The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2009.11.112

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions.

Full text of DOI:10.1016/j.bmcl.2009.11.112

Bioorganic & Medicinal Chemistry Letters 20 (2010) 493–498
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides
as allosteric modulators
a
a
a
a
a
Yingcai Wang a, , Xianyun Jiao , Frank Kayser , Jiwen Liu , Zhongyu Wang , Malgorzata Wanska ,
*
Joanne Greenberg b, Jennifer Weiszmann b, Hongfei Ge b, Hui Tian b, Simon Wong c, Ralf Schwandner d,
Taeweon Lee b, Yang Li b,
*
a Department of Chemistry, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
b Department of Metabolic Disorders, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
c Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
d Amgen Research GmbH, Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids
(SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of
phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis
in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as
pharmacological tools for exploring the potential function of FFA2 in various disease conditions.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 22 October 2009
Revised 18 November 2009
Accepted 20 November 2009
Available online 26 November 2009
Keywords:
FFA2
GPR43
Allosteric
Agonist
Phenylacetamide
Free fatty acid receptor 2 (FFA2), also known as GPR43, is a
member of a subfamily of closely related G-protein-coupled recep-
tors (GPCR) that can be activated by free fatty acids (FFA) of various
chain lengths.1,2 The other members of the subfamily, FFA1
(GPR40) and FFA3 (GPR41), as well as FFA2, cluster at chromosome
19q13.1 in humans and share ꢀ30–40% sequence identity.3 Each
subfamily member shows a distinct structure–activity relationship
(SAR) for FFAs, with short-chain fatty acids (SCFAs, six or fewer car-
bon molecules) activating FFA2 and FFA3, and medium- and long-
chain fatty acids activating FFA1.4 Acetate and propionate are the
that FFA2 expression is induced during adipocyte differentiation
and that activation of FFA2 by acetate resulted in inhibition of adi-
pocyte lipolysis in vitro and reduction of plasma FFA levels in
vivo.11 Given the similarity in effects on adipocyte function to
the nicotinic acid receptor, GPR109A, and the proposed mechanism
for the improvement in lipid profiles observed by niacin treatment
via inhibition of lipolysis,12 we were interested in the potential
utility of targeting FFA2 for the treatment of dyslipidemia and
other metabolic disorders. Clearly, the identification of pharmaco-
logical tools and better understanding of the receptor function will
facilitate the development of any potential therapies targeting this
receptor.
most potent natural ligands for FFA2 that can induce coupling to
5–7
both G and G .
aq
a
i
Since GPCRs have served as successful targets for a wide range
of therapeutic molecules,8,9 and given the important physiological
effects of FFAs, this subfamily of receptors has sparked great inter-
est in recent years as potential novel targets for various diseases.2
FFA2 expression has been reported to be enriched in islets, various
leukocyte populations, adipocytes, and the gastrointestinal tract
suggesting its potential role in conditions associated with inflam-
mation and metabolic disorders.5–7,10,11 We have recently shown
The potencies of SCFAs for FFA2 are low, in the high micromolar
to millimolar concentrations.2 In order to identify better pharma-
cological tools to study the physiological functions of the receptor
and its involvement in various diseases, we set out to search for
more potent FFA2 agonists. Here, the discovery, synthesis, and
SAR of phenylacetamides, a novel series of FFA2 agonists are re-
ported. The most promising compounds were also profiled for their
pharmacokinetic properties.
A high-throughput screening (HTS) campaign of our internal
sample collection led to the identification of compound 1, which
activated FFA2 with a better than 100-fold improvement in
* Corresponding authors.
E-mail addresses: yingcaiw@amgen.com (Y. Wang), yangl@amgen.com (Y. Li).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.112

494
Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 493–498
Table 1
FFA2 activities for SCFAs and compound 1 and its chiral components 2 and 3
chiral
H
H
H
N
S
N
N
S
S
+
separation
O
N
O
N
O
N
Cl
Cl
Cl
1
2
3
Compds
Human FFA2 cAMP IC50
,
l
Ma
Human FFA2 cAMP efficacyb (%)
Acetate
Propionate
1
2
3
120
130
0.8
0.7
>100
100
100
98
100
<30
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13 for assay protocol.
