Discovery of a brain-penetrant S1P3-sparing direct agonist of the S1P1 and S1P5 receptors efficacious at low oral dose

Article abstract of DOI:10.1021/jm200609t

2-Amino-2-(4-octylphenethyl)propane-1,3-diol 1 (fingolimod, FTY720) has been recently marketed in the United States for the treatment of patients with remitting relapsing multiple sclerosis (RRMS). Its efficacy has been primarily linked to the agonism on

Full text of DOI:10.1021/jm200609t

ARTICLE
pubs.acs.org/jmc
Discovery of a Brain-Penetrant S1P3-Sparing Direct Agonist
of the S1P1 and S1P5 Receptors Efficacious at Low Oral Dose
Emmanuel H. Demont,*,† Sandra Arpino,‡ Rino A. Bit,† Colin A. Campbell,† Nigel Deeks,† Sapna Desai,‡
Simon J. Dowell,‡ Pam Gaskin,† James R. J. Gray,† Lee A. Harrison,† Andrea Haynes,† Tom D. Heightman,§
Duncan S. Holmes,† Philip G. Humphreys,† Umesh Kumar,† Mary A. Morse,† Greg J. Osborne,‡
Terry Panchal,† Karen L. Philpott,§ Simon Taylor,† Robert Watson,† Robert Willis,† and Jason Witherington†
†Immuno Inflammation Center of Excellence for Drug Discovery, ‡Platform Technology and Science, GlaxoSmithKline,
Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom
§Neurology Center of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow,
Essex CM19 5AW, United Kingdom
S Supporting Information
b
ABSTRACT: 2-Amino-2-(4-octylphenethyl)propane-1,3-diol 1 (fingolimod, FTY720)
has been recently marketed in the United States for the treatment of patients with
remitting relapsing multiple sclerosis (RRMS). Its efficacy has been primarily linked to
the agonism on T cells of S1P1, one of the five sphingosine 1-phosphate (S1P) G-protein-
coupled receptors, while its cardiovascular side effects have been associated with activity
at S1P3. Emerging data suggest that the ability of this molecule to cross the blood-brain
barrier and to interact with both S1P1 and S1P5 in the central nervous system (CNS) may
contribute to its efficacy in treating patients with RRMS. We have recently disclosed the
structure of an advanced, first generation S1P3-sparing S1P1 agonist, a zwitterion with
limited CNS exposure. In this Article, we highlight our strategy toward the identification
of CNS-penetrant S1P3-sparing S1P1 and S1P5 agonists resulting in the discovery of
5-(3-{2-[2-hydroxy-1-(hydroxymethyl)ethyl]-5-methyl-1,2,3,4-tetrahydro-6-isoquinolinyl}-1,2,4-oxadiazol-5-yl)-2-[(1-methy-
lethyl)oxy]benzonitrile 15. Its exceptional in vivo potency and good pharmacokinetic properties translate into a very low predicted
therapeutic dose in human (<1 mg p.o. once daily).
’ INTRODUCTION
Understanding of the unique mode of action of 1 has triggered
intensive effort toward the discovery of S1P1 agonists with increased
2-Amino-2-(4-octylphenethyl)propane-1,3-diol 1 (Fingolimod,
FTY720, Figure 1)1 has been recently marketed in the United
States for the treatment of patients with remitting relapsing
multiple sclerosis (RRMS). Administration of 1 leads to the
sequestration of lymphocytes in secondary lymphoid organs and
consequently to a reduction of lymphocyte count in the periph-
eral blood. 1 is phosphorylated in vivo by sphingosine kinase-22,3
to form FTY720-P 2, a potent agonist of four of the five G-protein-
coupled receptors (S1P1, S1P3ꢀ5) associated with the lysolipid
sphingosine 1-phosphate (S1P) 3. Agonism of the S1P1 receptor
by S1P is required to induce egress of T cells from lymphoid
organs and 2 acts as a functional antagonist by internalizing the
receptor.4,5 The cardiovascular side effects observed in treated
patients (bradycardia and hypertension) have been linked to
partial agonism of the S1P3 receptor,6,7 although more recent
findings from human studies indicate that S1P1 may mediate the
transient effects on heart rate.8 Owing to its lipophilic nature, 1 is
able to cross the blood-brain barrier (BBB)9 where 2 interacts
with S1P receptors present on astrocytes (S1P1) and on oligo-
dendrocytes (S1P5). Recent publications suggest this may play a
role in fingolimod’s efficacy in the treatment of patients with
RRMS.10,11
selectivity versus S1P3,12 either as pro-drugs such as CS-077713
4
or direct agonists such as ACT-12880014 5 (Figure 2).
Excellent (>1000 fold) selectivity over S1P3 can be achieved
with agonists such as AMG 36915 6 or PF-99116 7, but these
molecules, as our own S1P3-sparing agonist 817 (Table 1), are
zwitterions and are therefore likely to have poor CNS penetra-
tion. 18 Typically, in our hands, 8 proved to be a P-gp substrate
(with an efflux ratio in a human MDR1 transfected MDCK type 2
cell line of 0.5 and 6.0 in the presence and absence of a P-gp
inhibitor, respectively). Interestingly, 8 shows no activity at S1P2
and S1P4, and is a partial agonist of the S1P5 receptor with similar
potency to that at S1P1.19
’ RESULTS AND DISCUSSION
Agonists such as 8 are interesting because molecules with an
identical triaryl core are in clinical trials20,21 (not as S1P1 agonists),
suggesting good developability properties. We decided to use this
template in order to identify agonists with increased brain
Received: May 13, 2011
Published: August 15, 2011
r
2011 American Chemical Society
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penetration versus 8. Due to the plethora of amines known to be
able to cross the BBB, and thanks to the intrinsic activity of
compounds such as 9 (Table 2) at S1P1, we decided to use 9 as
starting point, knowing that the removal of the acid functionality
should enhance CNS penetration.18 The profile of 9 suggested
the main liabilities associated with analogous molecules would be
related to (1) hERG inhibition, (2) risk of phospholipidosis22
with such cationic amphiphilic structure, and (3) long pharma-
cokinetic (PK) half-life of compounds with moderate clearance
and high volume of distribution. This could lead to accumulation
on chronic dosing and long pharmaco-dynamic (PD) half-life.
