Synthesis, biological evaluation, X-ray structure, and pharmacokinetics of aminopyrimidine c-jun-N-terminal kinase (JNK) inhibitors

Article abstract of DOI:10.1021/jm901351f

Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors with good cell potency and good brain penetration properties. The structure-activity relationships (SAR) around a series of aminopyrimidines were evaluated utilizing biochemical and cell-based assays to measure JNK inhibition and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC₅₀ values < 100 nM. Moreover, compound 9l showed an IC₅₀ = 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rats along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901351f

J. Med. Chem. 2010, 53, 419–431 419
DOI: 10.1021/jm901351f
Synthesis, Biological Evaluation, X-ray Structure, and Pharmacokinetics of Aminopyrimidine
c-jun-N-terminal Kinase (JNK) Inhibitors
Ted Kamenecka, Rong Jiang, Xinyi Song, Derek Duckett, Weimin Chen, Yuan Yuan Ling, Jeff Habel, John D. Laughlin,
Jeremy Chambers, Mariana Figuera-Losada, Michael D. Cameron, Li Lin, Claudia H. Ruiz, and Philip V. LoGrasso*
Department of Molecular Therapeutics, and Translational Research Institute, The Scripps Research Institute, Scripps Florida,
130 Scripps Way A2A, Jupiter, Florida 33458
Received September 14, 2009
Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in
neurodegenerative disorders, we set out to develop highly selective JNK inhibitors with good cell
potency and good brain penetration properties. The structure-activity relationships (SAR) around a
series of aminopyrimidines were evaluated utilizing biochemical and cell-based assays to measure JNK
inhibition and brain penetration in mice. Microsomal stability in three species, P450 inhibition,
inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also
measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based
IC50 values < 100 nM. Moreover, compound 9l showed an IC50 = 0.8 nM for inhibition of ROS and
had good pharmacokinetic properties in rats along with a brain-to-plasma ratio of 0.75. These results
suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first
small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.
Introduction
14 days and prevented behavioral consequences compared to
untreated mice.3 This profound protection correlated with a
decrease in c-jun phosphorylation and illustrated the benefit
of JNK inhibition as a potential neuroprotective agent for
stroke. Like the PD model, Jnk3 knockout mice also showed
protection against cerebral hypoxic ischemia injury in mice.
Jnk3 knockout mice showed only 28% neuronal tissue loss
compared to 48% for wild type mice subjected to unilateral
hypoxic-ischemia injury.4 Interestingly, JNK3 is almost ex-
clusively expressed in the brain, with only low level expression
seen in the heart and testis,5 suggesting a potential unique role
for this isoform in central nervous system (CNS) disorders.
Moreover, numerous reports have implicated JNK as a key
regulator of oxidative stress and neuronal death as a result of
reactive oxygen species generated in cell models of PD utiliz-
ing 6-hydroxy dopamine or MPTP/MPPþ.6-9 Combined, all
of these data are good validation for JNK as a target in CNS
disease.
From a chemistry perspective, numerous JNK selective
inhibitors have begun to emerge and include compounds
from classes such as indazoles,10,11 aminopyrazoles,11 amino-
pyridines,12,13 pyridine carboxamides,13,14 benzothien-2-yl-
amides and benzothiazol-2-yl acetonitriles,15,16 quinoline de-
rivatives,17 and aminopyrimidines.18,19 For a recent review of
all these classes, see LoGrasso and Kamenecka.20 All of these
compounds classes, with the exception of the indazoles, have
shown selectivity for JNK over p38, but few have demon-
strated good brain penetration, a feature essential for CNS
therapeutics. The well-described clinical toxicity of p38 in-
hibition necessitates this selectivity in any JNK inhibitor
program.21 The only compound class mentioned above to
show brain penetration was the benzothiazol-2-yl acetonitrile,
represented by AS601245, which was shown to be efficacious
Compelling evidence has surfaced over the past eight years
supporting JNK asa goodtherapeutic target for the treatment
of neurodegenerative disease. Indeed, numerous reports uti-
lizing either knockout mice or a peptide derived from the
JNK-interacting protein (JIPa) have shown that loss of JNK
activity is protective in animal models of neurodegeneration.
