Novel 5- and 6-subtituted benzothiazoles with improved physicochemical properties: Potent S1P1 agonists with in vivo lymphocyte-depleting activity

Article abstract of DOI:10.1016/j.bmcl.2011.10.069

An SAR campaign designed to increase polarity in the 'tail' region of benzothiazole 1 resulted in two series of structurally novel 5-and 6-substituted S1P₁ agonists. Structural optimization for potency ultimately delivered carboxamide (+)-11f,

Full text of DOI:10.1016/j.bmcl.2011.10.069

Bioorganic & Medicinal Chemistry Letters 22 (2012) 628–633
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 5- and 6-subtituted benzothiazoles with improved physicochemical
properties: Potent S1P1 agonists with in vivo lymphocyte-depleting activity
Mike Frohn a, , Victor J. Cee a, Brian A. Lanman a, Alexander J. Pickrell a, Jennifer Golden a,
⇑
Dalia Rivenzon-Segal e,à, Scot Middleton b, Mike Fiorino b, Han Xu c, Michael Schrag d,§
Yang Xu d, Michele McElvain c, Kristine Muller a, Jerry Siu b,–, Roland Bürli a,||
,
a Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Department of Inflammation Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Molecular Pharmacology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
d Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e EPIX Pharmaceuticals Inc., 167 Worcester Street, Suite 201, Wellesley Hills, MA 02481, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An SAR campaign designed to increase polarity in the ‘tail’ region of benzothiazole 1 resulted in two ser-
ies of structurally novel 5-and 6-substituted S1P1 agonists. Structural optimization for potency ultimately
delivered carboxamide (+)-11f, which in addition to possessing improved physicochemical properties rel-
ative to starting benzothiazole 1, also displayed good S1P3 selectivity and acceptable in vivo lymphocyte-
depleting activity.
Received 6 September 2011
Revised 18 October 2011
Accepted 20 October 2011
Available online 28 October 2011
Ó 2011 Published by Elsevier Ltd.
Keywords:
S1P1
Lymphocyte reduction
Physicochemical properties
G-protein coupled receptor
Sphingosine-1-phosphate (S1P, Fig. 1) is an endogenous lyso-
phospholipid that modulates various cellular processes, including
migration, adhesion, proliferation, and differentiation.1 The mole-
cule produces many of its effects via high affinity interaction(s)
with the sphingosine-1-phosphate (S1P1–5) G-protein coupled
receptors.2 The S1P1 receptor subtype, which is highly expressed
on lymphocytes, regulates lymphocyte egress from secondary lym-
phoid tissue. Therefore, the receptor has received considerable
attention as a potentially important target for therapeutic inter-
vention. Seminal studies with the aminodiol prodrug fingolimod
(FTY720, Fig. 1) have provided much of the validation for S1P1 as
a therapeutic target in autoimmunity and inflammation.3 Sphingo-
sine kinase 2 phosphorylates fingolimod in vivo,4 which generates
the corresponding (S)-phosphate (FTY720P); this active metabolite
has high affinity for S1P1,3–5. Upon binding S1P1, FTY720P causes
5
immunosuppression via S1P1 receptor internalization, which ulti-
mately results in lymphocyte sequestration in secondary lymphoid
tissue. Fingolimod has proven to be an effective treatment for
relapsing-remitting multiple sclerosis in clinical studies, and its
efficacy and safety profile has prompted the US Food & Drug
Administration to approve the agent for treatment of this disease.6
One of several adverse events observed with fingolimod therapy is
dose-dependent reduction of heart rate.7 Whereas this side effect
has been linked to S1P3 agonism in rodents,8 additional research
has indicated it is more likely S1P1- rather than S1P3-mediated in
humans.9 Nevertheless, we and others have aimed for the develop-
ment of selective S1P1 agonists to address the cardiovascular
findings.10
⇑
Corresponding author.
Benzothiazole 1 was first prepared during a structure–activity
relationship (SAR) study of benzannulated compounds that pro-
duced selective S1P1 receptor agonists.10e,f Although 1 was a potent
E-mail address: mfrohn@amgen.com (M. Frohn).