As previously highlighted,17 our strategy focused on drug-
ability, seeking CNS penetrant agonists in a chemical space
recently identified as minimizing the risk of toxicological findings
in the clinic (cLogP < 3, polar surface area (PSA) > 75).23 We
considered that lowering the pKa of our compounds compared to
9, while also lowering their lipophilicity (assessed by cLogP) with
the addition of hydrophilic polar substituents, was likely to
discharge the phospholipidosis and hERG-related cardiovascular
risks highlighted above, even if it may have an impact on CNS
penetration.18,24 Lowering pKa and cLogP should reduce the
volume of distribution (Vss) of our agonists; for a given in vivo
clearance, this would lead to a shorter PK half-life and lower the
risk of accumulation on chronic dosing, therefore offering better
reversibility of the lymphopenia in patients, in sharp contrast to 1.
We embarked on the generation of an array of S1P1 agonists
using a range of bicyclic amines (benzazepines, benzoxazepines,
THIQ, and aza-THIQ) of different pKa bearing hydrophilic
substituents such as amides or polyhydroxylated side chains.
The chemistry used is highlighted in Scheme 1: Protected
bicyclic amines bearing a nitrile substituent were reacted with
hydroxylamine, and the corresponding hydroxyamidines were
coupled to acid chloride 11 (for which the substitution pattern
was known to be the best compromise between MW, cLogP
and S1P1 activity from previous SAR)17 to form the central
Table 1. Activity of 2 and 8 at S1P1-5 Receptors
pEC50 (maximum activation %)
human receptor (assay)a
2b
8
S1P1 (β-arrestin)
S1P2 (yeast)
7.7 (99), n = 44
<4.5, n = 5
8.25 (94), n = 13
<4.48 (01), n = 6
<4.5 (35), n = 6
<4.38 (03), n = 4
6.79 (77), n = 6
S1P3 (GTPγS)
S1P4 (aequorin)
S1P5 (aequorin)
8.3 (62), n = 38
6.7 (48), n = 2
7.2 (62), n = 2
a See the Supporting Information for details. b For comparative pub-
lished values, see ref 35.
Table 2. Profile of Lead Amine 9
MW, cLogP, PSA
375, 3.6, 97
CHI LogD @ pH 2.0, 7.4, 10.5
CAD-likeness
1.34, 2.65, 3.73
91
measured pKa
9.69
7.7
S1P1 pEC50 (β-arrestin)
S1P3 pEC50 (GTPγS)
hERG pIC50 (Dofetalide)
fraction unbound (rat, %)
<4.5
5.5
0.25
rat PK
CLb (mL/min/kg)a
Vss (L/kg)a
t1/2 (h)a
20
9.7
6.3
70
F p.o. (%)b
Figure 1. Structure of FTY720 1, its phosphate 2, and S1P 3.
a 1 mg/kg i.v. b 3 mg/kg p.o.
Figure 2. Structure of known S1P1 agonists.
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Scheme 1. Synthesis of Nonacidic Biaryl Oxadiazole S1P1 Agonistsa
a Reagents and conditions: (a) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C; (b) aqueous NH2OH, EtOH, 80 °C; (c) 11, pyridine, toluene, 0 to 110 °C; (d)
HCl, dioxane, room temperature; (e) R3R4CO, CH2Cl2, room temperature then NaBH(OAc)3; (f) HCl, THF, room temperature; (g) Br-
(CH2)nCOOR5, K2CO3, CH3CN, 50 °C; (h) NaOH, EtOH, room temperature; (i) HATU, N-ethyl-N-isopropylpropan-2-amine (DIPEA), DMF/
NMP, HNR6R7, room temperature.
oxadiazole. After deprotection, well precedented chemistry (alkyla-
tion, saponification, amide coupling, or reductive amination)
allowed the introduction of the appropriate side chain (see
representative examples in Scheme 1).
The nature of the side chain had little impact on the S1P1
activity, and most of the compounds made had a pEC50 similar to
or better than that of 9 in our primary assay with good (>100
fold) or excellent (>1000 fold) selectivity against S1P3. The
diversity of the analogues made helped us to identify the drivers
of the three parameters we wanted to modify: (1) As shown in
Figure 3, lipophilicity (measured by chromatographic hydro-
phobicity index LogP (CHI LogP))25 had an impact on the
cationic amphiphilic drug (CAD)-likeness of our molecules, a
parameter linked to phospholipidosis,26 and lowering the lipo-
philicity tended to lower the CAD-likeness of our molecules.
However, the correlation was poor for molecules having a CHI
LogP in the area we were targeting (CHI LogP < 3).
The key parameter influencing CAD-likeness appeared to be
the basicity of the amines screened (Figure 4): the lower the pKa,
the lower the CAD-likeness, and amines with pKa < 7.5 were
therefore targeted.
(2) The basicity of the amine also had an impact on the ability
to distribute into tissue (Figure 5), and amines with a pKa < 7.5
had the highest probability of having a Vss in rats considered
unlikely to lead to accumulation on chronic dosing. Lipophilicity
(cLogP, CHI LogD) or PSA had lower impact on the tissue
Figure 3. Correlation between CHI LogP and CAD-likeness for a set of
benzazepine (blue), THIQ (green), AZA-THIQ (red), and benzoxaze-
pine (yellow) for CHI LogP < 5 and CAD-likeness <120.
distribution (see graphics in Experimental Section).
(3) Tuning the pKa and/or the lipophilicity had only a small,
nonpredictable effect on hERG inhibition (Figure 6), and most
compounds showed IC50 in a 1ꢀ20 μM range in a PatchXpress
assay; the good in vivo activity of these molecules (vide infra) and
their high protein binding (>95% in all cases; see 9, Table 2 as
example) led us to believe a significant safety margin might be
achieved.
Table 3 highlights the in vitro and in vivo potency and
selectivity as well as in vivo pharmacokinetics of key representa-
tives. Compounds with amide side chains such as 10 were
efficacious in our in vivo lymphopenia assay at an oral dose of
1 mg/kg, but further analysis showed that they were readily
hydrolyzed to the corresponding acid in vivo. Not only is this
metabolite responsible for part of the PD effect observed, but it
generated the same potential toxicity risk associated with our first
generation agonist 8 (formation of acyl-glucoronide).27 The
formation of the acid could be avoided with hindered amides,
but this led to molecules outside of the targeted chemical space
(cLogP < 3) and no further work was done in this area.
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Figure 4. Correlation between CAD-likeness and measured pKa.
Figure 6. Correlation between hERG PatchXpress pIC50 andmeasuredpKa.
Vss, and CAD-likeness). Introduction of a methyl substituent
ortho to the oxadiazole in this series gave improved S1P3
selectivity and lower in vivo clearance in rats (cf. 15 vs 14). Attempts
to further lower the pKa and lipophilicity by introduction of nitrogen
into the aromatic ring (13) led to compounds with lower Vss, but
all were less efficacious in vivo.
The synthesis of compound 15 is highlighted in Scheme 2:
enone 17 was obtained from commercial protected piperidone
16 via a Robinson annulation then was oxidized to phenol 18
using a Saegusa reaction. Palladium mediated cyanation via
triflate 19 gave 20 which was then reacted with hydroxylamine.
The hydroxyamidine 21 was then coupled to acid chloride 22
(obtained from the corresponding commercially available acid),
and oxadiazole 23 was obtained after dehydration of the
uncyclized intermediate. Deprotection in acidic conditions (24)
followed by reductive amination (25) and deprotection of the
acetal provided 15.
Similar chemistry was used to access compounds 9ꢀ14, using
the corresponding (hetero)aryl nitriles 28, 30, 33, and 38. Their
synthesis is highlighted in Scheme 3. Nitrile 28 and 30 could be
easily accessed via palladium mediated cyanation from commer-
cial bromide 26 and known28 phenol 29, respectively. The
synthesis of the benzoxazepine template started with known29
aldehyde 31, which after reductive amination using ethanol-
amine and protection of the secondary amine gave 32. The ring
was formed using a Mitsunobu reaction to give 33 after
cyanation. The aza-THIQ template was obtained following
amination of the beta-keto ester 35 (obtained from commercial
benzyl derivative 34) followed by imine formation and ring
closure in basic conditions which gave the aryl bromide 37.
The latter was converted to the desired intermediate 38 via a
Suzuki coupling.
Figure 5. Correlation between volume of distribution in rats and
measured pKa.
Derivatives with hydroxylated side chains, diols in particular,
proved interesting (see profiles of 11ꢀ15). Benzazepines (11)
had low in vivo clearance but were too basic, leading to a high Vss.
The less basic benzoxazepines had lower Vss but despite good
in vivo PK were less potent in our lymphopenia model (cf. 12 vs 11).
The analogous THIQ (15) had overall the best profile: It proved
more efficacious in vivo (at doses as low as 0.3 mg/kg p.o., vide
infra) and had appropriate intrinsic properties (CHI LogD, pKa,
Full lymphopenia could be achieved with 15 at doses as low as
0.3 mg/kg p.o. with reversibility within 24 h (Figure 7). PK/PD
modeling using an indirect response model estimated an in vivo
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Table 3. In Vitro and in Vivo Profile of Selected Agonists
compd
10
11
12
13
14
15
MW, cLogP, PSA
490, 2.7, 125
449, 3.3, 116
451, 2.7, 125
450, 2.2, 129
434, 2.5, 116
449, 3.0, 116
CHI LogD @ pH 7.4
3.04
2.74
2.41
2.00
2.49
2.64
CAD-likeness
50
64
45
44
60
55
measured pKa
6.07
7.81
6.03
6.22
7.28
7.30
S1P1 pEC50 (β-arrestin)
8.9 (2)
4.6 (1)
90 ( 3
<5 (43)
7.7 (8)
4.8 (3)
88 ( 3
4.75 (59)
7.7 (2)
<4.5 (2)
60 ( 13
5.17 (77)
7.7 (8)
<4 (10)
70 ( 3
4.9b
8.1 (6)
5.2 (4)
49 ( 12
4.7b
8.5 (54)
<4.5ꢀ5.5 (47)a
96 ( 4
S1P3 pEC50 (GTPγS)
maximum lymphopoenia ((SD) @ 1 mg/kg p.o.
hERG pIC50 (max inh % @ 30 μM, PatchXpress)
4.89 (58)
rat PK
(n = 1ꢀ3)
CLb (mL/min/kg)c
32
21 ( 3
14
21
87
60 ( 6
Vss (L/kg)c
t1/2 (h)c
4.4
7.6 ( 0.4
5.3 ( 0.3
68 ( 18c
3.3
3.5
3.3
3.7
0.6
4.1 ( 0.5
1.0 ( 0.1
52 ( 4d
2.0
38 ( 17e
2.1
37 ( 18e
F, p.o. (%)
ratio AUC acid:amide
2.3:1
a In 33 occasions, pEC50 < 4.5; in 14 occasions, 4.5 < pEC50 < 5.5; in all cases, maximum response <40% at 30 μM. b Dofetalide. c 1 mg/kg i.v. DMSO/10%
(w/v) kleptose HPB 0.9% saline (aqueous) (5%:95% v/v). d 3 mg/kg p.o. 1% (w/v) methylcellulose 400 (aqueous). e 1 mg/kg p.o. 1% methylcellulose.
IC50 of less than 0.1 nM. There were no active metabolites or
phosphorylated adducts observed systemically which could
explain such a level of potency. This increased in vivo potency
compared to our first generation zwitterionic agonists may be
attributed to an increased distribution into tissues: 15 not only
can desensitize T cells to S1P by internalizing S1P1 but it may
also play a role in the tightening of the lymphatic endothelial cell
junctions, a mode of action also hypothesized to explain fingo-
limod’s efficacy.30
from those of marketed CNS drugs (median MW = 305, number
of hydrogen bound donor = 1).32 The higher than expected CNS
penetration of 15 maybe in part due to its excellent permeability
which could be due to formation of an intramolecular hydrogen
bond. This phenomenon has already been observed with other
templates.33 Overall, a review of the literature shows that the “hot
spot” in the chemical space for CNS penetration is not exclusive
and that other molecules with similar profile to 15 can be CNS
penetrant.34
Due to these promising data, further profiling of 15 was imple-
mented. This compound has excellent intrinsic properties (Table 5),
solubility, and permeability. No significant CYP inhibition was
observed, and no covalent adducts were detected in glutathione
trapping experiments (nor time dependent inhibition of CYP
3A4). In vitro hepatocyte clearance correlated with in vivo
clearance in rat and dog. Oral bioavailability was good in both
species. As for 8, 15 proved to be a partial agonist at S1P5, with
similar EC50 as for S1P1. The permeability of 15 was assessed in
MDCKII-MDR1 cells. The basolateral-apical (BꢀA) to AꢀB
ratio was unaffected by the presence or absence of a P-gp
inhibitor, indicating that the transport of this agonist was not
affected by efflux transporters, in contrast to zwitterion 8. A CNS
penetration study in rats demonstrated that 15 was able to enter
the CNS with a brain to blood ratio concentration at steady state
of 1.85:1. The capacity of 15 to cross the BBB could be
considered as surprising given its PSA (116 Å2) is higher than
what is considered as upper limit for good CNS penetration
(PSA < 70 Å2).31 The intrinsic properties of 15 are also different
’ CONCLUSION
We have identified a druglike CNS-penetrant S1P3-sparing
direct (non-pro-drug) agonist of the S1P1 and S1P5 receptors
showing exceptional in vivo efficacy (IC50 < 0.1 nM). Its good
pharmacokinetic properties suggest very low human therapeutic
doses (<1 mg p.o. once daily).
’ EXPERIMENTAL SECTION
General. All solvents were purchased from Romil Ltd. (Hy-Dry
anhydrous solvents), and commercially available reagents were used as
received. Melting points were recorded on a Buchi B-545 apparatus and
are uncorrected. All reactions were followed by TLC analysis (TLC
plates GF254, Merck) or LCMS (liquid chromatography mass spectro-
metry) using a Waters ZQ instrument. NMR spectra were recorded on a
1
Bruker AVANCE 400 spectrometer and are referenced as follows: H
(400 MHz), internal standard TMS at δ = 0.00; 13C (100.6 MHz),
internal standard CDCl3 at δ = 77.23 or DMSO-d6 at δ = 39.70. Column
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Scheme 2. Synthesis of Compound 15a
a Reagents and conditions: (a) (i) Pyrrolidine, toluene, DeanꢀStark, reflux; (ii) 1-penten-3-one, hydroquinone, toluene, reflux. (b) (i) LiHMDS, THF,
ꢀ63 °C then TMSCl; (ii) CH3CN, Pd(OAc)2, T < 35 °C then TBAF, room temperature; (c) pyridine, (CF3SO2)2O, CH2Cl2, ꢀ35 °C. (d) Zn(CN)2,
Pd(PPh3)4, DMF, 100 °C. (e) NH2OH.HCl, K2CO3, ethanol, reflux; (f) 22, toluene/pyridine, room temperature to 110 °C; (g) HCl, 1,4-dioxane, room
temperature; (h) 2,2-dimethyl-1,3-dioxan-5-one, CH2Cl2, room temperature then NaHB(OAc)3; (i) HCl, THF/water, room temperature.
Scheme 3. Synthesis of Key Benzonitrile Intermediatesa
a Reagents and conditions: (a) Zn(CN)2, Pd(PPh3)4, DMF, microwave irradiations, 130 °C; (b) (Boc)2O, NEt3, CH2Cl2, room temperature; (c)
pyridine, (CF3SO2)2O, CH2Cl2, ꢀ35 °C; (d) Zn(CN)2, Pd(PPh3)4, DMF, 90 °C; (e) ethanolamine, THF, 0 °C then NaHB(OAc)3, 0 °C to room
temperature; (f) PPh3, DIAD, THF, 0 °C; (g) H2 (1 atm), Pd/C, ethanol, (Boc)2O, room temperature; (h) NH4OAc, ethanol, room temperature to 50 °C;
(i) 3,3-dimethoxypropionitrile, t-BuOK, THF, 0 to 70 °C then PBr3, DMF/CH2Cl2, 0 °C; (j) CH3BF3K, PdCl2(dppf), Cs2CO3, THF/water, 65 °C.
chromatography was performed on prepacked silica gel columns
(30ꢀ90 mesh, IST) using a biotage SP4. Mass spectra were recorded
on Waters ZQ (ESI-MS) and Q-Tof 2 (HRMS) spectrometers. Mass
Directed Auto Prep was performed on a Waters 2767 instrument with a
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Figure 7. Lymphocyte reduction over time in rats following oral administration of 15.
Table 5. In Vitro Profile of 15
MW, PSA, cLogP
449, 116, 3.0
CHI LogD @ pH 2.0, 7.4 and 10.5
0.98; 2.64; 2.99
7.7 ( 0.17 (65ꢀ67%)
>1000
S1P5 pEC50 (max response %; n = 5)
solubility (FeSSIF, ng/mL)
permeability (MDCK Type 2, nm/s)
hepatocyte CLi (mL/min/g; rat, dog, mouse, cyno, human)
CYP IC50 (μM, 2C9, 2C19, 2D6, 3A4VG, 3A4 VR, n = 2)
steady state rat [brain]:[blood]a,b (n = 3)
175
3; 30; 1.3; 1.3; 2
4.3; >50; >50; 8.4; 15.3
1.87:1 ( 0.02:1
dog PK (1 mg/kg i.v.b or p.o.c; n = 1)
CLb (mL/min/kg)b
Vss (L/kg)b
t1/2 (i.v., h)b
F, p.o. %c
29
7.0
3.0
45
a 7 h i.v. dose at 0.8 mg/kg/h. b DMSO/10% (w/v) Kleptose HPB 0.9% saline (aq) (2:98 v/v). c 1% (w/v) methylcellulose 400 (aq).
MicroMass ZQ mass spectrometer using a Supelco LCABZ++
column. GLOBAL gradients for chromatography are as follows (solvent
B polar component, CV = column volume). 10% GLOBAL: 3% B for 2
CV, 3 to 13% B over 10 CV then 13% B for 5 CV; 20% GLOBAL: 5% B
for 2 CV, 5 to 20% B over 10 CV then 20% B for 5 CV; 30% GLOBAL:
8% B for 2 CV, 8 to 38% B over 10 CV then 38% B for 5 CV;
40% GLOBAL: 10% B for 2 CV, 10 to 50% B over 10 CV then 50% B for
5 CV; 50% GLOBAL: 13% B for 2 CV, 13 to 63% B over 10 CV then
63% B for 5 CV. 100% GLOBAL: 25% B for 2 CV, 25 to 100% B over 10
CV then 100% B for 10 CV. Abbreviations for multiplicities observed in
NMR spectra: s; singlet; br s, broad singlet; d, doublet; t, triplet; q,
quadruplet; p, pentuplet; spt, septuplet; m, multiplet. All compounds
reported are of at least 95% purity according to LCMS (conditions
described in the Experimental Section).
1,1-Dimethylethyl 5-Methyl-6-oxo-3,4,6,7,8,8a-hexahydro-
2(1H)-isoquinolinecarboxylate (17). Compound 16 (70 g, 350
mmol, Aldrich) and pyrrolidine (43.6 mL, 530 mmol) were dissolved in
toluene (310 mL), and the resulting mixture was refluxed under Deanꢀ
Stark conditions for 24 h and then concentrated in vacuo. The residue was
dissolved in anhydrous toluene (270 mL) and treated with hydroquinone
(0.40 g) and 1-penten-3-one (29.6 g, 350 mmol). The resulting solution
was refluxed for 24 h and then diluted with AcOEt (300 mL). The mixture
was washed with HCl (0.5 N in water, 500 mL), and the aqueous phase
extracted with AcOEt (300 mL). The combined organic phases were dried
(MgSO4) and concentrated. Purification of the residue by flash chroma-
tography on a silica cartridge (1.5 kg) gave the title compound (55.2 g,
59.2%) as a pale yellow oil which crystallized on standing. LCMS (method
formate): Retention time 1.04 min, [M + H]+ = 266.24. 1H NMR (400
MHz, CDCl3) δppm 4.16ꢀ4.02 (m, 2H), 3.08ꢀ3.01 (m, 1H), 2.77ꢀ2.71
(m, 1H), 2.58ꢀ2.49 (m, 3H), 2.39ꢀ2.26 (m, 2H), 2.06ꢀ2.00 (m, 1H),
1.79 (s, 3H), 1.59ꢀ1.52 (m, 1H), 1.49 (s, 9H).
1,1-Dimethylethyl 6-Hydroxy-5-methyl-3,4-dihydro-2(1H)-
isoquinolinecarboxylate (18). Lithium bis(trimethylsilyl)amide
(1 M in THF, 246 mL, 250 mmol) was added dropwise to a solution
of compound 17 (54.4 g, 210 mmol) in THF (200 mL) at ꢀ63 °C, and
the mixture was stirred for an additional 30 min. Chloro(trimethyl)-
silane (31.4 mL, 250 mmol) was added dropwise, and the resulting
mixture was stirred for 2 h at ꢀ70 °C. The reaction was warmed to room
temperature over 20 min and diluted with Et2O (800 mL). The reaction
was added to a saturated Na2CO3 aqueous solution, and the phases were
separated. The aqueous phase was extracted with Et2O (300 mL) and
the combined organic phases were dried (Na2SO4) and concentrated in
vacuo. The residue was dissolved in acetonitrile (200 mL), and
palladium(II) acetate (46.0 g, 210 mmol) was added. The resulting
mixture was cooled (water bath) to maintain a reaction temperature
below 35 °C and stirred for 16 h. The reaction was filtered through
Celite, and the residue rinsed with AcOEt (3 ꢁ 300 mL). The filtrate was
further filtered through a 100 pad of silica gel and concentrated in vacuo.
The residue was dissolved in AcOEt (500 mL) and then treated with
tetrabutylammonium fluoride (1 M in THF, 200 mL, 200 mmol). The
resulting mixture was allowed to stand for 30 min, then was washed with
HCl (0.5 N in water, 300 mL) and a 10% sodium thiosulfate solution,
dried (MgSO4), and concentrated in vacuo. Purification of the residue
by flash chromatography on silica gel (300 g column) eluting with
0ꢀ60% gradient AcOEt/cyclohexane gave the title compound (29.9 g,
55%) as a white solid. LCMS (method formate): Retention time 1.07
min, [M + H]+ = 264.12. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.03
(s, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 4.36 (s, 2H),
3.52 (t, J = 6.1 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 2.01 (s, 3H), 1.41 (s,
9H). HRMS calculated for C15H22NO3: 264.1600. Found: 264.1605.
1,1-Dimethylethyl 5-Methyl-6-{[(trifluoromethyl)sulfonyl]-
oxy}-3,4-dihydro-2(1H)-isoquinolinecarboxylate (19). To a
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Journal of Medicinal Chemistry
ARTICLE
solution of compound 18 (3.16 g, 12 mmol) in dichloromethane (50 mL)
at room temperature under nitrogen was added pyridine (1.94 mL,
24 mmol), and the resulting solution was cooled to ꢀ30 °C before
trifluoromethanesulfonic anhydride (2.2 mL, 13.2 mmol) was added
dropwise. The resulting mixture was stirred for 40 min at this tempera-
ture, warmed to room temperature, and concentrated in vacuo. The
residue was diluted with AcOEt and washed sequentially with HCl (1 N
in water), a saturated NaHCO3 aqueous solution, and brine. The organic
phase was then dried over MgSO4 and concentrated in vacuo to give the
title compound (4.85 g, 102%) as a red oil which was used in the next
step without further purification. LCMS (method high pH): Retention
16 mmol) in toluene (15 mL). After 15 min, the resulting mixture was
refluxed for 90 min (internal temperature 110 °C) and then was cooled
to room temperature. The solution was decanted from the brown
precipitate, the precipitate washed with toluene, and the combined
organics concentrated in vacuo. The residue was dissolved in AcOEt,
and the resulting solution washed with HCl (2 N in water). The aqueous
phase was extracted with AcOEt, and the combined organic phases were
washed sequentially with a saturated NaHCO3 aqueous solution and
brine, dried over MgSO4, and concentrated in vacuo. Purification of the
residue on SP4 using a 30% GLOBAL gradient (AcOEt in hexanes) gave
the title compound (3.62 g, 51%) as a white foam. LCMS (method high
pH): Retention time 1.55 min, [M + H]+ = 475.31. 1H NMR (400 MHz,
CDCl3) δ ppm 8.42 (d, J = 2.3 Hz, 1H), 8.33 (dd, J = 8.8, 2.3 Hz, 1H),
7.76 (d, J = 8.1 Hz, 1H), 7.16ꢀ7.08 (m, 2H), 4.79 (spt, J = 6.1 Hz, 1H),
4.64 (s, 2H), 3.72 (t, J = 5.8 Hz, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.52 (s,
3H), 1.51 (s, 9H), 1.48 (d, J = 6.1 Hz, 6H).
2-[(1-Methylethyl)oxy]-5-[3-(5-methyl-1,2,3,4-tetrahydro-
6-isoquinolinyl)-1,2,4-oxadiazol-5-yl]benzonitrile hydro-
chloride (24). To a solution of compound 23 (3.4 g, 7.2 mmol) in
1,4-dioxane (20 mL) at room temperature under nitrogen was added
HCl (4 N in 1,4-dioxane, 18 mL, 72 mmol), and the resulting mixture
was stirred at this temperature for 5.5 h, stored in a freezer for 16 h, and
then concentrated in vacuo. The residue was coevaporated with Et2O to
give the title compound (2.88 g, 98%) as a white solid. LCMS (method
high pH): Retention time 1.21 min, [M + H]+ = 375.26. 1H NMR (400
MHz, DMSO-d6) δ ppm 9.54 (br s, 2H), 8.50 (d, J = 2.2 Hz, 1H), 8.39
(dd, J = 9.0, 2.2 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.56 (d, J = 9.0 Hz,
1H), 7.29 (d, J = 8.1 Hz, 1H), 4.98 (spt, J = 6.1 Hz, 1H), 4.35 (s, 2H),
3.45 (t, J = 6.1 Hz, 2H), 3.00 (t, J = 6.1 Hz, 2H), 2.47 (s, 3H), 1.39 (d, J =
6.1 Hz, 6H). HRMS calculated for C22H23N4O2: 375.1821. Found:
375.1817.
5-(3-(2-(2,2-Dimethyl-1,3-dioxan-5-yl)-5-methyl-1,2,3,4-
tetrahydroisoquinolin-6-yl)-1,2,4-oxadiazol-5-yl)-2-isopro-
poxybenzonitrile (25). Sodium triacetoxyborohydride (3.56 g, 16.8
mmol) was added portionwise over 30 min to a stirred supension of
compound 24 (1.5 g, 3.65 mmol) and 2,2-dimethyl-1,3-dioxan-5-one
(1.43 g, 11 mmol) in dry dichloromethane (50 mL). The reaction
mixture was then stirred at room temperature for 4 h. A saturated
aqueous NaHCO3 solution (50 mL) was added carefully (CARE: gas
evolved), and the resulting biphasic mixture stirred vigorously for 30
min. The layers were separated, and the aqueous phase was extracted
with DCM (2 ꢁ 20 mL). The combined organic phases were dried over
MgSO4 and concentrated in vacuo to give the title compound (1.80 g,
3.65 mmol, 100%) as a colorless solid which was used in next step
without further purification. LCMS (method high pH): Retention time
0.94 min, [M + H]+ = 488.9.
1
time 1.46 min, [M ꢀ H]ꢀ = 394.22. H NMR (400 MHz, CDCl3) δ
ppm 7.10 (d, J = 8.1 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 4.58 (s, 2H), 3.68
(t, J = 5.8 Hz, 2H), 2.76 (t, J = 5.8 Hz, 2H), 2.25 (s, 3H), 1.50 (s, 9H).