For example, in 2001, Xia et al. showed that stereotactic
adenoviral transfer of residues 127-281 from JIP into the
striatum prevented loss of dopaminergic neurons in the sub-
stantia nigra pars compacta (SNpc) and also increased levels
of striatal dopamine in mice subchronically treated with 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).1 Three
years later, Flavell and colleagues showed that Jnk2, Jnk3,
and especially Jnk2/3 knockout mice were resistant to acute
MPTP intoxication where these mice showed significantly less
loss of dopaminergic neurons in the SNpc and also increased
levels of striatal dopamine compared to wild type mice treated
with MPTP.2 In a similar fashion, Borsello et al. showed that a
20 amino acid JIP peptide fused to the 10-amino acid HIV Tat
transporter system delivered by intraventricular injection to
adult mice subjected to transient middle cerebral artery
occlusion (MCAO) reduced lesion volume by 90% for at least
*To whom correspondence should be addressed. Phone: 561-228-
2230. Fax: 561-228-3081. E-mail: lograsso@scripps.edu.
a Abbreviations: ATP, adenosine triphosphate; AMP-PCP, β,γ-
methyleneadenosine 50-triphosphate; CDK2, cyclin-dependent kinase
2; CM-H2DCFDA, 5-(and-6)-chloromethyl-20,70-dichlorofluorescin
diacetate, acetyl ester; CNS, central nervous system; IL-2, inteleukine-
2; INS-1, insulinoma-1; JIP, JNK-interacting protein; JNK, c-jun-N-
terminal kinase; MCAO, middle cerebral artery occlusion; MPTP, 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PSA, polar surface area;
PD, Parkinson’s disease; ROS, reactive oxygen species; SNpc, substan-
tia nigra pars compacta, STZ, streptozotocin.
r
2009 American Chemical Society
Published on Web 11/30/2009
pubs.acs.org/jmc

420 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Kamenecka et al.
Scheme 1. General Procedure for the Synthesis of Boronic
Scheme 2. General Procedure for the Synthesis of Anilinesa
Estersa
a Reagents and conditions: (a) morpholine, 120 °C; (b) Pd(dppf)Cl2-
(CH2Cl2), KOAc, bis(pinacolato)diboron, (OR0 = pinacol), DMSO.
a Reagents and conditions: (a) substituted triazole, K2CO3, DMF; (b)
H2 or SnCl2.
intransient globalischemia models in gerbils, albeit atip doses
g60 mg/kg.22,23 More recently, aminopyrimidines similar in
structure to those presented in our current work have been
reported for peripheral applications such as inflammatory
disorders18 and type II diabetes mellitus.19 In the study by
Alam et al., the key selectivity struggle was versus cyclin-
dependent-kinase-2 (CDK2), where phenyl-substituted pyr-
azolopyridines were single digit nanomolar JNK2 and JNK3
inhibitors showing no inhibition of CDK2 up to 10 μM.18
Thus, while these compounds are selective versus p38 and
potent JNK inhibitors, it is unclear if they are suitable for
CNS penetration, as they were not designed with these para-
meters in the desired compound profile.
The current study was designed to develop JNK3 inhibitors
which were selective over p38, had cell-based potency for
inhibition of phosphorylation of c-jun near 100 nM, showed
functional protection versus oxidative stress, had good phar-
macokinetic properties, and had a brain:plasma ration greater
than 0.5. These goals were achieved by biaryl substitution of
an aminopyrimidine core. Structural features which were
especially important for maintaining cellular potency and
achieving brain penetration were substitutions which included
1,2,4-morpholino substituted triazoles as represented by com-
pound 9l. The X-ray crystal structure of 9l revealed this class
of inhibitors to bind in the ATP pocket of JNK3.