Present address: Specialized Chemistry Center, University of Kansas, 2121 Simons
Drive, Lawrence, KS 66047, USA.
S1P1 agonist (EC50 = 0.041
(EC50 = 1.21 M), the overall physicochemical properties—low total
polar surface area (tPSA = 52.9 Å2) and relatively high lipophilicity
(clogP = 3.92)—predicted higher-than-average likelihood for
lM) and was selective against S1P3
à
Present address: Reinhold, Cohn and Partners, Tel-Aviv, Israel.
Present address: ProPharma Services, 3195 E. Yarrow Circle, Superior, CO 80301,
§
l
USA.
–
Present address: Autoimmune Inflammatory Disease Research, Biopharmaceuti-
a
cals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maalov, Denmark.
attrition during development because of an unfavorable safety pro-
||
Present address: Biofocus DPI, Chesterford Research Park, Saffron Walden, Essex
file.11 We therefore initiated an SAR campaign designed to increase
CB10 1XL, United Kingdom.
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.10.069

M. Frohn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 628–633
629
OH
OR
NH2
NH2
OH
OH
CH3(CH2)12
O
P
O
OH
S1P
R = H, fingolimod (FTY720)
R = PO(OH)2, FTY720P
O
OH
CO2H
"Tail"
"Core"
N
N
N
N
S
S
N
F
F
1
AMG 369
S1P1 EC50, µM = 0.041
S1P3 EC50, µM = 1.21
S1P1 EC50, µM = 0.002
S1P3 EC50, µM = 0.888
tPSA: 52.9Å2
clogP: 3.92
tPSA: 65.3Å2
clogP: 3.27
Figure 1. Sphingosine-1-phosphate (S1P), fingolimod (FTY720), FTY720P, benzothiazole 1, AMG 369.
the polarity of 1 to levels associated with a higher probability for
successful development. Specifically, our goal was to deliver a po-
tent, selective S1P1 agonist with acceptable in vivo lymphocyte-
depleting activity, lower clogP, and higher tPSA. One facet of this
strategy involved introducing polarity to the ‘core’ region of 1. That
work resulted in AMG 369, a potent and selective S1P1 agonist that
became a development candidate (Fig. 1).10f In parallel to these ef-
forts, we also attempted to increase polarity in the ‘tail’ region of
the molecule, and that is the focus of this manuscript.
Analogues for this study were prepared as shown in Schemes 1–
3. Compounds 6a and ( )-6b were constructed according to
Scheme 1. Aryl bromide 210e,10f was lithiated at ꢀ78 °C and then
exposed to 4-bromo-1-fluoro-2-isothiocyanatobenzene to give an
intermediate thioamide. That compound was subsequently cy-
clized under basic conditions at elevated temperature to give
benzothiazole 3 in good overall yield.12 Subsequent palladium-
catalyzed coupling with 2,4-dimethyl-3-(pyridin-2-ylmethyl)pen-
tan-3-ol resulted in picoline-substituted compound 4a.13 Methyl
substitution at the newly-formed methylene group was achieved
by deprotonation with lithium (bis)trimethylsilylamide followed
by alkylation with iodomethane, which produced compound
( )-4b. Removal of the 1,3-dioxane from both 4a and ( )-4b with
aqueous hydrochloric acid, reductive amination of the liberated
aldehydes with methyl azetidine-3-carboxylate, and saponification
of the resulting methyl esters with lithium hydroxide delivered
analogues 6a and ( )-6b.
Whereas C-6 substituted analogues 9a and ( )-9b (Scheme 2)
were constructed analogously to 6a and ( )-6b; the remaining C-
6 substituted analogues 9c–e were prepared slightly differently.
