Discovery and optimization of a novel series of GPR142 agonists for the treatment of type 2 diabetes mellitus

Article abstract of DOI:10.1016/j.bmcl.2012.07.063

The discovery and initial optimization of a series of phenylalanine based agonists for GPR142 is described. The structure-activity-relationship around the major areas of the molecule was explored to give agonists 90 times more potent than the initial HTS hit in a human GPR142 inositol phosphate accumulation assay. Removal of CYP inhibition by exploration of the pyridine A-ring is also described.

Full text of DOI:10.1016/j.bmcl.2012.07.063

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5942–5947
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a novel series of GPR142 agonists
for the treatment of type 2 diabetes mellitus
Mike Lizarzaburu a, , Simon Turcotte a, Xiaohui Du a, Jason Duquette a, Angela Fu a, Jonathan Houze a,
⇑
Leping Li a, Jinqian Liu a, Michiko Murakoshi b,c, Kozo Oda b,c, Ryo Okuyama b,c, Futoshi Nara b,
Jeff Reagan a, Ming Yu a, Julio C. Medina a
a Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
b Daiichi Sankyo Company Limited, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710, Japan
c Daiichi Sankyo Company Limited, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and initial optimization of a series of phenylalanine based agonists for GPR142 is described.
The structure-activity-relationship around the major areas of the molecule was explored to give agonists
90 times more potent than the initial HTS hit in a human GPR142 inositol phosphate accumulation assay.
Removal of CYP inhibition by exploration of the pyridine A-ring is also described.
Ó 2012 Published by Elsevier Ltd.
Received 7 June 2012
Revised 11 July 2012
Accepted 16 July 2012
Available online 23 July 2012
Keywords:
GPR142 agonist
Type 2 diabetes mellitus
Insulin secretagogue
CYP inhibition reduction
An estimated 135 million people worldwide are affected by
type 2 diabetes mellitus (T2DM), and the number of Americans af-
fected is estimated to range between 11.6 million and 14 million
people.1 Therapies for treating T2DM and T2DM-related conditions
are sought due to the increasing incidence of T2DM. Insulin secre-
tagogue therapy is recognized as an appropriate therapy for T2DM
management when diet and lifestyle modifications fail. Typically,
secretagogue therapy augments circulating insulin levels in pa-
tients with a moderate degree of b-cell dysfunction. Sulfonylureas,
which are prescribed as insulin secretagogues, reduce hyperglyce-
mia when administered to patients with T2DM. However, the long-
lasting effects of some sulfonylureas increase the risk of hypoglyce-
mia; therefore new insulin secretagogues are sought to avoid this
issue.2
With the recent sequencing of the human genome, several puta-
tive G Protein-coupled receptors (GPCRs) have been found in
which the endogenous ligand is unknown (orphan GPCRs). Re-
cently, it was reported that GPR142, highly expressed in pancreatic
b-cells, is a receptor for aromatic amino acids with tryptophan
being the most potent and efficacious.3 It was found that (1) the
receptor is coupled to the Gq signaling pathway, (2) tryptophan
dose-dependently stimulates insulin secretion from isolated
mouse islets in a glucose dependent manner, and (3) mice orally
dosed with tryptophan show improved glucose tolerance when
challenged with an oral glucose load. Based upon these findings,
it was surmised that synthetic agonists to GPR142 might represent
novel therapeutics for the treatment of T2DM. Because GPR142
will only stimulate insulin secretion under conditions of high blood
glucose, it was hypothesized that GPR142 agonists could provide a
benefit over existing therapies as the risk of hypoglycemia would
be greatly reduced.
High throughput screening4 (HTS) at Amgen identified several
GPR142 agonists with moderate potency, including compound 1
(Fig. 1). Compound 1 had an EC50 of 4.8
inositol phosphate (IP) accumulation assay and 1.4
l
M in a human GPR142
M in a mouse
l
GPR142 IP accumulation assay.5 The high level of efficacy (Emax of
100%) for human GPR142 along with the low molecular weight
(334.4) and low ClogP (0.33) made this an attractive lead for opti-
mization. Here, we describe the in vitro optimization of 1 resulting
in several potent GPR142 agonists with significantly reduced inhi-
bition of CYP 3A4 and 2D6.
H
N
O
O
NH2
N
H
N
1
⇑
Corresponding author.
E-mail address: mlizarza@amgen.com (M. Lizarzaburu).
Figure 1. Structure of GPR142 agonist from HTS.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.07.063

M. Lizarzaburu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5942–5947
5943
H
N
H
N
O
O
a
b
O
d or e
from 1
NH2
NH2
N
H
N
N
R
1a
1-12
c
H
N
H
N
O
O
O
O
H
N
R
N
H
N
H
N
N
13
14-18
Scheme 1. Reagents and conditions: (a) HBTU (1.5 equiv), DIEA (3 equiv), DMF, RCH(NHBoc)CO2H (1.2 equiv), 50 °C, 6 h, 55–77%; (b) TFA/DCM 1:1, rt, 2 h, 68–75%; (c) HBTU
(1.5 equiv), DIEA (1.5 equiv), DMF, 3-phenylpropanoic acid (1.2 equiv), 50 °C, 18 h, 67%; (d, 14) Acetic anhydride (5 equiv), DIEA (2 equiv), DCM, rt, 18 h, 79%; (e, 15–18)
RC(O)H (1.0 equiv), sodium triacetoxyborohydride, DCM, rt, 1 h, 25–87%.
Table 1
Table 2
SAR exploration with phenylalanine derivatives
SAR exploration of phenylalanine nitrogen
H
N
H
N
O
O
O
R
R
N
H
N
H
N
N
b
c
Compound
R
h-GPR142, IP EC50
(l
M)a,
Emax (%)
Compound
R
h-GPR142, IP EC50
(l
M)a,b
Emax (%)
c
O
O
NH2
Ph
1
13
NH2
H
4.8
>33
115
NAd
1
4.8
115
H
N
14
>33
NAd
NH2
Ph
O
2
3
13
93
H
N
15
16
0.76
0.83
106
97
O
O
H
N
NH2
>33
NAd
Ph
H
N
17
18
0.78
67
NH2
4
5
>33
>33
30
N
H
N
S
0.093
120
a
b
c
O
O
Ref. 5 for assay protocol.
NAd
Standard deviation for assay based on control compound was 30%.
%Fraction of maximal tryptophan response at 10 mM.
No data available.
NH2
d
X
NH2
6
7
8
9
10
11
12
X = 2-Cl
X = 3-Cl
X = 4-Cl
X = 4-F
X = 4-CN
X = 4-Me
X = 4-OMe
>33
>33
3.8
4.2
4.6
6.6
25
42
50
105
102
79
Compound 13 (Table 2) was synthesized by coupling 5-amino-
[3,40-bipyridin]-6(1H)-one (1a) with phenylpropanoic acid using
HBTU under standard conditions. N-substituted phenylalanine
derivatives 14–18 (Table 2) were synthesized, starting from 1, by
either acylation with acetic anhydride (14) or by reductive amina-
tion with the appropriate aldehyde under standard conditions
using sodium triacetoxyborohydride (15–18).
Synthesis of B-ring derivatives 20–24 (Table 3) began by con-
structing the amino biaryl cores (20b–24b) via a Suzuki coupling
of pyridine-4-boronic acid and the corresponding commercially
available aryl bromides 20a–24a followed by reduction of the nitro
group using Pd on carbon under an atmosphere of hydrogen
(Scheme 2). The amino biaryl intermediates 20b–24b were cou-
73
40
a
b
c
See Ref. 5 for assay protocol.
Standard deviation for assay based on control compound was 30%.
%Fraction of maximal tryptophan response at 10 mM.