available boronic acids proceeded uneventfully to provide
biaryl intermediates (8). In many instances, the crude pro-
ducts after aqueous workup were clean enough to proceed to
the final step in the synthesis; however, analytical samples
were purified by silica gel chromatography to obtain the
appropriate spectral data. Coupling of the 2-chloropyrimi-
dine products with 4-substituted anilines proceeded under
two different reaction conditions. The reactants could either
be heated in aqueous ethoxyethanol at 120 °C overnight or in
anhydrous ethoxyethanol at 190 °C for 1 h. Products were
typically precipitated out of the reaction mixture by addition
of excess water and were sufficiently pure for in vitro testing
or could be purified by silica gel chromatography or reverse-
phase preparative HPLC.
Structure-Activity Relationships Affecting Biochemical
and Cell-Based Potency for Aminopyrimidines. Table 1 high-
lights the structures for 12 compounds and presents the
biochemical IC50 values against JNK1, JNK3, and p38,
along with the cell-based IC50 for inhibition of c-jun phos-
phorylation in INS-1 cells treated with streptozotocin (STZ).
These 12 compounds were selected from among 500 amino-
pyrimidines synthesized in our program. All of the
compounds had biochemical IC50 values < 500 nM versus
JNK3. In addition, all 12 compounds had IC50 > 20 μM
versus p38. Substitution of the amide at R4 (compound 9a)
with a 1,2,4-triazole (compound 9b) showed no change in the
biochemical IC50 versus JNK3 (Table 1). The two most
potent compounds, 9d and 9e, had IC50 values = 7 and
4 nM, respectively. Key structure-activity relationship
(SAR) elements driving the in vitro potency of compounds
9d and 9e were the addition of methyl sulfonamide at the R2
position. Replacement of the sulfonamide moiety in the R4
position of compound 9d by a 1,2,3-triazole as seen in 9e had
no effect on JNK3 or JNK1 inhibition (Table 1). Converting
the 1,2,3-triazole at the R4 position of 9c to a 3-methyl-1,2,4-
triazole (9f) had no effect on JNK 3 and JNK1 biochemical
inhibition, nor did it affect cell-based inhibition of c-jun
phosphorylation (Table 1). When a nitrile group was added
to the R3 position (9g), an approximate 5-fold increase in
potency for JNK3 and JNK1 inhibition was seen compared
to 9f and an approximate 3-fold improved potency was
translated to the cell-based readout; inhibition of c-jun
phosphorylation (Table 1). When a 2-methyl-1,2,3,4,-tetra-
zole (9h) replaced the 3-methyl-1,2,4-triazole (9g), a less than
2-fold difference in IC50s was seen for JNK3, JNK1, and
c-jun phosphorylation (Table 1). Finally, 3-pyridyl (9i and
9j) or 3-morpholino (9l) substitutions to the 1,2,4-triazole
at position R4 had cell-based IC50s approximately 5-fold
more potent than N-methyl piperazine substituted at that
position (9k).
Results
Synthesis of Aminopyrimidine JNK Inhibitors. Disubsti-
tuted boronate esters (3) were prepared as described in
Scheme 1. Commercially available aryl fluorides (1) were
heated in neat morpholine to provide clean SNAr-substitu-
tion products (2). Quenching these reactions with water
typically resulted in precipitation of product in analytically
pure form. Conversion of the aryl bromides to boronate
esters was done under standard literature conditions as
shown in Scheme 1. Other boronic acids used were commer-
cially available from a variety of vendors.
Aniline synthesis was equally straightforward and is de-
scribed in Scheme 2. Treatment of 4-fluoro-1-nitrobenzene (4)
with substituted triazoles resulted in clean conversion to sub-
stitution products (5). Reduction of the nitro group to amino
was typically accomplished via hydrogenation in the presence
of a suitable palladium catalyst to provide anilines (6). Where
R = Br, nitro reduction was carried out by tin-mediated
reduction, without loss of the bromine atom. The bromine
atom then served as a functional group handle with which to
introduce additional groups (both aromatics and amines) later
in the synthesis as described in the Experimental Section.