Bromide 7 was first converted to the corresponding Weinreb amide
via palladium-catalyzed carbonylation14; subsequent treatment
with the organomagnesium reagent generated by addition of
iPrMgCl to 2-bromopyridine resulted in a versatile intermediate
ketone (8c). This compound could either be transformed into final
product 9c or converted to methylene-substituted intermediates
8d and ( )-8e (Scheme 2). The cyclopropyl unit of 8d was installed
Br
O
O
N
S
O
O
(c)
(a), (b)
Br
F
F
2
3
R
R
O
O
N
S
(e)
N
S
CHO
N
N
F
F
4a: R = H
5a: R = H
( )-5b: R = CH3
(d)
( )-4b: R = CH3
O
OH
R
N
N
(f), (g)
N
S
F
6a: R = H
( )-6b: R = CH3
Scheme 1. Reagents and conditions: (a) n-BuLi, 4-bromo-1-fluoro-2-isothiocyanatobenzene, THF, ꢀ78 °C, 63% (b) Na2CO3, DMF, 110 °C, 93% (c) 2,4-dimethyl-3-(pyridin-2-
ylmethyl)pentan-3-ol, Pd(OAc)2, PPh3, Cs2CO3, toluene, 120 °C, 76% (d) LiN(TMS)2, CH3I, THF, 71% (e) 5 N HCl (aq), THF, 65 °C, 68–99% (f) methyl azetidine-3-carboxylate
i
hydrochloride, AcOH, Pr2NEt, NaBH3CN, MeOH/CH2Cl2, 70–83% (g) NaOH, THF; HCl, then pH 6 sodium phosphate buffer, 11–59%.

630
M. Frohn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 628–633
(a) for 8a
O
N
S
O
O
N
S
(b), (c) for 8c
N
O
Br
F
F
R
N
7
8a: R = H
(d)
( )-8b: R = Me
8c: R = (=O)
8d: R = c-Pr
( )-8e: R = Et
(e), (f)
(g), (h), (i)
O
OH
N
S
(j), (k), (l)
N
F
9a: R = H
R
( )-9b: R = Me
9c: R = (=O)
9d: R = c-Pr
(+)-
(_)-9e
: R = Et
and
Scheme 2. Reagents and conditions: (a) 2,4-dimethyl-3-(pyridin-2-ylmethyl)pentan-3-ol, Pd(OAc)2, PPh3, Cs2CO3, toluene, 120 °C, 78% (b) N,O-dimethyl hydroxylamine,
Pd(OAc)2, XANTPHOS, Et3N, CO (1 atm), toluene, 80 °C, 86% (c) 2-bromopyridine, iPrMgCl, THF, 0 °C to RT, THF, 45% (d) LiN(TMS)2, CH3I, THF, 11% (e) n-BuLi,
methyltriphenylphosphonium bromide, THF, 56% (f) Me3SOI, tBuOK, DMSO/THF, 34% (g) EtMgCl, ZnCl2, THF, 0 °C, 84% (h) CH3SO2Cl, Et3N, CH2Cl2, 83% (i) H2, Pd-C, MeOH, 58%
i
(j) 5 N HCl (aq), THF, 65 °C, 88–98% (k) methyl azetidine-3-carboxylate hydrochloride, AcOH, Pr2NEt, NaBH3CN, MeOH/CH2Cl2, 34–91% (l) NaOH, THF/H2O; HCl, then pH 6
sodium phosphate buffer, 31–97%.
O
O
O
O
N
S
(a), (b), (c)
N
S
R1
N
Br
R
F
F
O
O
7
10
OH
(d), (e), (f)
N
N
S
R1
N
R
F
O
11a-h
Scheme 3. Reagents and conditions: (a) Pd(dppp)Cl2, Et3N, CO (20 psi), 1:2:1 DMF/THF/EtOH, 95 °C, 79% (b) NaOH, dioxane/H2O, 40 °C, 68% (c) HNRR1, HBTU, Et3N or iPr2NEt,
DMF, RT, 82–97% (d) 5 N HCl (aq), THF, 65 °C, 70–92% (e) methyl azetidine-3-carboxylate hydrochloride, AcOH, iPr2NEt, NaBH3CN, MeOH/CH2Cl2, 39–71% (f) NaOH, THF/H2O;
HCl, then pH 6 sodium phosphate buffer, 47–78%.