No data available.
d
Exploration of the lead structure started with phenylalanine
derivatives (Scheme 1). The synthesis of compounds 1–12 (Table 1)
began by coupling the appropriate Boc protected amino acid with
commercially available 5-amino-[3,40-bipyridin]-6(1H)-one (1a)
under standard HBTU coupling conditions. Deprotection of the
Boc group with TFA in DCM gave the final primary amine products.
pled to Boc-L-phenylalanine under standard HBTU coupling condi-
tions.6 The target compounds 20–24 were completed by TFA
mediated Boc deprotection of intermediates 20c–24c followed by
reductive amination using sodium triacetoxyborohydride with thi-

5944
M. Lizarzaburu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5942–5947
Table 3
B-ring SAR
a Suzuki coupling of 3-bromoaniline (Ar1) or 5-bromo-2-methoxy-
pyridin-3-amine (Ar2) with the appropriate boronic acid followed
by HBTU-mediated coupling with Boc-L-phenylalanine to provide
intermediates 26b–28b & 33b.
O
N
H
N
B
S
N
H
The second method started by first forming amide intermedi-
ates 25a and 34a by HBTU mediated coupling of Boc- -phenylala-
N
L
nine with 3-bromoaniline (Ar1) or 5-bromo-2-methoxypyridin-3-
amine (Ar2), followed by Suzuki coupling with the appropriate
boronic acid to provide intermediates 30b–32b and 34b–35b.
The intermediate, devoid of the A-ring (25b), was synthesized by
hydrogenolysis of aryl bromide 25a using Pd/C under an atmo-
sphere of hydrogen gas. The pyrazole intermediate 29b was con-
structed by reduction of the nitro group in commercially
available 5-(3-nitrophenyl)-1H-pyrazole (29a) using Pd/C under
an atmosphere of hydrogen gas, followed by standard HBTU cou-
a,b
Compound B-ring
h-GPR142, IP EC50
M)a,b
Human serum EC50
(lM)
(l
H
N
O
18
0.093
0.50
N
19
20
21
0.36
0.29
0.97
1.0
pling with Boc-L-phenylalanine as describe above. All the target
N
compounds 25–35 were completed by deprotection of intermedi-
ates 25b–35b under acidic conditions, followed by sodium triacet-
oxyborohydride mediated reductive amination with thiazole-4-
carbaldehyde.
0.088
0.089
OMe
With the understanding that GPR142 is a receptor for aromatic
amino acids, SAR exploration started with the
tion of the molecule (Table 1). The unnatural
was only three times less active than 1. However, other changes
to the -phenylalanine portion of the molecule lead to complete
loss in activity (>33 M), including the removal of the benzylic
methylene (3), saturation of the aromatic ring (4), and the com-
plete removal of the side-chain (5). Since the requirement for
phenylalanine within this series was now demonstrated, a focused
L
-phenylalanine por-
OMe
D-phenylalanine 2
22
23
0.053
0.24
0.81
0.98
L
l
MeO
L-
HO
O
effort was made on exploring substituted
tives. This exploration began with chloro
L
-phenylalanine deriva-
24
0.11
0.48
L-phenylalanine deriva-
tives. The 2-Cl (6) and the 3-Cl (7) derivatives had significantly
diminished potency (>33 M) relative to 1 whereas the potency
of the 4-Cl derivative (8) was slightly improved. With this informa-
tion, more 4-substituted -phenylalanine derivatives were ex-
l
a
Ref. 5 for assay protocol.
Standard deviation for assay based on control compound was 30%.
b
L
plored. Derivatives with an electron withdrawing substituent in
the 4-position maintained their potency as exemplified by the 4-
F derivative (9) and the 4-CN derivate (10). The lipophilic-electron
rich 4-Me derivative (11) also maintained its potency. In contrast,
the polar electron-rich 4-OMe derivative (12) had reduced po-
tency. From this exploration it was determined that 4-Cl and 4-F
azole-4-carbaldehyde. Compound 19 was synthesized in an analo-
gous fashion to compounds 20–24, starting with 4-(pyridin-4-
yl)pyrimidin-2-amine (19b) purchased from a commercial source.
The A-ring derivatives of compounds 18 and 20 (Table 4) were
synthesized via one of two methods (Scheme 3). The first method
began by constructing the amino biaryl cores (26a–28a & 33a) via
L-phenylalanine could be used in future analogs instead of L-
phenylalanine.
B(OH)2
1) a
R
NO2
c
R
N
2) b
Br
NH2
N
20a-24a
20b-24b
O
N
O
1) d
2) e
H
N
R
S
R
NHBoc
N
N
H
H
N
N
20-24
20c-24c
N
N
1) c
2) d
3) e
N
O
N
H
S
N
NH2
N
N
H
N
N
19b
19
Scheme 2. Reagents and conditions: (a) pyridin-4-ylboronic acid (2 equiv), Pd(PPh3)4 (0.2 equiv), DMF/NaHCO3 satd. aq. (4/1), Cs2CO3 (3.0 equiv), 70 °C, 16 h, 53–95%; (b)
10% Pd/C wet (0.5 equiv), H2 (1 atm), MeOH, 16 h, 95–100%; (c) HBTU (1.5 equiv), DIEA (3 equiv), DMF, Boc- -phenylalanine (1.2 equiv), 50 °C, 6 h 31–77%; (d) TFA/DCM 1:1,
rt, 2 h, 65–78%; (e) thiazole-4-carbaldehyde (1.0 equiv), sodium triacetoxyborohydride, DCM, rt, 1 h, 36–87%.
L

M. Lizarzaburu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5942–5947
5945
Table 4
SAR exploration of A-ring
ence of 100% human serum (502 nM, 100% Emax) and stimulated
insulin secretion from isolated mouse islets (2.7
M, 88% Emax).7
l
Next the influence of the pyridone ring on GPR142 activity in
buffer and in the presence of 100% human serum was investigated
(Table 3). Replacement of the pyridone with a pyrimidine ring (19)
provided a compound with only a fourfold reduction in in vitro po-
tency (360 nM). This indicated that the pyridone amide was not
essential for potency and would allow manipulations in this area
of the molecule. Next, the pyridone ring was replaced with a phe-
nyl ring (20). Although the phenyl ring retained a good level of po-
tency, it was observed that a reduction of polarity in this part of the
molecule resulted in a compound with a greater shift in the pres-
ence of 100% human serum. Addition of polarity to compound 20
in an attempt to reduce the serum shift was next explored. The
electron rich 6-methoxy phenyl 21 was equipotent to compound
20; and the 5-methoxyphenyl 22 was slightly more potent than
compound 20, however both of these derivatives had a high serum
shift when compared to compound 18. A methoxy group at the 4
position of the phenyl ring (23) proved to be detrimental to the po-
tency. Placing a carboxylic acid at the 5 position of the phenyl ring
(24) slightly diminished the potency but kept the shift in human
serum low, similar to that of compound 18.
It is known that unencumbered basic nitrogens of heterocycles,
in particular pyridines and imidazoles, bind to the heme portion of
CYP enzymes, causing inhibition which can lead to undesirable
drug-drug interactions.8 As suspected, compounds 18 and 20,
which both contained a sterically unencumbered pyridine, had
high levels of CYP inhibition (Table 4). With this in mind, it was
decided to look for a replacement of the A-ring pyridine first with
the synthetically more accessible phenyl B-ring and subsequently
combine the most interesting A-rings with the pyridone B-ring
(Table 4). To test the hypothesis that the pyridine ring was the ma-
jor cause of the CYP inhibition, it was replaced with a phenyl ring
(32) or completely removed (25). As predicted, compound 32 had a
dramatic reduction in CYP inhibition when compared to compound
20. Complete removal of the phenyl ring (25) reduced the CYP inhi-
bition even further when compared to compounds 20. While both
changes successfully reduced CYP inhibition, the GPR142 activity
was also significantly reduced.
Next, 2-substituted pyridines were investigated in the hope that
the steric bulk would reduce the CYP inhibition. Indeed, the 2-
methyl pyridine A-ring (26) not only improved the potency
(52 nM), but also led to a moderate reduction in the CYP inhibition.
The 2-amino pyridine A-ring 27 had a significant reduction in CYP
inhibition but also had a slight loss in potency. Next, a series of
compounds in which the pyridine A-ring was replaced with a pyr-
azole were evaluated. The 3-substituted 1-methyl-1H-pyrazole 28
had lower CYP inhibition, but was much less potent than 20. On the
other hand, the 5-substituted 1H-pyrazole 29 had a good balance
of potency (67 nM) and reduced CYP inhibition as compared to
compound 20. The 4-substituted 1-methyl-1H-pyrazole 30 was
three times less potent than 20, and, much like 3-substituted 1-
methyl-1H-pyrazole 28, had lower CYP inhibition than 20. The 4-
substituted 1H-pyrazole 31 was also three times less potent than
20, but in this case did not lower the CYP inhibition.
Next, the A-rings which gave the best balance of high potency
and low CYP inhibition in the phenyl B-ring series were combined
with the pyridone B-ring. It was gratifying to see that the 2-methyl
pyridine A-ring (33) improved the potency slightly (67 nM) com-
pared to compound 18. As expected, this change dramatically re-
duced the level of CYP inhibition. More interestingly, compound
33 had a significantly lower level of CYP inhibition than the corre-
sponding analogue with the phenyl B-ring (26). This is presumably
due to an increase in the polarity of the pyridone ring (ClogP of
33 = 1.39) versus the phenyl ring (ClogP of 26 = 3.6). The pyrazole
ring was also explored in combination with the pyridone B-ring,
O
N
H
N
S
B
N
H
A
Compound A–ring
B–ring
h-GPR142, IP
Cyp Inh. (3 lM)
3A4/2D6 (%)
a,b
EC50
(
l
M)
20
0.067
97/95
N
25
26
27
28
H
3.3
24/36
63/74
38/42
30/12
0.052
0.21
1.9
N
N
H2N
N
N
H
N
29
0.067
59/36
N
N
30
31
32
0.18
0.18
0.64
36/42
82/90
57/46
N
N
HN
H
N
O
O
O
O
18
33
34
35
0.093
0.067
0.20
97/95
22/20
18/29
24/21
N
N
H
N
H
H
N
N
N
H
N
N
0.23
N
a
Ref. 5 for assay protocol.
b
Standard deviation for assay based on control compound was 30%.
With the phenylalanine side chain now initially explored, atten-
tion was turned to the phenylalanine amine (Table 2). The impor-
tance of the phenylalanine amine was first explored by synthesis of
the desamino derivative (13) and by eliminating the basicity of the
amine via acetamide formation (14). Both of these changes lead to
a complete loss of potency (>33 lM). It was pleasing to find that a
mono alkyl substituent on the phenylalanine nitrogen gave a sig-
nificant improvement in activity, including derivatives with cyclo-
lM), benzyl (16, 0.83 lM) and phenethyl
(17, 0.78 M). Along these lines, and with a desire to incorporate
propylmethyl (15, 0.76
l
more polar substituents, N-methyl thiazole 18 was identified,
which provided an h-GRP142 agonist with potency of 93 nM and
an Emax of 120%. Compound 18 also had good potency in the pres-

5946
M. Lizarzaburu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5942–5947
O
a
b
Ar
Ar
NHBoc
R
NH2
R
N
H
Br
Br
NH2
Ar1
26a-28a; Ar1
26b-28b; Ar1
30b-32b; Ar1
33b-35b; Ar2
33a; Ar2
or
N
O
O
b
a or c
Ar
NHBoc
Br
N
H
NH2
Ar2
25a; Ar1
34a; Ar2
O
O
NHBoc
NHBoc
NHBoc
d
Br
N
H
N
H
25a
25b
1) d
2) b
O
H
H
N
N
NO2
N
H
N
R
N
29a
29b
H
N
O
O
N
O
N
H
S
H
1) e or f
2) g
N
S
N
N
H
R
N
H
25-32
33-35
Scheme 3. Reagents and conditions: (a) ArB(OH)2 (2 equiv), Pd(PPh3)4 (0.2 equiv), DMF/NaHCO3 satd aq (4/1), Cs2CO3 (3 equiv), 70 °C, 16 h, 40–95%; (b) HBTU (1.5 equiv),
DIEA (3 equiv), DMF, Boc- -phenylalanine (1.2 equiv), 50 °C, 6 h, 40–75%; (c) ArB(OR)2 (1.2 equiv), Pd2(dba)3 (0.05 equiv), Xphos (0.2 equiv), t-AmylOH, K3PO4 (3.0 equiv),
L
100 °C, 16 h, 50%; (d) 10% Pd/C wet (0.5 equiv), H2 (1 atm), MeOH, 16 h, 95–97%; (e, 25–32) TFA/DCM 1:1, rt, 2 h, 65–83%; (f, 33–35) 1 N HCl/dioxane 1:1, 80 °C, 2 h; (g)
thiazole-4-carbaldehyde (1.0 equiv), sodium triacetoxyborohydride, DCM, rt, 1 h, 25–83%.
