4-methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide: A potent and selective agonist of S1P1

Article abstract of DOI:10.1021/ml2001399

The sphingosine-1-phosphate-1 receptor (S1P₁) and its endogenous ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking from the lymphatic system. Herein, we disclose 4-methoxy-N-[2- (trifluoromethyl)biphenyl-4-ylcar

Full text of DOI:10.1021/ml2001399

LETTER
pubs.acs.org/acsmedchemlett
4-Methoxy-N-[2-(trifluoromethyl)biphenyl-4-
ylcarbamoyl]nicotinamide: A Potent and Selective Agonist of S1P1
Lewis D. Pennington,*,† Kelvin K. C. Sham,† Alexander J. Pickrell,† Paul E. Harrington,† Michael J. Frohn,†
Brian A. Lanman,† Anthony B. Reed,† Michael D. Croghan,† Matthew R. Lee,‡ Han Xu,§ Michele McElvain,§
Yang Xu,^ Xuxia Zhang,|| Michael Fiorino,|| Michelle Horner,# Henry G. Morrison,+ Heather A. Arnett,||
Christopher Fotsch,† Min Wong,|| and Victor J. Cee†
†Medicinal Chemistry, ‡Molecular Structure, §HTS and Molecular Pharmacology, Inflammation Research, ^Pharmacokinetics and
Drug Metabolism, #Toxicology, and +Pharmaceutics, Amgen, One Amgen Center Drive, Thousand Oaks, California 91320-1799,
United States
S Supporting Information
b
ABSTRACT: The sphingosine-1-phosphate-1 receptor (S1P1) and its endogenous
ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking
from the lymphatic system. Herein, we disclose 4-methoxy-N-[2-(trifluoromethyl)biphenyl-
4-ylcarbamoyl]nicotinamide (8), an uncommon example of a synthetic S1P1 agonist
lacking a polar headgroup, which is shown to effect dramatic reduction of circulating
lymphocytes (POC = À78%) in rat 24 h after a single oral dose (1 mg/kg). The excellent
potency that 8 exhibits toward S1P1 (EC50 = 0.035 μM, 96% efficacy) and the >100-fold
selectivity that it displays against receptor subtypes S1P2À5 suggest that it may serve as a valuable tool to understand the clinical
relevance of selective S1P1 agonism.
KEYWORDS: Sphingosine-1-phosphate-1 receptor agonist, peripheral lymphocyte count, immunosuppression, multiple sclerosis
phingosine-1-phosphate (1, S1P) and its affiliated G-protein-
coupled receptors S1P1À5 are involved in modulating myriad
S
physiological processes including cardiovascular function, pulmonary
endothelium integrity, and cell motility (Figure 1).1,2 S1P (1) is
derived biosynthetically from sphingosine (2) via phosphoryla-
tion mediated by either sphingosine kinase 1 or sphingosine
kinase 2 (SphK1 or SphK2).3 Several years ago, S1P (1) and S1P1
were shown to regulate lymphocyte trafficking from the lymphatic
system.4,5 At high concentrations of 1 or upon exposure to syn-
thetic agonists of S1P1, binding to S1P1 located on the cell surface of
lymphocytes in lymphatic tissues induces receptor internalization
(RI) and prevents lymphocyte egress to the periphery and the central
nervous system(CNS).6,7 Synthetic agonists of S1P1 are under intense
investigation as potential therapies for medical conditions that may
benefit from immunosuppression, such as multiple sclerosis (MS).8,9
Fingolimod (3, FTY720)10,11 bears a dihydroxyamino polar
headgroup akin to that of 2 and is likewise phosphorylated by
SphK2 to give its bioactive congener 4 (FTY720-P),12,13 the first
non-natural agonist of S1P1 to be reported (Figure 1). Notably, 4
displays agonist activity toward several S1P receptor subtypes,14
and until recently, its activation of S1P3 was believed to be exclu-
sively responsible for its bradycardia side effects in humans and
preclinical animal models.15,16 A short time ago, our laboratory
disclosed 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]-
pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5, AMG369), a
potent and selective dual agonist of S1P1/S1P5 bearing a polar head-
group that does not require bioactivation.17,18 Much more rare are
S1P1 agonists that do not contain a polar headgroup,8,9 such as
5-(4-phenyl-5-(trifluoromethyl)thiophen-2-yl)-3-(3-(trifluoro-
Figure 1. Endogenous and synthetic agonists of S1P1.
methyl)phenyl)-1,2,4-oxadiazole (6, SEW2871),19,20 a compound
that is exquisitely selective for S1P1 versus the other S1P recep-
tor subtypes.21 The agonism of S1P1 by 4, 5, and 6 has been shown
to effect reduction of circulating lymphocytes in vivo,11,17,22 and
3 (FTY720) was recently approved by the U.S. Food and Drug
Administration for the treatment of relapsing forms of MS.23,24
As part of an effort to discover structurally novel agonists of S1P1,
we set out to search for atypical chemotypes like 6 that lack a
polar headgroup.25,26
During a high-throughput screening (HTS) campaign, we
identified N-(3-chloro-4-(piperidin-1-yl)phenylcarbamothioyl)-
2-methoxybenzamide (7, Scheme 1).27 The auspicious potency
Received: June 10, 2011
Accepted: July 29, 2011
Published: July 29, 2011
r
2011 American Chemical Society
752
dx.doi.org/10.1021/ml2001399 ACS Med. Chem. Lett. 2011, 2, 752–757
|

ACS Medicinal Chemistry Letters
LETTER
of 7 toward S1P1 (EC50 = 0.43 μM, 87% efficacy),28 coupled with
its excellent selectivity against S1P3 (EC50 > 25 μM)29 and reason-
able molecular and physicochemical properties (MW = 404,
PSA = 54 Å2, cLogP = 4.5, and cLogD7.4 = 4.7),30 prompted us to
investigate analogues that might exhibit improved physicochem-
ical and biological profiles. The highlights of this lead optimiza-
tion effort and its fruition in the discovery of a potent, selective,
and orally bioavailable agonist of S1P1 lacking a polar headgroup,
4-methoxy-N-(2-(trifluoromethyl)biphenyl-4-ylcarbamoyl)nico-
tinamide (8) are the subject of this report (Scheme 1).
was treated with phosgene at elevated temperature to afford
isocyanate 10,32 which was used immediately in the next step of
the synthesis without purification. Deprotonation of known
4-methoxynicotinamide (11)33 with NaH followed by addition
of 10 gave carbamoylnicotinamide 8 in 35% overall yield for the
two-step sequence.34 This expedient route provided a multigram
quantity of 8 that was sufficiently pure (>99%, HPLC) for an
in vivo toxicology study (vide infra).
Table 1 summarizes the lead optimization endeavor through
which carbamothioylbenzamide 7 evolved to carbamoylnicoti-
namide 8.35 Although initially wary of the potential chemical
lability of the carbamothioylbenzamide moiety of 7,36 we specu-
lated that its intramolecular hydrogen-bonding network37 might
help impart hydrolytic stability and conformational rigidity to
it and its carbamoylbenzamide analogues.38À40 However, the
potential toxicity of the carbamothioylbenzamide moiety or its pos-
sible metabolites41 prompted us to exchange the sulfur atom with
oxygen to give carbamoylbenzamide 12. Whereas12 exhibits reason-
able potency toward S1P1 (EC50 = 0.23 μM, 120% efficacy), its
cell permeability is diminished (Papp < 1.0 Â 10À6 cm/s).
Postulating that the chloro and piperidinyl substituents of 12
may bind to the same region of S1P1 as do the trifluoromethyl
and phenyl groups on the thiophene ring of 6,25 we replaced the
Carbamoylnicotinamide 8was prepared as detailed in Scheme 2.
Commercially available 2-(trifluoromethyl)biphenyl-4-amine (9)31
Scheme 1. EvolutionofHTSHit7toCarbamoylnicotinamide8
Scheme 2. Synthesis of Carbamoylnicotinamide 8
Table 2. S1P Receptor Subtype Selectivity of 8a
EC50 (μM)
hS1P2
>25
hS1P3
>25
hS1P4
>50
hS1P5 (% eff.)
4.3 (58)
a See the Supporting Information for experimental details (data repre-
sent an average of at least two determinations; see ref 44).