by Wittig olefination of the ketone followed by cyclopropanation
with trimethylsulfoxonium ylide. The ethyl group of ( )-8e was
produced via nucleophillic addition of ethylmagnesium bromide,
dehydration of the resulting tertiary alcohol, and palladium-
catalyzed hydrogenation of the olefins. Both 8d and ( )-8e supplied
9d, (+)-9e, and (ꢀ)-9e using the same end game sequence as de-
picted in Scheme 1.15 Carboxamide analogues 11a–h were synthe-
sized from 7 by palladium-catalyzed carbonylation to form the
corresponding ethyl ester, saponification of the ester, and amide
coupling (Scheme 3). Completion of the analogues proceeded as
in Schemes 1 and 2.16,17
substitution at the methylene linker was examined next. Unfortu-
nately, even replacement of a single hydrogen atom with a methyl
group had a negative impact ([ ]-6b, S1P1 EC50 = 2.37 lM). There-
fore, the study turned to compounds with the ‘tail’ region extending
from C-6 of the benzothiazole core. Despite being over twofold less
active than the analogous C-5 substituted analogue 6a, derivatizing
C-6 substituted picoline 9a ultimately provided access to com-
pounds with improved potency. Immediate gains were realized with
methyl-substituted compound ( )-9b (EC50 = 0.57 lM), and addi-
tional improvements were obtained with slightly larger alkyl sub-
stituents. Cyclopropyl derivative 9d was roughly twofold more
Analogue effect on S1P1 was evaluated by measuring receptor
internalization (RI) of an hS1P1–GFP fusion protein in U2 osteosar-
coma (U2OS) cells. Selectivity against S1P3 was measured using a
Ca2+ flux assay in CHO cells that stably co-expressed hS1P3 receptor
and a chimeric Gq/i5 G-protein (Tables 1 and 2).18 The SAR study be-
gan by evaluating single N for C–H replacements of the terminal
phenyl ring. Prior SAR from a related benzofuranyl series indicated
that of the three possible N for C–H substitutions, replacement of
the 2-C–H was tolerated the best (data not shown). The related 2-
active than ( )-9b (EC50 = 0.24
ethyl-substituted derivative (ꢀ)-9e increased potency even further
(EC50 = 0.10 M); however, ethyl enantiomer (+)-9e displayed sig-
nificantly lower activity (EC50 = 0.515 M) than its isomer. Whereas
additional improvements were not achieved in this series, ketone 9c
(S1P1 EC50 = 1.30 M, S1P3 EC50 >25 M) was also considered a
promising analogue in its own right. Since options within the 2-pic-
oline series appeared exhausted, we adjusted our SAR strategy to-
wards finding high-affinity agonists that incorporated the
carbonyl subunit.
lM, >105-fold selectivity), and
l
l
l
l
pyridyl benzothiazole analogue 6a (S1P1 EC50 = 0.68
>25 M, >37-fold selectivity) exhibited reasonable potency for
S1P1 and became the starting point for further optimization. Alkyl
lM, S1P3 EC50
l
It was immediately clear that the linker carbonyl could be
incorporated into a carboxamide function, so we began an SAR

M. Frohn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 628–633
631
Table 1
SAR of 2-picoline substituted benzothiazole analoguesa
CO2H
R1
5
6
N
N
S
R2
F
Cmpd
R1
R2
H
hS1P1 RI EC50
0.042 (102)
,
lM (% efficacy)
hS1P3 Ca2+ EC50
1.21 (24)
,
lM (% efficacy)
clog Pb
tPSA (Å2)
1
Bn
3.92
52.9
66.3
6a
H
0.68 (94)
2.37 (62)
1.96 (71)
0.57 (79)
>25
>25
>25
>25
2.42
2.82
2.42
2.82
N
N
( )-6b
9a
H
66.3
66.3
66.3
N
N
H
H
( )-9b
N
N
9d
H
H
0.24 (90)
0.10 (95)
>25
2.86
3.35
66.3
66.3
(ꢀ)-9e
2.06 (82)
Et
Et
O
N
N
(+)-9e
H
H
0.52 (93)
1.30 (50)
9.75 (30)
3.35
2.25
66.3
83.4
9c
>25
a
Data reported as an average of at least two experimental determinations. S1P1 efficacy was measured relative to 0.200 lM S1P.