in vivo clearance (Table 5). It should be noted that the issue of low
stability in liver microsomes was addressed by the incorporation of
a carboxylic acid in the B-ring (24, Table 3) but this compounds
had very low bioavailability and high in vivo clearance due to glu-
curonidation of the carboxylic acid.11
In summary, a novel series of GPR142 agonists was identified.
Initial efforts improved the potency of the HTS hit from 4.8 lM
to 67 nM in a human GPR142 IP accumulation assay. Furthermore,
the inhibition of CYP 3A4 and 2D6 was significantly reduced.
Although low in vivo exposure has precluded the use of these com-
pounds in proof-of-concept experiments, their promising in vitro
properties has justified further exploration.12
Table 5
Rat pharmacokinetic properties of key analogues
Compound
Cl (h/L/kg)a
Vss (L/kg)a
MRT (h)a
%Fb
RLMc (%)
24a,b
33ab
2.9
2.6
0.5
0.75
0.2
0.3
0
19
74
11
a
b
c
Compounds were dosed iv at 0.5 mg/kg.
Compounds were dosed po at 2.0 mg/kg.
See Ref. 10 for assay protocol.
but proved to be less effective. The 5-substituted 1H-pyrazole 34
and the 4-substituted 1-methyl-1H-pyrazole 35 both had lower
CYP inhibition with the pyridone B-ring versus the phenyl B-ring,
but none of these derivatives were as potent as the 2-methyl pyr-
idine A-ring making their utility limited. These pyrazole deriva-
tives also illustrate that the increase in polarity of the pyridone
ring versus the phenyl ring leads to a reduction in CYP inhibition.
It is interesting to note that the incorporation of the carboxylic acid
on the B-ring (24, Table 3) was another method of lowering the
CYP inhibition.9
References and notes
1. Koopman, R. J.; Mainous, A. G., III; Diaz, V. A.; Geeseey, M. E. Ann. Fam. Med.
2005, 3, 60–63.
2. Gangji, A. S.; Goldsmith, C. H.; Cukierman, T.; Clase, C. M.; Gerstein, H. C.
Diabetes Care 2007, 2, 389.
3. Xiong, Y.; Motani, A.; Reagan, J.; Gao, X.; Yang, H.; Ma, J.; Schwandner, R.;
Zhang, Y.; Liu, Q.; Miao, L.; Luo, J.; Tian, H.; Chen, J-L.; Murakoshi, M.; Nara, F.;
Yeh, W.-C.; Cao, Z. manuscript is in preparation.
4. Xiao, S.-H.; Reagan, J. D.; Lee, P. H.; Fu, A.; Schwandner, R.; Zhao, X.; Knop, J.;
Beckmann, H.; Young, S. W. Comb. Chem. High Throughput Screening 2008, 11(3),
195–215.
From the early optimization of this lead, compound 33 emerged
as the top compound in this series. This compound had good po-
tency for h-GPR142 in both buffer (67 nM, 90% Emax) and in the
presence of 100% human serum (290 nM, 97% Emax). The CYP inhi-
5. Inositol Phosphate Accumulation Assay-HEK293 cells were dispensed into a
poly-D-lysine tissue culture treated 96 well plate at a density of 25,000 cells per
well. The next day, the cells (ꢀ80–90% confluent) were transfected with 100 ng
receptor plasmid per well using Lipofectamine2000 according the
manufacturer’s instructions. Six hours after transfection the media was
replaced with inositol free DMEM/10% dialyzed FCS supplemented with
bition IC50 was >30 lM for isoforms 3A4 and 2D6. A major issue
with 33 was poor stability in human/rat liver microsomes (10%/
11% remaining after 30 min incubation)10 which translated to high
1 lCi/mL tritiated inositol. After incubation overnight, the cells were washed

M. Lizarzaburu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5942–5947
5947
once in HBSS and then treated with the 100
l
L HBSS/0.01% BSA containing
Elkins, P. A.; Gardiner, C-M.; Garver, E.; Gilbert, S. A.; Gontarek, R. R.; Jackson, J.
R.; Kershner, K. L.; Luo, L.; Raha, K.; Sherk, C. S.; Sung, C. M.; Sutton, D.;
Tummino, P. J.; Wegrzyn, R. J.; Auger, K. R.; Dhanak, D. ACS Med. Chem. Lett.
2010, 1, 39–43.
various concentrations of test compounds (prepared as above in DMSO),
10 mM LiCl and incubated at 37 °C for 1 h. The media was aspirated and the
cells were lysed with ice cold 20 mM formic acid. After incubation at 4 °C for
5 h, the lysate were added to yttrium silicate SPA beads, allowed to settle
overnight and read on a Beckman TopCount scintillation counter. In measuring
the EC50 with serum, HBSS/0.01% BSA was replaced with100% human serum.
6. Protection of the carboxylate in 24b as the methyl ester was necessary to
prevent homocoupling in the next step. The methyl ester in 24b was
deprotected by standard saponification conditions using LiOH in THF/water,
9. IC50 >30
10. Microsome stability were measured in a high throughput format by incubating
inhibitors at 1 m concentration with rat(r) or human (h) microsomes and the
lM for all isoforms tested (3A4, 2D6 and 2C9).
l
percentage of the parent compounds remaining were measured after 30 min of
incubation by liquid chromatography/mass spectrometry analysis.
11. Measured clearance in rat hepatocytes was 18 (ml/min/10ꢁ6 cells) which
equals a half-life of 39 min.
after it was coupled to Boc-L-phenylalanine.
7. Mouse islets were run with 16.7 mM Glucose, 0.625% HAS and 1% DMSO. For
details of assay conditions and mouse islet isolation see Lin, DC -H; Zhang, R.;
Li, F.; Nguyen, K.; Chen, M.; Tran, T.; Lopez, E.; Lu, J. Y. L.; Li, X. N.; Tang, L.;
Tonn, G. R.; Swaminath, G.; Reagan, J. D.; Tian, H.; Lin, Y.-J.; Houze, J. B.; Luo, J
PLoS ONE 2011, 6, e27270. http://dx.doi.org/10.1371/journal.pone.0027270.
8. Knight, S. D.; Adams, N. D.; Burgess, J. L.; Chaudhari, A. M.; Darcy, G. M.;
Donatelli, A. C.; Luengo, J. I.; Newlander, K. A.; Parrish, C. A.; Ridgers, L. H.;
Sarpong, M. A.; Schmidt, S. J.; Van Aller, G. S.; Carson, J. D.; Diamond, M. A.;
12. (a) Du, X.; Kim, Y-J; Lai, S-J.; Chen, X.; Lizarzaburu, M.; Turcotte, S.; Fu, Z.; Liu,
Q.; Zhang, Y.; Motani, A.; Oda, K.; Okuyama, R.; Nara, F.; Murakoshi, M.; Fu, A.;
Reagan, J.D.; Fan, P.; Xiong, Y.; Shen, W.; Li, L.; Houze, J.; Medina, J.C. Bioorg.
Med. Chem. Lett. accepted for publication; (b) Yu, M.; Lizarzaburu, M.; Motani,
A.; Fu, Z.; Du, X.; Liu, J.; Jiao, X.; Lai, S-J.; Fu, A.; Lin, D.; Liu, Q.; Murakoshi, M.;
Nara, F.; Oda, K.; Okuyama, R.; Reagan, J.; Turcotte, S.; Zhang, Y.; Houze, J.;
Medina, J.C.; Li, L. manuscript in preparation to ACS Med. Chem. Lett.