Table 1. S1P1 StructureÀActivity Relationship, Physicochemical and Microsomal Stability Profiles, and PLC Activitya
P
app (ER)d
CLint, RLMe
PLC, POCf
X
Y
R1
R2
hS1P1, (μM)b EC50 (% eff.) sol. (μg/mL)c HCl, PBS, SIF (Â 10À6 cm/s) (μL/min/mg) (Cplasma, ng/mL)
7
12
13
14
15
16
17
18
19
8
S
CH Cl
CH Cl
N-piperidinyl
N-piperidinyl
0.43 (87)
0.23 (120)
<1.0, < 1.0, 27
3.6, < 1.0, 8.5
3.0, < 1.0, 4.1
<1.0, < 1.0, 23
<1.0, < 1.0, 24
28, 19, 8.5
1.8 (1.0)
<1.0 (NR)h
<1.0 (NR)h
<1.0 (NR)h
1.2 (1.0)
29
36
NDg
NDg
NDg
À20 (4.9)i,j
+3.0 (7.0)i
À14 (13)i
À18 (1.0)i
À16 (BQL)i,k
+4.0 (1.0)i
À67 (64)l
O
O
O
O
O
O
O
O
O
CH CF3 N-piperidinyl
0.013 (120)
0.068 (110)
0.0054 (120)
0.013 (110)
0.038 (130)
0.014 (110)
0.0068 (83)
0.035 (96)
26
CH CF3 phenyl
15
CH i-Pr
phenyl
<14
15
CH CF3 2-pyridinyl
CH CF3 2-thiazolyl
CH CF3 isopropyl
CH CF3 CF3CH2O
7.4 (1.1)
1.1, < 1.0, 12
<1.0, < 1.0, 6.9
<1.0, < 1.0, 2.2
17, 6.6, 16
12 (1.4)
33
2.1 (1.0)
<1.0 (NR)h
<14
25
N
CF3 phenyl
2.3 (1.0)
<14
a See the Supporting Information for experimental details. b Data represent an average of at least two determinations; see ref 28. c Solubility in 0.010 M
aqueous hydrogen chloride (HCl), phosphate-buffered saline (PBS) at pH 7.4, or simulated intestinal fluid (SIF). d Apparent permeability (Papp) through
porcine proximal tubule cells (LLC-PK1 cell line) and efflux ratio (ER). e Estimated intrinsic clearance (CLint) determined by incubation of test compound
with rat liver microsomes (RLM) for30 min and measurement of% turnover. f Percent-of-control (POC) reduction vs vehicle inPLC 24 h after a single oral
dose (1 mg/kg; vehicle = 20% captisol, 1% HPMC, and 1% pluronic F68, pH 2.1 with MSA) administered to female Lewis rats (N = 5/group) and total
compound concentration in plasma (Cplasma) at the 24 h time point. g ND, not determined. h NR, not reportable. i The measured POC reduction in PLC
is not statistically significant (P > 0.05 vs vehicle by the ANOVA/Dunnett's multiple comparison test). j See ref 43. k BQL, below the quantifiable limit.
l The measured POC reduction in PLC is statistically significant (P < 0.05 vs vehicle by the ANOVA/Dunnett's multiple comparison test).
753
dx.doi.org/10.1021/ml2001399 |ACS Med. Chem. Lett. 2011, 2, 752–757

ACS Medicinal Chemistry Letters
LETTER
Table 3. Pharmacokinetic Profile of Carbamoylnicotinamide 8a
1 mg/kg iv
c
AUC0f48 h
AUC0f48 hd (ng h/mL)
3 mg/kg po
AUC0f48 hf (ng h/mL)
100 mg/kg po
PPBb (%) rat
95
CLc (L/h/kg)
0.25
Vdssc (L/kg)
T1/2c (h)
(ng h/mL)
Fe (%)
6.3
19
3400
13000
140
350000
a In vivo experiments were conducted using male SpragueÀDawley rats (N = 3/group). b Percent rat plasma protein binding (PPB) of 8 measured in
vitro following separation by ultracentrifugation. c Clearance (CL), volume of distribution (Vdss), half-life (T1/2), and area under the plasma
concentrationÀtime curve (AUC0f48 h) determined following a single intravenous dose (1 mg/kg; vehicle = DMSO). d AUC0f48 h determined
e
following a single oral dose (3 mg/kg; vehicle = 20% HPBCD, 1% HPMC, and 1% pluronic F68, pH 2.1, with MSA). Percent bioavailability (F)
calculated using AUC0f∞ values determined from the 1 (iv) and 3 mg/kg (po) doses. f AUC0f48 h determined following a single oral dose (100 mg/kg;
vehicle = 20% HPBCD, 1% HPMC, and 1% pluronic F68, pH 2.1, with MSA).
19 also shows potent activity toward S1P1 (EC50 = 0.0068 μM,
83% efficacy) but has lower cell permeability and microsomal
stability (Papp < 1.0 Â 10À6 cm/s; CLint = 25 μL/min/mg).
Finally, inspired by 2-pyridinyl analogue 16, we desired to probe
the effect of placing a polar N atom into the right-hand ring of
carbamoylbenzamide 14. Capitalizing on the ready availability of
4-methoxynicotinamide (11),33 carbamoylnicotinamide 8 was
prepared (Scheme 2). Indeed, 8 displays good potency toward
S1P1 (EC50 = 0.035 μM, 96% efficacy), excellent stability against
both human and rat liver microsomes (CLint < 14 μL/min/mg),
reasonable cell permeability (Papp = 2.3 Â 10À6 cm/s), and
moderate solubility (Sol. = 6.6À17 μg/mL).
Figure 2. Female Lewis rats (vehicle, 0.30, and 1.0 mg/kg dose groups:
We desired next to examine the effect of the S1P1 agonists in
Table 1 on circulating lymphocyte levels in vivo. Female Lewis
rats were administered a single oral dose of each compound
(1 mg/kg); after 24 h, lymphocyte counts in blood and com-
pound concentrations in plasma were measured. The 24 h time
point was selected for our studies, as it was found to mitigate the
effects of dosing on lymphocyte counts.17 Notably, carbamoyl-
nicotinamide 8 reaches >5-fold higher concentration in plasma
than the other compounds tested in Table 1 (64 ng/mL) and is
also the only one that effects statistically significant (P < 0.05 vs
vehicle by the ANOVA/Dunnett's multiple comparison test) reduc-
tion in circulating lymphocytes (POC = À67%). Given their
comparable in vitro profiles, it is unclear why compounds 14À16
and 18 do not achieve higher plasma levels and realize statistically
significant reduction of peripheral lymphocyte counts (PLCs).43
Because of its robust activity in our preliminary in vivo study,
carbamoylnicotinamide 8 was selected for further examination.
As mentioned previously, compound 8 exhibits potent activa-
tion of S1P1 internalization (EC50 = 0.035 μM, 96% efficacy;
Table 1);28 gratifyingly, it does not display appreciable agonism
of receptor subtypes S1P2À4 (EC50 > 25 μM)44 and shows only
weak activation of S1P5 (EC50 = 4.3 μM, 58% efficacy; Table 2).44
As the agonism of both S1P1 and S1P5 in oligodendrocytes by 4
has been suggested to contribute to its efficacy in the treatment of
MS,45 S1P1-selective agonists such as 8 may serve as valuable tool
compounds to examine the clinical relevance of S1P5 agonism.
A pharmacokinetic profile of carbamoylnicotinamide 8 was
undertaken in preparation for more detailed studies in vivo (Table 3).
In male SpragueÀDawley rats (N = 3) given a single intravenous
dose (1 mg/kg), 8 displays low clearance (CL = 0.25 L/h/kg),
moderate volume of distribution (Vdss = 6.3 L/kg), and a long
half-life (T1/2 = 19 h). Although the plasma protein binding of 8
was found to be moderately high (PPB = 95%), its excellent bio-
availability (% F = 140) and dose-proportional plasma exposure over
a wide range of doses (AUC0f48 h, 1 mg/kg iv =3400 ng h/mL;
N = 5/group; 3.0 mg/kg dose group: N = 3; vehicle = 20% HPBCD, 1%
HPMC, and 1% pluronic F68, pH 2.1, with MSA) administered a single
oral dose of carbamoylnicotinamide 8 (0.30, 1.0, or 3.0 mg/kg) showed
dose-proportional plasma exposure (black circles represent average
plasma concentration ( SE) and dose-dependent reduction in circulating
lymphocytes (gray bars represent average blood lymphocyte counts (
SE) at the 24 h time point (statistical significance: ***P < 0.001 and
**P < 0.01 vs vehicle by the ANOVA/Dunnett's multiple comparison test,
respectively); see the Supporting Information for experimental details.
Cl atom in 12 with a CF3 group to afford 13. Although the
improved potency of 13 was encouraging (EC50 = 0.013 μM,
120% efficacy), we desired to improve its stability against rat liver
microsomes (CLint = 26 μL/min/mg). Suspecting that the
piperidine ring of 13 may be the primary site of its metabolism,42
we replaced this ring with a phenyl group to provide 14.
Gratifyingly, carbamoylbenzamide 14 displays enhanced micro-
somal stability (CLint = 15 μL/min/mg) and exhibits good potency
toward S1P1 (EC50 = 0.068 μM, 110% efficacy). In an attempt
improve the potency of 14, we replaced the CF3 substituent in 14
with an isopropyl group to give 15, which does indeed exhibit
potent agonism of S1P1 (EC50 = 0.0054 μM, 120% efficacy). We
also sought to improve the solubility and cell permeability of 14
by inserting a polar N atom into the scaffold, exemplified by
2-pyridinyl derivative 16, which shows the desired improvements
(Sol. = 8.5À28 μg/mL; Papp = 7.4 Â 10À6 cm/s), in addition to
excellent potency on S1P1 (EC50 = 0.013 μM, 110% efficacy).
Interestingly, 2-thiazolyl congener 17 displays greater cell per-
meability as compared to 16 (Papp = 12 Â 10À6 cm/s) but has
lower solubility and microsomal stability (Sol. = < 1.0À12 μg/mL;
CLint = 33 μL/min/mg). To examine the effect of replacing
the left-hand heterocyclic and (hetero)aromatic rings present
in 7 and 12À17 with acyclic groups, isopropyl analogue 18 was
prepared. Analogue 18 exhibits both excellent potency toward
S1P1 (EC50 = 0.014 μM, 110% efficacy) and microsomal stability
(CLint < 14 μL/min/mg). The 2,2,2-trifluoroethoxy compound
754
dx.doi.org/10.1021/ml2001399 |ACS Med. Chem. Lett. 2011, 2, 752–757

ACS Medicinal Chemistry Letters
LETTER
AUC0f48 h, 3 mg/kg po =13000 ng h/mL; AUC0f48 h, 100 mg/kg
po = 350000 ng h/mL) enabled a subsequent in vivo toxicology
study (vide infra).
recommendations of the Guide for the Care and Use of Labora-
tory Animals. All work using research animals was conducted
under Institutional Animal Care and Use Committee (IACUC)
approved protocols.
We next sought to evaluate the pharmacodynamic effects of 8
in more detail (Figure 2). Following a single oral dose in female
Lewis rats (0.30, 1.0, or 3.0 mg/kg), 8 exhibits dose-proportional
plasma exposure (17, 63, and 160 ng/mL, respectively) and
concomitant dose-dependent reduction in circulating lympho-
cytes at the 24 h time point. Statistically significant lymphocyte
depletion is realized for the 1.0 and 3.0 mg/kg doses (78 and 81%
reduction in PLCs; P < 0.001 and P < 0.01 vs vehicle by the
ANOVA/Dunnett's multiple comparison test, respectively). This
dose-dependent decrease in circulating lymphocytes reaching a
plateau at 81% maximal reduction is consistent with the S1P1
agonist activity of 8.19,22
The potential safety of carbamoylnicotinamide 8 was initially
examined in vitro. Encouragingly, 8 displays no appreciable
activity against either hERG (IC50 > 10 μM) or hCYPs 3A4
and 2D6 (IC50 > 30 μM) and exhibits negligible induction of
hPXR (POC = +3.7%, 10 μM). An in vivo toxicology study of
8 was subsequently conducted. In male SpragueÀDawley rats
(N = 3/group) dosed orally at 50 or 200 mg/kg (qd) for 4 days,
no mortality, adverse clinical signs, or changes in body weight
were observed. However, increases in total bilirubin (both doses)
and cholesterol (high dose only) suggest altered bile uptake,
processing, or excretion.
’ ABBREVIATIONS
EC50, molar concentration of compound that produces half
maximal response; RI, receptor internalization; PSA, polar
surface area; cLogP, calculated log P (logarithm of the octanol/
water partition coefficient); cLogD7.4, calculated log D (logarithm
of the octanol/water distribution coefficient at pH 7.4); Papp
,
apparent permeability; CLint, intrinsic clearance; PLC, peripheral
lymphocyte count; HPMC, hydroxypropyl methylcellulose;
MSA, methanesulfonic acid; Cplasma, total compound concentra-
tion in plasma; HPBCD, hyroxypropyl β-cyclodextrin; CL, clear-
ance; Vdss, volume of distribution at steady state; T1/2, half-life of
compoundin plasma; AUC, area under the plasmaconcentrationÀ
time curve; F, bioavailability; hERG, human ether-a-go-go related
gene product (Kv11.1 potassium ion channel); hCYP 3A4, human
cytochrome P450 subtype 3A4; hCYP 2D6, human cytochrome
P450 subtype 2D6; IC50, molar concentration of compound that
produces half maximal inhibition; hPXR, human pregnane X recep-
tor;qd, quaque die (once per day dosing)
’ REFERENCES
In conclusion, carbamoylnicotinamide 8 is a rare example of
an S1P1 agonist lacking a polar headgroup. Despite its modest
solubility and cell permeability, 8 is orally bioavailable and induces
dose-dependent reduction in lymphocyte count in rat 24 h after a
single oral dose. The excellent potency that 8 exhibits toward
S1P1 and the remarkable selectivity that it displays against receptor
subtypes S1P2À5 suggest that it may serve as a valuable tool to
examine the proposed clinical relevance of S1P5 agonism for
treating MS and the cardiovascular safety issues associated with
S1P3 agonism.
(1) For a recent review, see Rosen, H.; Gonzalez-Cabrera, P. J.;
Sanna, M. G.; Brown, S. Sphingosine-1-phosphate receptor signaling.
Annu. Rev. Biochem. 2009, 78, 743–768.
(2) For a review, see Takabe, K.; Paugh, S. W.; Milstien, S.; Spiegel,
S. Inside-out signaling of sphingosine-1-phosphate: Therapeutic targets.
Pharmacol. Rev. 2008, 60, 181–195.
(3) For a review, see Kihara, A.; Mitsutake, S.; Mizutani, Y.; Igarashi,
Y. Metabolism and biological functions of two phosphorylated sphingo-
lipids, sphingosine 1-phosphate and ceramide 1-phosphate. Prog. Lipid
Res. 2007, 46, 126–144.
(4) For a review, see Cyster, J. G. Chemokines, sphingosine-1-
phosphate, and cell migration in secondary lymphoid organs. Annu.
Rev. Immunol. 2005, 23, 127–159.
’ ASSOCIATED CONTENT
(5) S1P (1) hS1P1À5 EC50, μM (% efficacy): hS1P1 = 0.018 (91%),
hS1P2 = 0.022 (91%), hS1P3 = 0.0094 (79%), hS1P4 = 0.16 (57%), and
hS1P5 = 0.012 (63%); see the Supporting Information for experimental
details.
(6) For a review, see Rivera, J.; Proia, R. L.; Olivera, A. The alliance of
sphingosine-1-phosphate and its receptors in immunity. Nat. Rev.
Immunol. 2008, 8, 753–763.
S
Supporting Information. Experimental procedures and
b
characterization data for compounds 8 and 12À19, as well as
experimental details and statistical analysis for key biological
assays. This material is available free of charge via the Internet at
http://pubs.acs.org.
(7) Another possible mechanism involves S1P1-mediated lymphatic
endothelium barriermodification; forareview, seeMarsolais, D.; Rosen, H.
Chemical modulators of sphingosine-1-phosphate receptors as barrier-
oriented therapeutic molecules. Nat. Rev. Drug Discovery 2009, 8, 297–307.
(8) For a recent review, see 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 auto-
immune disorders. Expert Opin. Ther. Patents 2008, 18, 1141–1159.
(9) For a review, see Cooke, N.; Zꢀecri, F. Sphingosine 1-phosphate
type 1 receptor modulators: Recent advances and therapeutic potential.
Annu. Rep. Med. Chem. 2007, 42, 245–263.
(10) 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 recep-
tor agonists. Science 2002, 296, 346–349.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 805-447-0048. Fax: 805-480-1337. E-mail: lewisp@amgen.com.
’ ACKNOWLEDGMENT
We thank Laura Scott (HTS and Molecular Pharmacology,
Amgen) for executing the HTS campaign that identified carba-
mothioylbenzamide 7, Kevin Turney (Analytical Research and
Development, Amgen) and Christopher Wilde (Molecular
Structure, Amgen) for confirmation of the structure assignment
of carbamoylnicotinamide 8 usingHRMS and NMR methods, res-
pectively, and Ronya Shatila and Elizabeth Tominey (Pharmacokinetics
and Drug Metabolism, Amgen) for in vivo pharmacokinetic
studies. All live animal studies were conducted in an AAALAC-
accredited facility and husbandry procedures met all of the
(11) 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
755
dx.doi.org/10.1021/ml2001399 |ACS Med. Chem. Lett. 2011, 2, 752–757

ACS Medicinal Chemistry Letters
LETTER
targets sphingosine 1-phosphate receptors. J. Biol. Chem. 2002, 277,
21453–21457.
(25) Fujiwara, Y.; Osborne, D. A.; Walker, M. D.; Wang, D.-a.;
Bautista, D. A.; Liliom, K.; Van Brocklyn, J. R.; Parrill, A. L.; Tigyi, G.
Identification of the hydrophobic ligand binding pocket of the S1P1
receptor. J. Biol. Chem. 2007, 282, 2374–2385.
(26) Gonzalez-Cabrera, P. J.; Jo, E.; Sanna, M. G.; Brown, S.; Leaf, N.;
Marsolais, D.; Schaeffer, M.-T.; Chapman, J.; Cameron, M.; Guerrero,
M.; Roberts, E.; Rosen, H. Full pharmacological efficacy of a novel
S1P1 agonist that does not require S1P-like headgroup interactions. Mol.
Pharmacol. 2008, 74, 1308–1318.
(12) Kharel, Y.; Lee, S.; Snyder, A. H.; Sheasley-O’Neill, S. L.;
Morris, M. A.; Setiady, Y.; Zhu, R.; Zigler, M. A.; Burcin, T. L.; Ley,
K.; Tung, K. S. K.; Engelhard, V. H.; Macdonald, T. L.; Pearson-White,
S.; Lynch, K. R. Sphingosine kinase 2 is required for modulation of
lymphocyte traffic by FTY720. J. Biol. Chem. 2005, 280, 36865–36872.
(13) Albert, R.; Hinterding, K.; Brinkmann, V.; Guerini, D.; M€uller-
Hartwieg, C.; Knecht, H.; Simeon, C.; Streiff, M.; Wagner, T.; Welzenbach,
K.; Zꢀecri, F.; Zollinger, M.; Cooke, N.; Francotte, E. Novel immuno-
modulator FTY720 is phosphorylated in rats and humans to form a
single stereoisomer. Identification, chemical proof, and biological
characterization of the biologically active species and its enantiomer.
J. Med. Chem. 2005, 48, 5373–5377.
(14) rac-FTY720-P (rac-4) hS1P1À5 EC50, μM (% efficacy):
hS1P1 = 0.0020 (115%), hS1P2 > 1.0, hS1P3 = 0.027 (35%), hS1P4 =
0.17 (25%), and hS1P5 = 0.022 (36%); see the Supporting Information
for experimental details.