ACD clogP.
b
study of C-6 substituted amides (Table 2). We began with the
homologous amides 11a–c. Whereas piperidine amide 11a (S1P1
taken 4 h post dose (Table 3).20 Gratifyingly, analogue (+)-11f dis-
played statistically significant reduction of circulating lymphocytes
after a single 10 mg/kg dose (55%, P <0.05). Measurement of total
plasma concentration showed systemic exposure was 10-fold
higher than the S1P1 receptor internalization EC50. Conversely,
(ꢀ)-11f did not show significant lymphocyte sequestration, most
likely because of insufficient plasma exposure. Although the lym-
phocyte-depleting activity of (+)-11f was not superior to 1 (58%
lymphocyte depletion vs vehicle at 24 h post dose, 1 mg/kg dose,
N = 5), this carboxamide achieved several goals of the current
study: it is a potent S1P1 agonist in vitro, selective against S1P3,
displays acceptable in vivo lymphocyte-depleting activity, and
has improved physicochemical properties relative to 1. Therefore,
this molecule is attractive not only because of its in vivo activity,
but also because its improved physicochemical profile positions
it as a suitable template for further SAR study in the ‘core’ and
‘head’ regions of the molecule.
In conclusion, a novel series of selective S1P1 benzothiazole
agonists with polar functionality in the ‘tail’ region of the molecule
was discovered. Starting from benzothiazole 1, an SAR study led to
the preparation of carboxamide (+)-11f, which displayed potent
S1P1 receptor internalization, moderate S1P3 selectivity, acceptable
in vivo lymphocyte-depleting activity, and improved physico-
chemical properties.
EC50 = 1.17
EC50 = 2.72
l
l
M) and homopiperidine amide 11b (S1P1
M) both had reasonable activity, the pyrrolidine
amide 11c did not (EC50 >20
lM). Thiazolidine amide 11d
(EC50 = 2.71 M), a structural isostere of 11a, restored S1P1 activ-
l
ity; and we therefore focused on this structural unit for the
remainder of the study. Additional potency increases were realized
from alkyl substitution at the C-2 position of the thiazolidine ring.
For
example,
methyl-substituted
thiazolidine
( )-11e
(EC50 = 0.042
l
M) was fivefold more potent than 11d; and the
slightly larger ethyl-containing derivative (+)-11f ultimately be-
came the most potent compound in either the picoline or amide
series (EC50 = 0.017
Ethyl enantiomer (ꢀ)-11f had much lower S1P1 agonistic activity
(EC50 = 0.51 M). A number of additional compounds with other
lM, S1P3 EC50 = 0.39 lM, 23-fold selectivity).
l
substituents were also prepared and tested (e.g., ( )-11g and
11h), but none were more active and selective than (+)-11f.
Since (+)-11f and (ꢀ)-11f were moderately selective against
S1P3, showed divergent pharmacology on S1P1, and possessed im-
proved physicochemical properties relative to 1—clogP (2.58) and
tPSA (73.7 Å2)—the in vivo lymphocyte-depleting activity of the
two compounds was evaluated.19 Each compound was orally dosed
in female Lewis rats, and total blood lymphocyte counts were

632
M. Frohn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 628–633
Table 2
SAR of ketone and carboxamide substituted benzothiazole analoguesa
CO2H
N
N
S
R
F
O
Cmpd
R
hS1P1 RI EC50
1.30 (50)
,
lM (% efficacy)
hS1P3 Ca2+ EC50
>25
,
lM (% efficacy)
clogPb
tPSA (Å2)
83.4
N
N
9c
2.25
11a
11b
1.17 (95)
>25
>25
1.79
2.34
73.7
73.7
2.72 (108)
N
11c
11d
>20
>25
1.23
1.53
73.7
73.7
N
S
2.71 (110)
7.42 (21)
N
Me
S
S
S
( )-11e
(ꢀ)-11f
(+)-11f
0.042 (90)
0.15 (106)
2.04 (71)
6.24 (36)
0.39 (82)
2.05
2.58
2.58
73.7
73.7
73.7
N
Et
N
Et
0.017 (107)
0.049 (96)
0.43 (80)
N
( )-11g
0.90 (87)
2.68 (72)
2.98
2.57
73.7
73.7
S
S
N
N
11h
a
Data reported as an average of at least two experimental determinations. S1P1 efficacy was measured relative to 0.200 lM S1P.