Efficacy relative to acetate.
b
H
H
H
HO
N
N
N
S
S
S
H
OH
OH
OH
GS
N
N
O
N
O
N
O
N
Cl
Cl
Cl
O
S
Gluc
OH
OH
Cl
M1
M2
M3
M4
Figure 1. Metabolic profiling of compound 2.
potency in a forskolin-induced cAMP assay (Table 1).13 Chiral
separation of racemic 1 into its enantiomers 2 and 3 revealed that
the activity resides in the (S) configuration (compound 2).14
Unfortunately, compound 2 displayed a poor pharmacokinetic
profile in male Sprague-Dawley rats (2: Cl = 6.4 L/h/kg, AUC =
Table 2
FFA2 activities of amide derivatives
79 lg h/L; 0.5 mg/kg iv) and poor microsomal stability (2% remain-
R
ing at 30 min with rat liver microsomes). Metabolic profiling of
compound 2 in rat and human liver microsomes identified two
metabolic soft spots (Fig. 1). First, hydroxylation was seen on the
isopropyl group (M1). Oxidation of the C@C bond followed by
hydrolysis (M2), glucuronidation (M3) and glutathione conjuga-
tion (M4) were identified on the thiazole.
To quickly identify good in vivo tool compounds, we set out to
improve the PK profiles by focusing our chemistry efforts on mod-
ifications in the amide region and the isopropyl group. The SAR is
described here first, followed by the discussion on improvement
of PK profiles.
Cl
Compds
R
Human FFA2 cAMP
IC50
Human FFA2 cAMP
efficacyb (%)
, l
Ma
H
N
S
S
2
4
0.7
100
37
O
O
N
N
N
O
84
S
S
A brief survey of the amide region was conducted, including re-
versed amide (5), sulfonamides (9 and 10), and acid 8 (Table 2).
However, none of the efforts yielded a replacement for the amide.
Compound 4 showed that N-methylation of the amide resulted in a
significant loss of activity, thus we focused on secondary amides
for our following efforts.
Subsequently, the SAR of the thiazolylamine portion was stud-
ied in more detail (Table 3). The primary amide (11) and alkyl
amides (12–14, and analogs not shown) lose most of the activity.
Phenyl amides (15 and analogs not shown) are generally not very
active. However, 2-pyridyl amides (18–20) as well as the pyrida-
zin-3-yl amide (23) proved to be notably potent.
Exploration of five-membered heterocycles resulted in many
interesting findings (Table 4). Incorporating thiadiazoles (26 and
27) and a few benzofused heterocycles (35–37) yielded compounds
with good activities. Notably a CF3 group can enhance the activity by
sixfold on the 1,3,4-thiadizole (compound 26 vs 25). An aromatic
ring is not required, as exemplified by compound 33.
5
>100
<30
N
H
N
N
6
>100
>100
>100
>100
<30
<30
<30
<30
OMeN
H
N
S
7
N
OH
8c
9c
O
H
N
S
S
S
O O
N
O O
S
10
>100
<30
N
H
N
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
for assay protocol.
b
The effect of substituents on the thiazole was studied in some-
what more detail (Table 5). The 4-position appears to be less toler-
Efficacy relative to acetate.
c
Racemic mixture.

Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 493–498
495
Table 3
Table 4
Amide SAR I: alkyl and six-membered aromatic rings
Amide SAR II: five-membered rings
H
N
H
N
R
R
O
O
Cl
Cl
Compds
R
Human FFA2 cAMP
IC50
Human FFA2 cAMP
efficacyb (%)
Compds
R
Human FFA2 cAMP
IC50
Ma
Human FFA2 cAMP
efficacyb (%)
, l
Ma
,
l
11c
12
13
14
15
H
>100
11
>100
>100
>100
<30
66
<30
<30
<30
S
O
S
Me
t-Bu
Bn
Ph
2
0.7
100
92
N
N
N
24
25
25
12
16
17c
18
19
56
40
73
92
84
115
N
N
N
S
49
26
27
28
CF3 1.9
115
99
N
N
N
N
N
N
N
N
N
N
S
1.4
1.1
2.0
20
N
H
N
F
F
55
H
N
29c
>100
<30
N
20
1.3
53
93
N
30
31c
32
33
>100
36
<30
90
N
N
21c
22c
23
50
NH
N
O
5.7
2.3
115
101
S
N
N
N
N
57
48
N
S
2.1
109
N
N
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
S
for assay protocol.
34
20
96
91
b
Efficacy relative to acetate.
Racemic mixture.
c
O
S
35c
3.0
N
N
ant of substituents, especially those of bigger size (39–41). How-
ever, the 5-position can tolerate many groups, irrespective of their
electronic properties (43–48).
Although compounds 49 and 50 (Table 5) may suggest that po-
lar groups are not preferred, a more careful study revealed that
many polar groups are tolerated (Table 6, compounds 57, 60–63).
O
36
4.3
5.2
88
84
Cl
H
N
37c
Turning our attention to the a-substitution revealed that small
N
alkyl groups are preferred (Table 7). The most desired alkyl groups
have three or four carbons. The (R)-configured isomers are gener-
ally much less active than the corresponding (S)-enantiomers.
Geminal dialkyl groups were explored in an effort to remove the
stereocenter (Table 8). Notably, cyclobutyl (77) appears to be a
good replacement and demonstrated that a stereocenter is not re-
quired to maintain agonist activity.
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
for assay protocol.
b
Efficacy relative to acetate.
Racemic mixture.
c
The phenyl SAR was briefly studied as shown in Table 9.
Replacement of the chlorine with a sulfone (81) was not tolerated
and some pyridines (82 and 83) are less active.
By blocking the two metabolic soft spots of compound 2,
namely the isopropyl group and the amide region, we were able
to improve the PK properties significantly (Table 10). The AUC in-
creased steadily in both rat and mouse as X was changed from H to
F then Cl and Ph, and when R was changed from an isopropyl to a t-
butyl group.
Compounds15 were synthesized as illustrated by synthesis of
compound 58 (Scheme 1). Peterson olefination of (1,3-dithian-2-
yl)trimethylsilane16 with 1-(4-chlorophenyl)-2,2-dimethylpropan-
1-one (84) delivered 2-(4-chlorophenyl)-3,3-dimethylbutanoic acid
(86) as a racemic mixture after methanolysis/hydrolysis.17 Chiral

496
Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 493–498
Table 5
Table 7
Amide SAR III: thiazoles
a-Substitution SAR
R
H
N
H
S
N
S
R1
X
O
N
O
N
Cl
Cl
R2
Compds
R
X
Human FFA2 cAMP
IC50
Ma
Human FFA2 cAMP
efficacyb (%)
,
l
Compds
R1
R2
Human FFA2 cAMP
Human FFA2 cAMP
efficacyb (%)
IC50, l
Ma
65
66
67
68
2
69
51
70
71
72
73
74
H
Me
Et
n-Pr
i-Pr
c-Pr
t-Bu
i-Bu
c-Pr-CH2
H
H
H
H
H
F
>100
46
<30
83
2
H
H
0.7
1.4
17
100
87
123
45
<30
97
99
103
100
95
105
98
5.0
5.5
0.7
1.2
0.7
0.6
8.8
170
1.1
105
108
100
103
91
68
72
53
88
38
39
40
41c
42
43
44
45
46
47
48
49
50
H
H
H
H
Me
Me
F
Me
CF3
t-Bu
3-Pyr
Me
H
H
H
H
H
7.9
>100
1.8
0.8
0.5
0.7
1.5
2.0
2.0
79
F
H
H
H
H
H
MeOCH2CH2
allyl
Cl
CF3
CN
CO2Me
CO2H
CONH2
4-Cl-Ph
>100
<30
H
H
H
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
42
59
for assay protocol.
71
b
Efficacy relative to acetate.
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
for assay protocol.
b
Efficacy relative to acetate.
Racemic mixture.
Table 8
Gemi-dialkyl substitutions
c
R
R
H
N
S
X
O
N
Cl
Table 6
Amide SAR IV: 5-thiazoles
Compds
R, R
X
Human FFA2 cAMP
IC50
Human FFA2 cAMP
efficacyb (%)
, l
Ma
H
N
75
76
77
78
79
Me, Me
c-Pr
c-Bu
c-Pentyl
c-Hexyl
F
H
F
H
H
50
>100
1.5
5.0
8.5
57
<30
89
88
81
S
R
O
N
Cl
a
Compds
R
Human FFA2 cAMP
Human FFA2 cAMP
Values are means of four experiments, standard deviation is 30%. See Ref. 13
IC50
,
l
Ma
efficacyb (%)
for assay protocol.
b
Efficacy relative to acetate.
51
52
53
54
55
56
57
58
59
60
61
62
F
Cl
Br
CN
CF3
SMe
SO2Me
Ph
4-Cl-PhCH2
NMe2
Morpholine
4-
0.7
1.0
0.7
1.5
1.5
0.5
3.4
0.7
25
92
97
95
106
95
109
107
111
100
112
111
113
ligands bind to the receptor at a site distinct from binding site of
SCFAs.20 Because of the structural difference of compound 58 com-
pared to compounds 2 and 44, we also tested the combination of
acetate and compound 58 in forskolin-induced cAMP assays, and
found that the addition of 58 significantly left-shifted acetate dose
responses (Fig. 2). On the other hand, the addition of 58 did not
change the IC50 of compound 2 in the cAMP assay (Fig. 3).
These findings suggest that these phenylacetamides bind to an
allosteric site on the receptor and induce positive cooperativity
with natural ligands. Molecular modeling analysis of FFA2 based
on the human b2-adrenergic receptor structure and subsequent
mutagenesis studies revealed potential non-overlapping binding
sites for the endogenous and synthetic ligands, further providing
insight into the nature of the allosteric interactions.20,21 Allosteric
modulators present a new avenue for potential therapeutic inter-
vention on FFA2. They may potentially offer greater receptor selec-
tivity by binding to non-conserved regions of the receptor among a
family of related receptors and may offer other potential advanta-
ges such as differential effects on tachyphylaxis and efficacy.22,23
Therefore, positive allosteric agonists offer an attractive therapeu-
tic approach for the activation of GPCRs.
1.6
1.6
3.8
Methylpiperazine
63
64
2.4
1.2
118
113
N
SO2
OH
CF3
CF3
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
for assay protocol.
b
Efficacy relative to acetate.
separation gave the (S)-isomer 87.18 The acid 87 was then coupled
with 5-phenylthiazol-2-yl amine through its acid chloride to obtain
compound 58, under a condition (step d) where no racemization was
observed.19
Since our compounds can also activate mouse FFA2, with the
We have previously shown that compounds 2 and 44 are allo-
steric agonists of FFA2, and we have suggested that these synthetic
potency of 1.0, 1.4, and 0.7
respectively, in cAMP response in CHO cells stably expressing
lM for compounds (2, 43 and 44),

Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 493–498
497
Table 9
Phenyl SAR
a
b
S
OH
O
H
S
O
Cl
Cl
Cl
O
N
S
R
F
84
85
86
O
N
H
N
c
d
Compds
R
Human FFA2 cAMP
Human FFA2 cAMP
OH
S
IC50
,
l
Ma
efficacyb (%)
O
N
Cl
Cl
87
58
69
80
1.2
103
108
<30
Cl
Scheme 1. Reagents and conditions: (a) (1,3-dithian-2-yl)trimethylsilane, butyl-
lithium, THF, 0–23 °C, 3 h, 95%; (b) (i) mercury(II) chloride, MeOH/H2O, 85 °C, 2 h;
(ii) LiOH, EtOH/H2O, 23 °C, 16 h, 60%, two steps; (c) chiral separation on OJ-H, 45%;
(d) DMF, oxalyl chloride, 2,4,6-collidine, 5-phenylthiazol-2-amine, À15 °C, 2 h, 23%,
>99% ee.
1.1
F3C
81
82
>100
MeO2S
Compound 58
10 µM
50000
40000
30000
20000
10000
0
Cl
N
16
45
120
75
3.33 µM
1.11 µM
0.37 µM
0.12 µM
0.041 µM
control
83
N
a
Values are means of four experiments, standard deviation is 30%. See Ref. 13
for assay protocol.
b
Efficacy relative to acetate.
-8
-7
-6
-5
-4
-3
-2
Table 10
Pharmacokinetic properties
Log[Acetate] M
R
Figure 2. Acetated-mediated cAMP responses in the presence of 58. Values are
means of two experiments, standard deviation is 30%.
H
N
S
X
O
N
Cl
Compound 58
50000
40000
30000
20000
10000
0
Compds
R
X
2
i-Pr
H
44
i-Pr
F
45
i-Pr
Cl
52
t-Bu
Cl
58
t-Bu
Ph
3.33 µM
1.11 µM
0.37 µM
0.12 µM
0.041 µM
control
Rat iva
CL (L/h/kg)
AUC (lg h/L)
MRT (h)
Vdss (L/kg)
6.4
79
1.1
7.0
2.6
194
3.8
9.8
2.0
249
4.2
8.3
0.48
1030
9.0
0.32
1670
9.1
4.4
2.7
Mouse POb
Cmax
(
lg/L)
5
83
62
0.93
0.33
Not tested
569
4550
7.6
1871
18585
9.2
AUC (
l
g h/L)
10
1.8
1.0
MRT (h)
Tmax (h)
2.0
2.0
-10 -9
-8
-7
-6
-5
-4
a
Dose at 0.5 mg/kg.
Dose at 5 mg/kg.
Log[Compound 2] M
b
Figure 3. Compound 2-mediated cAMP responses in the presence of 58. Values are
means of two experiments, standard deviation is 30%.
mouse FFA2, we next tested compound effect on adipocyte lipoly-
sis. Our compounds inhibited lipolysis in 3T3L1 adipocytes in a
concentration-dependent manner (Fig. 4). Such inhibition was sen-
sitive to pertussis toxin treatment,20 indicating that this was the
20
18
16
14
12
10
8
6
4
2
0
result of activating the G coupled signaling pathway, similar to
a
i
our previously reported effects of acetate and propionate on FFA2
in adipocytes.11
The acute effects of compound 44 on plasma FFA were tested in
C57BL6 mice (Fig. 5).24 Compound 44 reduced the plasma FFA level
in wild type (WT) mice but not FFA2 knock-out (KO) mice25 similar
to our previous observation on the effects of acetate on FFA levels
in mice,11 suggesting that these synthetic ligands could serve as
in vivo pharmacological tools to probe the receptor function as
well. More importantly, we showed11 that the activation of FFA2
by acetate did not induce flushing, an adverse effect associated
with the activation of GPR109A.26
Compound 2
Compound 43
Compound 44
-1
0
1
2
Log[Compounds] µM
Figure 4. Inhibition of lipolysis in adipocytes. Values are means of three experi-
ments, standard deviation is 30%.

498
Y. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 493–498
WT
FFA2 KO
140
120
100
80
140
120
100
80
**
60
60
40
40
0 min
20
20
15 min
0
0
Figure 5. Compound 44 reduces plasma FFA levels in WT but not KO mice. Ten animals per group. **p Value less than 0.01 using T test.
11. Ge, H.; Li, X.; Weiszmann, J.; Wang, P.; Baribault, H.; Chen, J.-L.; Tian, H.; Li, Y.
Sustained reduction in plasma FFA was reported to improve
Endocrinology 2008, 149, 4519.
glucose tolerance in patients.27 Given the similarity in activity on
adipocytes to GPR109A, and the beneficial effects of nicotinic acid
treatment on raising high-density lipoprotein (HDL) levels,
improvements in multiple cardiovascular risk factors, and overall
reduction in mortality,12 FFA2 could also potentially function to
modulate aspects of metabolic disorders. These results suggest a
potential role and possible advantage for FFA2 in regulating plasma
lipid profiles and perhaps aspects of the metabolic syndrome.
In summary, we discovered phenylacetamides as the first class
of non-SCFA agonists of FFA2. These novel FFA2 allosteric modula-
tors induce positive cooperativity with natural SCFAs. Our com-
pounds inhibit adipocyte lipolysis in vitro and reduce plasma FFA
levels in vivo, suggesting a similar role of FFA2 to GPR109A. Signif-
icant improvement in rodent PK profiles was achieved through
optimization focusing on metabolic soft spots. These compounds
(e.g., 52 and 58) may serve as good tools for further unraveling
the physiological functions of the receptor and its involvement in
various diseases.
12. Carlson, L. A. J. Intern. Med. 2005, 258, 94.
13. Inhibition of cAMP response was measured in CHO cells stably expressing
human FFA2 via HitHunter cAMP XS assay kit (GE Healthcare). In brief, cells re-
suspended (10,000 in 10
ll/well) in Hank’s balanced salt solution (137 mM
NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, and
1.0 mM MgSO4) with 25 mM HEPES and 0.01% (w/v) BSA were stimulated with
forskolin (5
lM final in 5 ll/well) in the presence of serially diluted test ligands
(5 l/well) in a 384-well Optiplate (Perkin–Elmer) at room temperature for
l
30 min before adding antibody and lysis reagents according to manufacturer’s
protocol. The plates were further incubated in the dark overnight after adding
detection solution, and read in CLIPR (Molecular Devices) for 1 min per plate.
Data were expressed as Relative Luminescence Unit (RLU).
14. Separated on ChiralPak AD-H column using 5% 2-propanol in hexanes.
Retention times are 10.4 min for compound 3 and 13.8 min for compound 2.
15. The purity of final compounds was evaluated by HPLC and determined to be
>95%. The structures were confirmed by 1H NMR and LC–MS.
16. (a) Seebach, D.; Kolb, M.; Grobel, B.-T. Chem. Ber. 1973, 106, 2277–2290; (b)
Seebach, D.; Kolb, M.; Grobel, B.-T. Tetrahedron Lett. 1974, 36, 3171.
17. Chamberlin, A. R.; Nguyen, H. D.; Chung, J. Y. L. J. Org. Chem. 1984, 49, 1682.
18. Separated on ChiralCel OJ-H using 15% 2-propanol in hexanes. Retention times
are 11.8 min for (S)-enantiomer 87 and 17.2 min for its (R)-enantiomer.
19. The chiral center was preserved under the coupling condition (step d in
Scheme 1). The ee (enantiomeric excess) of 58 was determined to be >99%, by
comparing to a mixture of S,R enantiomers (S/R = 3:1) that was obtained by
running the same reaction using DBU (instead of 2,4,6-colidine) at room
temperature. Retention times are 19.7 min for (S)-enantiomer 58 and
35.5 min for its (R)-enantiomer on ChiralPak AD-H using 5% 2-propanol in
hexanes.
Acknowledgments
The authors are grateful to Dr. Randall Hungate and Dr. Julio
Medina for many helpful discussions of this work and to Mr. Scott
Silbiger for editing the manuscript.
20. Lee, T.; Schwandner, R.; Swaminath, G.; Weiszmann, J.; Cardozo, M.;
Greenberg, J.; Jaeckel, P.; Ge, H.; Wang, Y.; Jiao, X.; Liu, J.; Kayser, F.; Tian, H.;
Li, Y. Mol. Pharmacol. 2008, 74, 1599.
21. Swaminath, G.; Jaeckel, P.; Guo, Q.; Cardozo, M.; Weiszmann, J.; Lindberg, R.;
Wang, Y.; Schwandner, R.; Li, Y., manuscript in preparation.
22. Langmead, C. J.; Christopoulos, A. Trends Pharmacol. Sci. 2006, 27, 475.
23. May, L. T.; Leach, K.; Sexton, P. M.; Christopoulos, A. Annu. Rev. Pharmacol.
Toxicol. 2007, 47, 1.
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