1,1-Dimethylethyl 6-Cyano-5-methyl-3,4-dihydro-2(1H)-
isoquinolinecarboxylate (20). A solution of compound 19 (26.1
g, 66 mmol) in DMF (200 mL) was degassed for 10 min under vacuum
and then flushed with nitrogen. The solution was treated with tetrakis-
(triphenylphosphine)palladium (7.6 g, 6.6 mmol) and zinc cyanide
(10.1 g, 86 mmol), and the resulting mixture was stirred at 100 °C under
nitrogen for 6 h and then was cooled to room temperature. The mixture
was filtered, the residue washed with AcOEt, and most of the solvent
evaporated in vacuo. The residue was dissolved in AcOEt, and the
organic phase was washed twice with a saturated NaHCO3 aqueous
solution. The combined aqueous phases were extracted twice with
AcOEt, and the combined organic phases were washed with brine, dried
over MgSO4, and concentrated in vacuo. Purification of the residue by
flash chromatography on silica gel eluting with a 0ꢀ50% AcOEt/
cyclohexane gradient gave the title compound (16.6 g, 92%) as a white
solid. LCMS (method high pH): Retention time 1.24 min, [M + H]+ =
273.25. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.57 (d, J = 8.1 Hz, 1H),
7.22 (d, J = 8.1 Hz, 1H), 4.56 (s, 2H), 3.59 (t, J = 6.1 Hz, 2H), 2.72 (t, J =
6.1 Hz, 2H), 2.39 (s, 3H), 1.44 (s, 9H). HRMS calculated for
C16H21N2O2: 273.1603. Found: 273.1608.
1,1-Dimethylethyl 6-[(Hydroxyamino)(imino)methyl]-5-
methyl-3,4-dihydro-2(1H)-isoquinolinecarboxylate (21). A
mixture of compound 20 (16.6 g, 61 mmol), NaHCO3 (30.7 g, 370
mmol), and hydroxylamine hydrochloride (25.4 g, 370 mmol) in
ethanol (250 mL) was refluxed for 28 h and then was allowed to cool
to room temperature. The reaction was filtered, and the residue washed
with ethanol. The combined filtrate and washings were concentrated in
vacuo. The residue was poured into water (100 mL) and stirred at room
temperature for 20 min. The precipitated solid was isolated by filtration
and dried under vacuum at 40 °C for 16 h to give the title compound
(16 g, 86%) as a white solid. LCMS (method high pH): Retention time
1
0.93 min, [M + H]+ = 306.17. H NMR (400 MHz, DMSO-d6) δ
ppm 9.21 (s, 1H), 7.07 (d, J = 8.9 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 5.65
(s, 2H), 4.48 (s, 2H), 3.58 (t, J = 5.9 Hz, 2H), 2.67 (t, J = 5.9 Hz, 2H),
2.20 (s, 3H), 1.43 (m, 9H). HRMS calculated for C16H24N3O3:
306.1818. Found: 306.1817.
3-Cyano-4-[(1-methylethyl)oxy]benzoyl Chloride (22). Ox-
alyl chloride (6.4 mL, 73 mmol) was added to a solution of 3-cyano-
4-[(1-methylethyl)oxy]benzoic acid (10.7 g, 52 mmol, Biopharma Inc.)
in CH2Cl2 (100 mL) followed by the addition of DMF (0.044 mL, 0.57
mmol), and the resulting mixture was stirred at room temperature for
4 h. The reaction mixture was filtered and concentrated in vacuo. The
residue was coevaporated with cyclohexane (2 ꢁ 50 mL) to give the
title compound (11.7 g, 100%) as a pale yellow oil which solidified on
standing.
5-(3-(2-(1,3-Dihydroxypropan-2-yl)-5-methyl-1,2,3,4-tet-
rahydroisoquinolin-6-yl)-1,2,4-oxadiazol-5-yl)-2-isopropox-
ybenzonitrile hydrochloride (15). A solution of compound 25
(1.78 g, 3.65 mmol) in THF (50 mL) was treated with HCl (2 N in
water, 50 mL, 100 mmol), and the resulting mixture was stirred at room
temperature for 16 h. Most of the THF was removed in vacuo. The
residue was neutralized by the portionwise addition of solid NaHCO3
(CARE: gas evolved). During neutralization, a colorless solid precipi-
tated which was filtered off and dissolved in 10% methanol in DCM
(50 mL). The solution was dried over MgSO4 then concentrated in
vacuo. The residue was dissolved in methanol (∼10 mL) and treated
with HCl (1 N in Et2O, 4 mL, 4 mmol). Et2O (100 mL) was then added
to the solution. The precipitated formed was filtered off, washed with
Et2O, and dried under vacuum to give the title compound (1.47 g, 3
mmol, 83%) as a colorless solid. LCMS (method high pH): Retention
time 0.89 min, [M + H]+ = 449.05. 1H NMR (400 MHz, DMSO-d6) δ
ppm 10.58 (br s, 1H), 8.52 (d, J = 2.2 Hz, 1H), 8.42 (dd, J = 9.2, 2.2 Hz,
1H), 7.81 (d, J = 8.1 Hz, 1H), 7.59 (d, J = 9.2 Hz, 1H), 7.31 (d, J = 8.1 Hz,
1,1-Dimethylethyl 6-(5-{3-Cyano-4-[(1-methylethyl)oxy]-
phenyl}-1,2,4-oxadiazol-3-yl)-5-methyl-3,4-dihydro-2(1H)-
isoquinolinecarboxylate (23). To a suspension of compound 21
(4.6 g, 15 mmol) in toluene (30 mL) and pyridine (30 mL) at room
temperature under nitrogen was slowly added compound 22 (3.5 g,
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Journal of Medicinal Chemistry
ARTICLE
1H), 5.54 (br s, 2H), 5.01 (spt, J = 6.1 Hz, 1H), 4.78 (dd, J = 15.8, 8.3 Hz,
1H), 4.62 (d, J = 15.8 Hz, 1H), 4.01ꢀ3.90 (m, 5H), 3.65ꢀ3.55 (m, 1H),
3.47ꢀ3.43 (m, 1H), 3.28ꢀ3.19 (m, 1H), 3.18ꢀ3.09 (m, 1H), 2.50 (s,
3H), 1.42 (d, J = 6.1 Hz, 6H). 13C NMR (DMSO-d6) δ ppm 173.0,
169.0, 162.5, 136.0, 134.5, 133.7, 132.1, 131.9, 128.0, 125.2, 124.6, 115.9,
115.2, 114.9, 102.4, 72.5, 66.6, 56.9, 56.7, 50.7, 47.4, 23.9, 21.4, 16.2.
HRMS calculated for C25H29N4O4: 449.2189. Found: 449.2194.
(6) Forrest, M.; Sun, S.-Y.; Hajdu, R.; Bergstrom, J.; Card, D.;
Doherty, G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.; Mills, S.;
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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.
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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.
(8) Gergely, P.; Wallstr€om, E.; Nuesslein-Hildesheim, B.; Bruns, C.;
Zꢀecri, F.; Cooke, N.; Traebert, M.; Tuntland, T.; Rosenberg, M.;
Saltzman, M. Phase I study with selective S1P1/S1P5 receptor modulator
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modeling of FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-
1,3-diol hydrochloride) in rats after oral and intravenous doses. Drug
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures for
b
the synthesis of compounds 9ꢀ14, in vitro assay protocols for
the determination of EC50, and protocols for in vivo studies and
data correlations. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: + 44 1438 764319. Fax: + 44 1438 768302. E-mail:
emmanuel.h.demont@gsk.com.
(10) Brinkmann, V. FTY720 (fingolimod) in Multiple Sclerosis:
therapeutic effects in the immune and the central nervous system. Br. J.
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in multiple sclerosis. Crit. Rev. Biochem. Mol. Biol. 2011, 46, 2–10.
(12) Buzard, D. J.; 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 disor-
ders. Expert Opin. Ther. Pat. 2008, 18, 1141–1159.
(13) Nishi, T.; Miyazaki, S.; Takemoto, T.; Suzuki, K.; Iio, Y.;
Nakajima, K.; Ohnuki, T.; Kawase, Y.; Nara, F.; Inaba, S.; Izumi, T.;
Yuita, H.; Oshima, K.; Doi, H.; Inoue, R.; Tomisato, W.; Kagari, T.;
Shimozato, T. Discovery of CS-0777: A Potent, Selective, and Orally
Active S1P1 Agonist. ACS Med. Chem. Lett. 2011, 2, 368–372.
(14) Bolli, M. H.; Abele, S.; Binkert, C.; Bravo, R.; Buchmann, S.;
Bur, D.; Gatfield, J.; Hess, P.; Kohl, C.; Mangold, C.; Mathys, B.;
Menyhart, K.; M€uller, C.; Nayler, O.; Scherz, M.; Schmidt, G.; Sippel, V.;
Steiner, B.; Strasser, D.; Treiber, A.; Weller, T. 2-Imino-thiazolidin-4-
one Derivatives as Potent, Orally Active S1P1 Receptor Agonists. J. Med.
Chem. 2010, 53, 4198–4211.
(15) Cee, V. J.; Frohn, M.; Lanman, B. A.; Golden, J.; Muller, K.;
Neira, S.; Pickrell, A.; Arnett, H.; Buys, J.; Gore, A.; Fiorino, M.; Horner,
M.; Itano, A.; Lee, M. R.; McElvain, M.; Middleton, S.; Schrag, M.;
Rivenzon-Segal, D.; Vargas, H. M.; Xu, H.; Xu, Y.; Zang, X.; Siu, J.;
Wong, M.; B€urli, R. W. Discovery of AMG 369, a Thiazolo[5,4-
b]pyridine Agonist of S1P1 and S1P5. ACS Med. Chem. Lett. 2011,
2, 107–112.
(16) Walker, J. K. Discovery of a potent and selective S1P1 receptor
agonist for the treatment of rheumatoid arthritis. ACS meeting, 21ꢀ25
March, 2010, San Francisco, CA.
(17) Demont, E. H.; Andrews, B. I.; Bit, R. A.; Campbell, C. A.;
Cooke, J. W. B.; Deeks, N.; Desai, S.; Dowell, S. J.; Gaskin, P.; Gray,
J. R. J.; Haynes, A.; Holmes, D. S.; Kumar, U.; Morse, M. A.; Osborne,
G. J.; Panchal, T.; Patel, B.; Perboni, A.; Taylor, S.; Watson, R.;
Witherington, J.; Willis, R. Discovery of a selective S1P1 receptor agonist
efficacious at low oral dose and devoid of effects on heart rate. ACS Med
Chem. Lett. 2011, 2, 444–449.
(18) Didziapetris, R.; Japertas, P.; Avdeef, A.; Petrauskas, A. Classi-
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(19) Such a selectivity profile has already been reported with this
biaryl oxadiazole template. See: Li, Z.; Chen, W.; Hale, J. J.; Lynch, C. L.;
Mills, S. G.; Hajdu, R.; 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. Discovery of Potent 3,5-Diphenyl-1,2,4-oxadiazole
’ ACKNOWLEDGMENT
The authors thank Dr. Richard Upton and Mr. Nick Waite for
NMR support, Dr. Bill Leavens for recording HRMS spectra,
Mrs. Helen Tracey for conducting P-gp and permeability experi-
ments, Mrs. Catherine Cartwright and Miss Aarti Patel for
determining intrinsic clearance, and Mrs. Karen Leavens for
supporting the lymphopenia studies. Mrs. Shenaz Nunhuck and
Mr. Alan Hill are also acknowledged for pKa and CHI determina-
tion, and Miss Helen Garden for solubility measurements.
’ ABBREVIATIONS
RRMS, remitting relapsing multiple sclerosis; CNS, central ner-
vous system; BBB, blood-brain barrier; PSA, polar surface area;
CHI, chromatographic hydrophobicity index; CAD, cationic
amphiphilic drug; LiHMDS, lithium bis(trimethylsilyl)amide;
TMSCl, chloro(trimethyl)silane; DIAD, diisopropyl diazene-
1,2-dicarboxylate; GPCR, G-protein-coupled receptor
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