ACD clogP.
b
Table 3
Effects of (+)-11f and (ꢀ)-11f on circulating lymphocytes 4 h post dose in female lewis rats
Compd
hS1P1 RI EC50
,
lM
Dose (mg/kg)
Lymphocyte reduction (%)
Plasma concentration (ng/mL, lM)
(ꢀ)-11f
(+)-11f
0.151
0.017
10
10
24
65 (0.13)
92 (0.19)
55*
*
P <0.05 versus vehicle by ANOVA/Dunnett’s multiple comparison test. (N = 5/group. Vehicle = 20% HPBCD, 1% HPMC, 1% pluronic F68 w/MSA).
5. Brinkmann, V.; Davis, M. D.; Heise, C. E.; Albert, R.; Cottens, S.; Hof, R.; Bruns,
C.; Prieschl, E.; Baumruker, T.; Hiestand, P.; Foster, C. A.; Zollinger, M.; Lynch, K.
R. J. Biol. Chem. 2002, 277, 21453.
6. (a) Cohen, J. A.; Barkhof, F.; Comi, G.; Hartung, H. –P.; Khatri, B. O.; Mantalban,
X.; Pelletier, J.; Capra, R.; Gallo, P.; Izquierdo, G.; Tiel-Wilck, K.; de Vera, A.; Jin,
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Supplementary data
Supplementary data (synthesis and characterization of (+)-11f
and (ꢀ)-11f, and statistical analysis of S1P1 and S1P3 data) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.10.069.
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containing 0.2% diethylamine, 70 mL/min flow rate. (+)-9e: Rt = 3.10 min,
½
a 2D5
ꢂ
= +19.4°, (c 0.12, MeOH); (ꢀ)-9e: Rt = 3.65 min, ½a D25
= ꢀ16.8°, (c 0.18,
ꢂ
MeOH).
10. For recent work in this area see: (a) Bolli, M. H.; Lescop, C.; Naylor, O. Curr. Top.
Med. Chem. 2011, 11, 726; (b) Demont, E. H.; Arpino, S.; Bit, R. A.; Campbell, C.
A.; Deeks, N.; Desai, S.; Dowell, S. J.; Gaskin, P.; Gray, J. R. J.; Harrison, L. A.;
Haynes, A.; Heightman, T. D.; Holmes, D. S.; Humphreys, P. G.; Kumar, U.;
Morse, M. A.; Osborne, G. J.; Panchal, T.; Philpott, K. L.; Taylor, S.; Watson, R.;
Willis, R.; Witherington, J. J. Med. Chem. 2011, 54, 6724; (c) Pennington, L. D.;
Sham, K. K. C.; Pickrell, A. J.; Harrington, P. E.; Frohn, M. J.; Lanman, B. A.; Reed,
A. B.; Croghan, M. D.; Lee, M. R.; Xu, H.; McElvain, M.; Xu, Y.; Zhang, X.; Fiorino,
M.; Horner, M.; Morrison, H. G.; Arnett, H. A.; Fotsch, C.; Wong, M.; Cee, V. J.
ACS Med. Chem. Lett. 2011, 2, 752; (d) 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. ACS Med. Chem. Lett. 2011, 2, 368; (e) Lanman, B. A.; Cee, V. J.; Cheruku, S. R.;
Frohn, M.; Golden, J.; Lin, J.; Lobera, M.; Marantz, Y.; Muller, K. M.; Neira, S. C.;
Pickrell, A. J.; Rivenzon-Segal, D.; Schutz, N.; Sharadendu, A.; Yu, X.; Zhang, Z.;
Buys, J.; Fiorino, M.; Gore, A.; Horner, M.; Itano, A.; McElvain, M.; Middleton, S.;
Schrag, M.; Vargas, H. M.; Xu, H.; Xu, Y.; Zhang, X.; Siu, J.; Bürli, R. ACS Med.
Chem. Lett. 2011, 2, 102; (f) 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.;
Bürli, R. ACS Med. Chem. Lett. 2011, 2, 107.
16. Substituted thiazolidines required for 11e–h were synthesized using a
literature procedure: Lalezari, I.; Schwartz, E. L. J. Med. Chem. 1988, 31, 1427.
17. Please refer to the Supplementary data for the synthesis and chiral separation
of (+)-11f and (ꢀ)-11f. Separation conditions: chiralcel OJ-H column
(250 ꢁ 21 mm), mobile phase: 40% IPA containing 0.2% diethylamine, 65 mL/
min flow rate.
18. The hS1P1 receptor internalization assay was performed using a U2OS cell line
expressing hS1P1–eGFP chimeric protein (Thermo Scientific, Søborg,
Denmark). Upon compound treatment, the hS1P1 receptor was internalized
into the cytoplasm, forming GFP-containing-endosomes. This event was
detected using an ArrayScan automated microscope (Thermo Scientific
Cellomics, Pittsburg, PA), and the degree of receptor internalization was
quantitated by counting the number of GFP-containing endosomes per cell.
hS1P1–eGFP expressing U2OS cells were starved in serum free media for two
hours prior to compound treatment. Compounds were incubated with the
starved cells at 37 °C for one hour. Compound-treated cells were subsequently
fixed using 4% formaldehyde, and nuclei were stained using Hoechst dye
(Invitrogen/Molecular Probes, Cat. #H3570). The cells were then imaged by
ArrayScan, and the potency and efficacy of the compounds were determined by
plotting the number of GFP-containing endosomes per cell against
corresponding compound concentration.
19. The measured solubility in 0.1 N HCl, PBS buffer, and simulated intestinal fluid
was >200 ng/mL Tan, H.; Semin, D.; Wacker, M.; Cheetham, J. J. Assoc. Lab
Autom. 2005, 364.
20. Female Lewis rats (250 g, 6–8 wks) were received from Charles River
Laboratories (Wilmington, MA) and allowed to acclimatize for at least one
week before being placed on study. Rats (N = 5/group) were administered
vehicle (20% captisol in water), (+)-11f or (ꢀ)-11f (10 mg/kg in 20% captisol/
water) by oral gavage (10 mL/kg). 4 h Post-dose, animals were sacrificed by
CO2 inhalation, and blood was collected by cardiac puncture. Approximately
1 mL of blood was transferred to a MicrotainerÒ hematology tube containing
11. A study of 245 compounds that progressed through rat or dog tolerability
studies suggested a higher likelihood of attrition, due to an unfavourable safety
profile, for compounds with relatively low tPSA (<75 Å2) and high lipophilicity
(clogP >3) Hughes, J. D.; Blagg, J.; Price, D. A.; Bailey, S.; DeCrescenzo, G. A.;
Devraj, R. V.; Ellsworth, E.; Fobian, Y. M.; Gibbs, M. E.; Gilles, R. W.; Greene, N.;
Huang, E.; Krieger-Burke, T.; Loesel, J.; Wager, T.; Whiteley, L.; Zhang, Y. Bioorg.
Med. Chem. Lett. 2008, 18, 4872.
12. Yoshino, K.; Hori, N.; Hori, M.; Morita, T.; Tsukamoto, G. J. Heterocycl. Chem.
1989, 1039.
13. Niwa, T.; Yorimitsu, H.; Oshima, K. Angew. Chem., Int. Ed. 2007, 46, 2643.
14. Martinelli, J. R.; Watson, D. A.; Freckmann, D. M. M.; Barder, T. E.; Buchwald, S.
L. J. Org. Chem. 2008, 73, 7102.
EDTA (Becton Dickinson, #365973) for CBC analysis and 500 lL of plasma was
placed in a MicrotainerÒ tube containing heparin (Becton Dickinson, #365958)
for subsequent pharmacokinetic analysis (plasma exposure). Differential cell
counts were obtained using an AdviaÒ 120 hematology system (Bayer
Diagnostics).
15. Chiral-phase SFC of (+/ꢀ)-9e separately provided (+)-9e and (ꢀ)-9e. Separation
conditions: Chiralpak AD-H column (21 ꢁ 250 mm), mobile phase: 40% IPA