(15) 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 and references cited therein.
(16) Hamada, M.; Nakamura, M.; Kiuchi, M.; Marukawa, K.;
Tomatsu, A.; Shimano, K.; Sato, N.; Sugahara, K.; Asayama, M.; Takagi,
K.; Adachi, K. Removal of sphingosine 1-phosphate receptor-3 (S1P3)
agonism is essential, but inadequate to obtain immunomodulating
2-aminopropane-1,3-diol S1P1 agonists with reduced effect on heart
rate. J. Med. Chem. 2010, 53, 3154–3168.
(17) 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.; Zhang, 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 and
references cited therein.
(18) AMG369 (5) hS1P1À5 EC50, μM (% efficacy): hS1P1 = 0.0020
(98%), hS1P2 > 2.5, hS1P3 = 0.89 (26%), hS1P4 > 2.5, and hS1P5 =
0.029 (50%); see the Supporting Information for experimental details.
(19) Hale, J. J.; Lynch, C. L.; Neway, W.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei, G.-J.; Parent,
S. A.; Chrebet, G.; Bergstrom, J.; Card, D.; Ferrer, M.; Hodder, P.;
Strulovici, B.; Rosen, H.; Mandala, S. A rational utilization of high-
throughput screening affords selective, orally bioavailable 1-benzyl-3-
carboxyazetidine sphingosine-1-phosphate-1 receptor agonists. J. Med.
Chem. 2004, 47, 6662–6665.
(27) By measurement of β-galactoside activity resulting from
β-arrestin binding to hS1P1 in CHO-K1 cells.
(28) By measurement of RI of an hS1P1ÀeGFP fusion protein in
U2OS cells (% efficacy is reported relative to known S1P1 agonist
1-(4-(6-benzylbenzofuran-2-yl)-3-fluorobenzyl)azetidine-3-carboxylic
acid at a concentration of 1.0 μM; see ref 17); see the Supporting
Information for experimental details.
(29) By measurement of Ca2+ mobilization in CHO-K1 cells
expressing hS1P3 and a chimeric Gq/i5 G-protein (% efficacy is reported
relative to S1P at a concentration of 0.20 μM; > [highest concentration
tested] is reported for compounds that do not achieve >10% of control
activity); see the Supporting Information for experimental details.
(30) PSA and cLogP values were calculated using Daylight Chemical
Information Systems software (web site: www.daylight.com); the
cLogD7.4 value was calculated using Advanced Chemistry Development
(ACD/Labs) software (web site: www.acdlabs.com).
(31) Pan, S.; Mi, Y.; Pally, C.; Beerli, C.; Chen, A.; Guerini, D.;
Hinterding, K.; Nuesslein-Hildesheim, B.; Tuntland, T.; Lefebvre, S.;
Liu, Y.; Gao, W.; Chu, A.; Brinkmann, V.; Bruns, C.; Streiff, M.; Cannet,
C.; Cooke, N.; Gray, N. A monoselective sphingosine-1-phosphate
receptor-1 agonist prevents allograft rejection in a stringent rat heart
transplantation model. Chem. Biol. 2006, 13, 1227–1234.
(32) For a review, see Twitchett, H. J. Chemistry of the production
of organic isocyanates. Chem. Soc. Rev. 1974, 209–230.
(33) Ross, W. C. J. The preparation of some 4-substituted nicotinic
acids and nicotinamides. J. Chem. Soc. C 1966, 1816–1821.
(34) The standard thermal conditions for coupling amides and
isocyanates (toluene, 110 ꢀC; Wiley, P. F. The reaction of amides with
isocyanates. J. Am. Chem. Soc. 1949, 71, 1310–1311) resulted in
migration of the methyl group to the pyridyl nitrogen atom in 8; for
the synthesis of compounds 12À19, see the Supporting Information.
(35) None of the compounds in Table 1 displays appreciable
agonism of hS1P3 (EC50 > 25 μM); see ref 29.
(36) Douglass, I. B.; Dains, F. B. The preparation and hydrolysis of
mono- and disubstituted benzoylthioureas. J. Am. Chem. Soc. 1934,
56, 1408–1409.
(20) Jo, E.; Sanna, M. G.; Gonzalez-Cabrera, P. J.; Thangada, S.;
Tigyi, G.; Osborne, D. A.; Hla, T.; Parrill, A. L.; Rosen, H. S1P1-selective
in vivo-active agonists from high-throughput screening: Off-the-shelf
chemical probes of receptor interactions, signaling, and fate. Chem. Biol.
2005, 12, 703–715.
(21) SEW2871 (6) hS1P1À5 EC50, μM (% efficacy): hS1P1 = 0.27
(86%) and hS1P3 > 25 (see the Supporting Information for experimental
details); hS1P2À5 EC50 > 10 μM (see ref 22).