Synthesis of final compounds as described in Table 1 is
described in Scheme 3. Suzuki coupling of 2,4-dichloropyri-
midine with synthesized boronate esters or commercially

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 421
Table 1. Biochemical IC50s for Inhibition of JNK1, JNK3, p38, and Cell-Based Inhibition of c-jun for Aminopyrimidinesa
a The biochemical IC50 values are the averages of three or more experiments. The cell-based IC50 values are the averages of two or more experiments.
All standard deviations e39%.
Table 2. X-ray Crystallography Data Collection and Refinement Statistics
Scheme 3. General Procedure for the Synthesis of Substituted
Pyrimidinesa
data collection
space group
resolution range (A)
model refinement
˚
C2221
resolution range (A) 24-2.4
22.0
26.9
˚
68-2.4
Rworkb(%)
Rfreeb(%)
98.6(87.5) rmsd bonds (A)
no. of unique reflections 13329
˚
completeness (%)
Rsyma (%)
0.021
2.107
31.973
6.2
44.7 (2.4)
P
rmsd angles (deg)
2
mean B value (A )
˚
I/σ
P
P
P
a Rsym
Rfree represents a 5% subset of reflections excluded from refinement.
=
hkl (|(Ii - I)|)/ (I). b R = (||Fo| - |Fc||)/ (|Fo|) where
a Reagents and conditions: (a) Pd(PPh3)4, 2 M K2CO3 and DME,
90 °C, 10 h, (OR = pinacol or OH); (b) 4-substituted aniline and
2-ethoxyethanol, 120 °C, 10 h.
Values in parentheses represent the highest resolution shell.
lack of potential donor and acceptor groups on the ligand.
In addition, the highly planar nature of 9l fit nicely into
a groove largely composed of hydrophobic residues but
did not cause movement of the Met146 side chain to the
back of the ATP binding pocket as seen for other JNK
inhibitors.12,26 A final feature of this structure was that the
Crystallography of JNK3-Compound 9l Complex. Utiliz-
ing steady state kinetics we had previously shown compound
9a to be an ATP competitive inhibitor of JNK3,24 and
preliminary unpublished crystallography data from our lab
at the time indicated compound 9a bound in the ATP pocket
˚
(unpublished results). In this work, the structure of 9l-soaked
˚
JNK3 crystals were solved at 2.4 A and refined to final Rwork
Gly-rich loop appeared to drop by ∼2.5 A, forming a more
/
compressed active site compared to Merck compound 1
(PDB ID 1PMN) from Scapin et al.26 The rmsd for this
Rfree of 22.0/26.9 with further refinement statistics presented
in Table 2. Figure 1a presents the crystal structure of JNK3
with 9l bound in the ATP pocket and highlights eight key
amino acid residue side chains previously shown to have
essential binding contributions for JNK inhibitors.11,12,25,26
The aminopyrimidine portion of 9l overlaid nicely with the
˚
˚
region of the protein was 1.95 A compared to 1.39 A for the
entire protein. To further illustrate this difference, Figure 1b
presents the backbone trace of JNK3 (gray) complexed with
compound 9l overlaid with the backbone of JNK3 (green)
from Scapin et al.26 The glycine-rich loop containing residues
˚
˚
adenosine portion of AMP-PCP (rmsd = 0.51 A), and the
I70-V78 was collapsed inward by ∼2.5 A compared to the
morpholino group at R1 was found to overlay with the ribose
of AMP-PCP as well. A striking feature about this structure
was that 9l did not appear to have any hydrogen bond
interactions with any residues in JNK3 due to the relative
glycine-rich loop (green) of JNK 3 from 1PMN.26 The space
filling model presented in Figure 1c further illustrates the
planar nature of compound 9l and the hydrophobic interac-
tions which dominate compound 9l binding to JNK3.

422 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Kamenecka et al.
Figure 1. X-ray structure of JNK3 with compound 9l. (A) The ATP binding pocket of JNK3 (gray) is shown in schematic ribbon form and key
residues are shown stick form. