1 H), 9.44 (br s, 1 H), 12.62 (br s, 1 H). 13C NMR (100
MHz, DMSO-d6) δ 185.0, 174.6, 174.1, 149.0, 141.5, 135.7, 132.8,
131.8, 131.5, 130.2, 128.5, 126.2, 117.2, 113.2, 51.1, 42.7, 38.0, 29.8,
26.6, 25.5, 22.4, 22.0, 15.8. HRMS C29H35N4O2S (M + H)+ calcd
503.2481, found 503.2491. LC/MS: tR = 4.15 min, >95%, m/z 503.2
(M + H)+.
5-(3-(2-Ethyl-3-(2-(3-hydroxyazetidin-1-yl)ethyl)phenyl)-
1,2,4-thiadiazol-5-yl)-2-isobutylbenzonitrile (17i). To a solution
of 3-azetidinol (38 mg, 0.51 mmol) and 15c (100 mg, 0.26 mmol) in
DCM (10 mL), which was prestirred at room temperature for 20 min,
was added sodium triacetoxyborohydride (108 mg, 0.51 mmol). The
reaction mixture was stirred at room temperature for 2 h. Afterward,
the reaction was quenched with water. The reaction mixture was
concentrated in vacuo, and the residue was purified by MDAP to give
20 mg (17%) of 17i as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ ppm 0.93 (d, J = 6.53 Hz, 6 H), 1.11 (t, J = 7.40 Hz, 3 H), 1.98 (dt, J
= 13.36, 6.53 Hz, 1 H), 2.57−2.64 (m, 2 H), 2.64−2.79 (m, 6 H), 2.90
(q, J = 7.40 Hz, 2 H), 3.52−3.58 (m, 2 H), 4.11−4.20 (m, 1 H), 5.30
(d, J = 5.52 Hz, 1 H), 7.26 (t, J = 7.65 Hz, 1 H), 7.35 (d, J = 7.03 Hz, 1
H), 7.67 (dd, J = 7.40, 3.89 Hz, 2 H), 8.31 (dd, J = 8.28, 1.51 Hz, 1 H),
8.50 (s, 1 H). HRMS C26H31N4OS (M + H)+ calcd 447.2219, found
447.2217. LC/MS: tR = 3.29 min, >95%, m/z 447.2 (M + H)+.
5-(3-(2-Ethyl-3-(2-(piperidin-1-yl)ethyl)phenyl)-1,2,4-thiadia-
zol-5-yl)-2-isobutylbenzonitrile (17j). Compound 17j (48 mg,
32%) was prepared from 15c (100 mg, 0.26 mmol) and piperidine
(109 mg, 1.28 mmol) in the same manner as described for 17i as a
1
white solid. H NMR (400 MHz, DMSO-d6) δ ppm 0.94 (d, J = 6.53
Hz, 6 H), 1.15 (t, J = 7.40 Hz, 3 H), 1.34−1.48 (m, 1 H), 1.59−1.79
(m, 3 H), 1.88 (d, J = 14.31 Hz, 2 H), 1.99 (dt, J = 13.36, 6.53 Hz, 1
H), 2.78 (d, J = 7.28 Hz, 2 H), 2.90−3.00 (m, 4 H), 3.07−3.16 (m, 2
H), 3.27 (dt, J = 12.05, 4.77 Hz, 2 H), 3.61 (d, J = 11.54 Hz, 2 H),
7.33−7.40 (m, 1 H), 7.40−7.45 (m, 1 H), 7.70 (d, J = 8.03 Hz, 1 H),
7.76 (dd, J = 7.53, 1.25 Hz, 1 H), 8.33 (dd, J = 8.16, 1.88 Hz, 1 H),
8.53 (d, J = 1.76 Hz, 1 H), 9.39 (br s, 1 H). 13C NMR (100 MHz,
DMSO-d6) δ 184.9, 174.1, 158.2, 157.9, 149.0, 141.5, 135.9, 132.8,
129.9, 128.5, 126.2, 118.6, 117.2, 115.6, 113.1, 56.4, 52.1, 42.7, 29.7,
26.5, 22.7, 22.3, 21.9, 21.4, 15.7. HRMS C28H35N4S (M + H)+ calcd
459.2582, found 459.2592. LC/MS: tR = 3.55 min, >95%, m/z 459.3
(M + H)+.
CNS Penetration Study (BBR) in Male Mice after ip
Administration. Six male C57BL/6 mice received an ip injection
of the test compound at 2 mg/kg. The dose was prepared on the day
of administration in a suspension (DMSO/1% methylcellulose = 1:99
as the vehicle) at a concentration of 0.2 mg/mL (dose volume of 10
mL/kg). At 1, 2, and 4 h after ip administration, blood samples (50 μL
per animal) from two of the animals were collected. An amount of 30
μL of blood was then diluted with 90 μL of water. Immediately after
blood collection, whole brains of the two animals were quickly
removed and rinsed with cold H2O and surface vasculature was
ruptured and blotted with dry tissue. Brain tissue was homogenized
with 3× PBS (w/v). Blood and brain samples were analyzed by LC/
MS/MS. The measured concentrations were corrected for dilution
factor, and then the brain-to-blood ratio (BBR) was determined based
on the ratio of brain concentration and blood concentration at each
time point (1, 2, and 4 h):
Cbrain
BBR =
Cblood
Lymphopenia Study in Male Mice after po Administration.
Animals (n = 3/group) were treated once with various doses of the
compounds (and vehicle) via oral gavage. At predetermined time
points (including immediately prior to compound administration), 50
μL of peripheral blood was obtained and the number of lymphocytes
therein was determined by flow cytometry and expressed as relative
percentages of the baseline value at time 0. For repeat dose studies,
animals were dosed daily for a week and lymphocyte counts were
determined either just prior to or 4 h after dosing every day except on
days 5 and 6, which were the weekend.
1-(3-(5-(3-Cyano-4-isobutylphenyl)-1,2,4-thiadiazol-3-yl)-2-
ethylphenethyl)azetidine-3-carboxylic Acid (17h). Compound
17h (24 mg, 14%) was prepared from 15c (110 mg, 0.28 mmol) and
methyl azetidine-3-carboxylate, HCl salt (45 mg, 0.30 mmol) in the
same manner as described for 12c, giving a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 0.94 (d, J = 6.78 Hz, 6 H), 1.13 (t, J = 7.28
Hz, 3 H), 1.99 (dt, J = 13.49, 6.78 Hz, 1 H), 2.78 (d, J = 7.28 Hz, 2 H),
2.87−2.97 (m, 4 H), 3.38−3.44 (m, 2 H), 3.57−3.68 (m, 1 H), 4.18−
4.34 (m, 4 H), 7.33−7.39 (m, 1 H), 7.42−7.47 (m, 1 H), 7.70 (d, J =
8.03 Hz, 1 H), 7.74−7.79 (m, 1 H), 8.33 (dd, J = 8.16, 1.88 Hz, 1 H),
EAE Induction and Treatment. For induction and treatment of
EAE, male C57BL/6 mice (6−8 weeks old) were purchased from the
I
dx.doi.org/10.1021/jm2016107 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Shanghai Laboratory Animal Center and were maintained under
pathogen-free conditions. Mice were immunized subcutaneously with
a synthetic peptide (300 μg) of myelin oligodendrocyte glycoprotein
(MOG) corresponding to residues 35−55 (GL Biochem, Shanghai,
China). Immunization was performed by mixing MOG peptide in
complete Freund adjuvant containing 5 mg/mL heat-killed H37Ra,
strain of Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI).
Pertussis toxin (200 ng, List Biological Laboratories, Campbell, CA) in
PBS was administered iv on the day of immunization and 48 h later.
For treatment of EAE, compounds or vehicle (DMSO in PBS) was
administered at 1 mg/kg po daily from day 10 after immunization
onward. Mice were examined daily and scored for disease severity
using the following standard scale: 0, no clinical signs; 1, limp tail; 2,
paraparesis (weakness, incomplete paralysis of one or two hind limbs);
3, paraplegia (complete paralysis of two hind limbs); 4, paraplegia with
forelimb weakness or paralysis; 5, moribund or death. The animal use
protocol was approved by Institutional Animal Care and Use
Committee of GSK R&D China.
(5) Brinkmann, V.; Cyster, J. G.; Hla, T. FTY720: Sphingosine 1-
Phosphate Receptor-1 in the Control of Lymphocyte Egress and
Endothelial Barrier Function. Am. J. Transplant. 2004, 4, 1019−1025.
(6) Miron, V. E.; Ludwin, S. K.; Darlington, P. J.; Jarjour, A. A.;
Soliven, B.; Kennedy, T. E.; Antel, J. P. Fingolimod (FTY720)
Enhances Remyelination following Demyelination of Organotypic
Cerebellar Slices. Am. J. Pathol. 2010, 176, 2682−2694.