(22) 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. S1P1 agonists have been shown to effect ∼70%
maximal reduction in circulating lymphocytes; see ref 19.
(23) Kappos, L.; Radue, E.-W.; O'Connor, P.; Polman, C.; Hohlfeld,
R.; Calabresi, P.; Selmaj, K.; Agoropoulou, C.; Leyk, M.; Zhang-
Auberson, L.; Burtin, P. A placebo-controlled trial of oral fingolimod
in relapsing multiple sclerosis. N. Engl. J. Med. 2010, 362, 387–401.
(24) Cohen, J. A.; Barkhof, F.; Comi, G.; Hartung, H.-P.; Khatri,
B. O.; Montalban, X.; Pelletier, J.; Capra, R.; Gallo, P.; Izquierdo, G.;
Tiel-Wilck, K.; de Vera, A.; Jin, J.; Stites, T.; Wu, S.; Aradhye, S.; Kappos,
L. Oral fingolimod or intramuscular interferon for relapsing multiple
sclerosis. N. Engl. J. Med. 2010, 362, 402–415.
(37) For a review, see Aly, A. A.; Ahmed, E. K.; El-Mokadem, K. M.;
Hegazy, M. E.-A. F. Update survey on aroyl substituted thioureas and
their applications. J. Sulfur Chem. 2007, 28, 73–93.
(38) Gasteiger, J.; Hondelmann, U.; R€ose, P.; Witzenbichler, W.
Computer-assisted prediction of the degradation of chemicals: Hydro-
lysis of amides and benzoylphenylureas. J. Chem. Soc. Perkin Trans. 2
1995, 193–204.
(39) Sluiter, C.; Kettenes-van den Bosch, J. J.; Hop, E.; van der Houwen,
O. A. G. J.; Underberg, W. J. M.; Bult, A. Degradation study of the
investigational anticancer drug clanfenur. Int. J. Pham. 1999, 185, 227–235.
(40) Quantum mechanics calculations using density functional theory
at the B3LYP/6-31G* level (gas phase) indicate that 2-methoxy-
N-(phenylcarbamoyl)benzamide prefers the bis-keto tautomeric form
having the exchangeable protons attached to the nitrogen atoms; see
Gaussian 03, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2004.
(41) For an investigation of the metabolism and toxicity of ar-
ylthioureas and their metabolites, seeScheline, R. R.; Smith, R. L.;
Williams, R. T. The metabolism of arylthioureas—II. The metabolism
of 14C- and 35S-labelled 1-phenyl-2-thiourea and its derivatives. J. Med.
Pharm. Chem. 1961, 4, 109–135.
(42) A preliminary metabolite identification study of a closely
related scaffold using rat liver microsomes revealed the metabolic
instability of the piperidine ring.
756
dx.doi.org/10.1021/ml2001399 |ACS Med. Chem. Lett. 2011, 2, 752–757

ACS Medicinal Chemistry Letters
LETTER
(43) In aninitial study, carbamoylbenzamide14achieveda 9.6ng/mL
plasma concentration and effected a statistically significant (P < 0.05 vs
vehicle by the ANOVA/Dunnett's multiple comparison test) 48%
reduction in circulating lymphocytes (1.0 mg/kg, po, 24 h). A subse-
quent doseÀresponse study (0.30, 1.0, and 3.0 mg/kg, po, 24 h)
indicated that higher doses were necessary to achieve greater plasma
levels and statistically significant lymphocyte count reduction; see the
Supporting Information for experimental details.
(44) By measurement of Ca2+ mobilization in CHO-K1 cells
expressing the target hS1P receptor and a chimeric Gq/i5 G-protein
(for hS1P2, hS1P3, and hS1P5, % efficacy is reported relative to S1P at a
concentration of 0.20 μM and > [highest concentration tested] is
reported for compounds that do not achieve >10% of control activity;
for hS1P4, % efficacy is reported relative to known S1P4 agonist (2R)-2-
amino-3-(4-(2-benzylphenyl)-1H-indole-2-carboxamido)propanoic
acid at a concentration of 0.40 μM and > [highest concentration tested]
is reported for compounds that do not achieve >10% of control
activity, see Azzaoui, K.; Bouhelal, R.; Buehlmayer, P.; Guerini, D.;
Koller, M. Indol-alanine derivatives as selective S1P4-agonists. World
Intellectual Property Organization Patent Application WO 2005070886,
2005); see the Supporting Information for experimental details.
(45) For a review, see Brinkmann, V. FTY720 (fingolimod) in
multiple sclerosis: Therapeutic effects in the immune and the central
nervous system. Br. J. Pharmacol. 2009, 158, 1173–1182.
757
dx.doi.org/10.1021/ml2001399 |ACS Med. Chem. Lett. 2011, 2, 752–757