(7) Choi, J. W.; Herr, D.; Kennedy, G.; Chun, J. Astrocytic
Sphingosine 1-Phosphate (S1P) Receptor Subtype 1 Signalling
Influences Levels of S1P and Cytokines during Experimental
Autoimmune Encephalomyelitis and Fingolimod (FTY720) Inter-
vention. Mult. Scler. 2009, 15, S58.
(8) (a) Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie,
J.; Milligan, J.; Thornton, R.; Shei, G.-J.; Card, D.; Keohane, C.;
Rosenbach, M.; Hale, J.; Lynch, C. L.; Rupprecht, K.; Parsons, W.;
Rosen, H. Alteration of Lymphocyte Trafficking by Sphingosine-1
Phosphate Receptor Agonists. Science 2002, 296, 346−349.
(b) 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. The Immune Modulator FTY720
Targets Sphingosine-1-Phosphate Receptors. J. Biol. Chem. 2002, 277,
21453−21457. (c) Verzijl, D.; Peters, S. L. M.; Alewijnse, A. E.
Sphingosine-1-Phosphate Receptors: Zooming in on Ligand-Induced
Intracellular Trafficking and Its Functional Implications. Mol. Cell
2010, 29, 99−104.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis procedures and spectral data for compounds 3, 4a−u,
and 5a−l. This material is available free of charge via the
Internet at http://pubs.acs.org.
(9) (a) Matloubian, M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.; Xu,
Y.; Brinkmann, V.; Allende, M. L.; Proia, R. L.; Cyster, J. G.
Lymphocyte Egress from Thymus and Peripheral Lymphoid Organs Is
Dependent on S1P Receptor 1. Nature 2004, 427, 355−360.
(b) Allende, M. L.; Dreier, J. L.; Mandala, S.; Proia, R. L. Expression
of the Sphingosine-1-Phosphate, S1P1, on T-Cells Controls Thymic
Emigration. J. Biol. Chem. 2004, 279, 15396−15401.
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-021-61590718. Fax: +86-021-61590730. E-mail:
Xichen.2.Lin@gsk.com.
■
Notes
The authors declare no competing financial interest.
(10) Mizugishi, K.; Yamashita, T.; Olivera, A.; Miller, G. F.; Spiegel,
S.; Proia, R. L. Essential Role for Sphingosine Kinases in Neural and
Vascular Development. Mol. Cell. Biol. 2005, 25, 11113−11121.
(11) (a) Kovarik, J. M.; Lu, M.; Riviere, G.; Barbet, I.; Maton, S.;
Goldwater, D. R.; Schmouder, R. L. The Effect on Heart Rate of
Combining Single-Dose Fingolimod with Steady-State Atenolol or
Diltiazem in Healthy Subjects. Eur. J. Clin. Pharmacol. 2008, 64, 457−
463. (b) Schmouder, R.; Serra, D.; Wang, Y.; Kovarik, J. M.; Dimarco,
J.; Hunt, T. L.; Bastien, M.-C. FTY720: Placebo-Controlled Study of
the Effect on Cardiac Rate and Rhythm in Healthy Subjects. J. Clin.
Pharmacol. 2006, 46, 895−904. (c) Forrest, M.; Sun, S.-Y.; Hajdu, R.;
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.; Tiam, M.; West, S.; White, V.; Xie, J.; Proia, R. L.;
Mandala, S. Immune Cell Regulation and Cardiovascular Effects of
Sphingosine-1-Phosphate Receptor Agonists in Rodents Are Mediated
via Distinct Receptor Subtypes. J. Pharmacol. Exp. Ther. 2004, 309,
758−768. (d) Sanna, M. G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.;
Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H.
Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3,
Respectively, Regulate Lymphocyte Recirculation and Heart Rate. J.
Biol. Chem. 2004, 279, 13839−13848.
(12) Shimizu, H.; Takahashi, M.; Kaneko, T.; Murakami, T.;
Hakamata, Y.; Kudou, S.; Kishi, T.; Fukuchi, K.; Iwanami, S.;
Kuriyama, K.; Yasue, T.; Enosawa, S.; Matsumoto, K.; Takeyoshi, I.;
Morishita, Y.; Kobayashi, E. KRP-203, a Novel Synthetic Immuno-
suppressant, Prolongs Graft Survival and Attenuates Chronic Rejection
in Rat Skin and Heart Allografts. Circulation 2005, 111, 222−229.
(13) Li, W.; Luo, S.; Smith, H. T.; Tse, F. L. S. Quantitative
Determination of BAF312, a S1P-R Modulator, in Human Urine by
LC−MS/MS: Prevention and Recovery of Lost Analyte Due to
Container Surface Adsorption. J. Chromatogr. B 2010, 878, 583−589
and references therein.
ACKNOWLEDGMENTS
We thank Drs. Eric Y. Yang, Hong Lu, and Zongping Zhang for
their helpful comments and discussions.
■
ABBREVIATIONS USED
■
AI, aromatic index; AUC, area under curve; BBR, brain to
blood ratio; CNS, central nervous system; CYP, cytochrome
P450; DMSO, dimethylsulfoxide; EAE, experimental auto-
immune encephalomyelitis; F, oral bioavailability; FRET,
fluorescence resonance energy transfer; Fu, free unbound
fraction; GSH, glutathione; HP-β-CD, 2-hydroxypropyl-β-
cyclodextrin; ip, intraperitoneal; MC, methylcellulose; MS,
multiple sclerosis; PD, pharmacodynamic; PdCl2(dppf), 1,1′-
bis(diphenylphosphino)ferrocenepalladium(II) dichloride; Pd-
(PPh3)4, tetrakis(triphenylphosphine)palladium(0); PK, phar-
macokinetic; po, oral; RRMS, relapsing-remitting multiple
sclerosis; S1P1, sphingosine 1-phosphate receptor 1; S1P3,
sphingosine 1-phosphate receptor 3; S1P4, sphingosine 1-
phosphate receptor 4; S1P5, sphingosine 1-phosphate receptor
5; SAR, structure−activity relationship; T1/2, half-life
REFERENCES
■
(1) Compston, A.; Coles, A. Multiple Sclerosis. Lancet 2002, 359,
1221−1231.
(2) Sorbera, L. A.; Dulsat, C.; Rosa, E. Therapeutic Targets for
Multiple Sclerosis. Drugs Future 2009, 34, 405−417.
(3) Brinkmann, V.; Billich, A.; Baumruker, T.; Heining, P.;
Schmouder, R.; Francis, G.; Aradhye, S.; Burtin, P. Fingolimod
(FTY720): Discovery and Development of an Oral Drug to Treat
Multiple Sclerosis. Nat. Rev. 2010, 9, 883−897.
(14) Bolli, M. H.; Abele, S.; Binkert, C.; Bravo, R.; Buchmann, S.;
Bur, D.; Gatfield, J.; Hess, P.; Kohl, C.; Mangold, C.; Mathys, B.;
Menuhart, K.; Muller, C.; Nayler, O.; Scherz, M.; Schmidt, G.; Sippel,
V.; Steiner, B.; Strasser, D.; Treiber, A.; Weller, T. 2-Imino-thiazolidin-
(4) Lublin, F. D.; Baier, M.; Cutter, G. Effect of Relapses on
Development of Residual Deficit in Multiple Sclerosis. Neurology 2003,
61, 1528−1532.
J
dx.doi.org/10.1021/jm2016107 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4-one Derivatives as Potent, Orally Active S1P1 Receptor Agonists. J.
Med. Chem. 2010, 53, 4198−4211.
(15) Buzard, D.; Thatte, J.; Lerner, M.; Edwards, J.; Jones, R. M.
Recent Progress in the Development of Selective S1P1 Receptor
Agonists for the Treatment of Inflammatory and Autoimmune
Disorders. Expert Opin. Ther. Pat. 2008, 18, 1141−1159 and
references therein.
Qin, X.; Lu, L.; Li, R.; Pan, H.; Song, M.; Liu, A.; Hong, J.; Lu, H.;
Zhang, J. Z. Crucial Role of Interleukin-7 in T Helper Type 17 Survival
and Expansion in Autoimmune Disease. Nat. Med. 2010, 16, 191−197.
(26) Lin, X.; Ren, F.; Zhang, H. Pyrimidine Derivatives for Use as
Sphingosine 1-Phosphate
WO2011113309(A1), 2011.
1 (S1P1) Receptor Agonists.
(16) (a) Yan, L.; Budhu, R.; Huo, P.; Lynch, C. L.; Hale, J. J.; Mills, S.
G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei,
G.-J.; Chrebet, G.; Bergstrom, J.; Card, D.; Mandala, S. M. 2-
Aryl(pyrrolidin-4-yl)acetic Acids Are Potent Agonists of Sphingosine-
1-Phosphate (S1P) Receptors. Bioorg. Med. Chem. Lett. 2006, 16,
3564−3568. (b) Yan, L.; Huo, P.; Doherty, G.; Toth, L.; Hale, J. J.;
Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J.
A.; Shei, G.-J.; Chrebet, G.; Bergstrom, J.; Card, D.; Quackenbush, E.;
Wickham, A.; Mandala, S. M. Discovery of 3-Arylpropionic Acids as
Potent Agonists of Sphingosine-1-Phosphate Receptor-1 (S1P1) with
High Selectivity against All Other Known S1P Receptor Subtypes.
Bioorg. Med. Chem. Lett. 2006, 16, 3679−3683. (c) Vachal, P.; Toth, L.
M.; Hale, J. J.; Yan, L.; Mills, S. G.; Chrebet, G. L.; Keohane, C. A.;
Hajdu, R.; Milligan, J. A.; Rosenbach, M. J.; Mandala, S. Highly
Selective and Potent Agonists of Sphingosine-1-Phosphate 1 (S1P1)
Receptor. Bioorg. Med. Chem. Lett. 2006, 16, 3684−3687. (d) Yan, L.;
Huo, P.; Hale, J. J.; Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach,
M. J.; Milligan, J. A.; Shei, G.-J.; Chrebet, G.; Bergstrom, J.; Card, D.;
Mandala, S. M. SAR Studies of 3-Arylpropionic Acids as Potent and
Selective Agonists of Sphingosine-1-Phosphate Receptor-1 (S1P1)
with Enhanced Pharmacokinetic Properties. Bioorg. Med. Chem. Lett.
2007, 17, 828−831.
(17) Deng, G.; Lin, X.; Meng, Q.; Ren, F.; Xiang, J.; Xu, H.; Zhao, B.;
Zhang, H. Preparation of 5-Membered Heteroaryl Derivatives as
Sphingosine 1-Phosphate Receptor Agonists. WO2010148649(A1),
2010. Mouse experimental autoimmune encephalomyelitis (EAE) data
are unpublished results from this laboratory.
(18) Unpublished results from this laboratory. See Supporting
Information for experimental procedure for the mouse CNS
penetration study.
(19) (a) Lan, S. J.; Weliky, I.; Schreiber, E. C. Metabolic Studies with
trans-5-Amino-3-[2-5-nitro-2-furyl]vinyl]-1,2,4-[5-14C]oxadiazole
(SQ18,506): 1. Reductive Cleavage of the 1,2,4-Oxadiazole Ring.
Xenobiotica 1973, 3, 97−102. (b) Dalvie, D. K.; Kalgutkar, A. S.;
Khojasteh-Bakht, S. C.; Obach, R. S.; O’Donnell, J. P. Biotransforma-
tion Reactions of Five-Membered Aromatic Heterocyclic Rings. Chem.
Res. Toxicol. 2002, 15, 269−299. (c) Sendzik, M.; Hui, H. C.
Environmentally Friendly and Efficient: Iron-Mediated Reduction of
3-Methyl-5-aryl-1,2,4-oxadiazoles to Benzamidines. Tetrahedron Lett.
2003, 44, 8697−8700.
(20) (a) Bird, C. W. A New Aromaticity Index and Its Application to
Five-membered Ring Heterocycles. Tetrahedron 1985, 41, 1409−1414.
(b) Bird, C. W. The Application of a New Aromaticity Index to Six-
Membered Ring Heterocycles. Tetrahedron 1986, 42, 89−92.
(21) S1P1 FRET assay was conducted according to the
manufacturer’s protocol (catalog no. K1520, Invitrogen, Carlsbad,
CA).
(22) S1P3 FRET assay was conducted according to the
manufacturer’s protocol (catalog no. K1713, Invitrogen, Carlsbad,
CA).
(23) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and Computational Approaches To Estimate Solubility
and Permeability in Drug Discovery and Development Settings. Adv.
Drug Delivery Rev. 1997, 23, 3−25.
(24) Fessey, R. E.; Austin, R. P.; Barton, P.; Davis, A. M.; Wenlock,
M. C. The Role of Plasma Protein Binding in Drug Discovery. In
Pharmacokinetic Profiling in Drug Research: Biological, Physicochemical,
and Computational Strategies, LogP2004: 3rd Lipophilicity Symposium,
Zurich, Switzerland, Feb 29−Mar 4, 2004; Testa, B., et al.et al., Eds.;
Wiley-VCH: Weinheim, Germany, 2006; pp 119−141.
(25) For EAE protocol, refer to the following: Liu, X.; Leung, L.;
Wang, C.; Tan, Z.; Wang, J.; Guo, T. B.; Fang, L.; Zhao, Y.; Wan, B.;
K
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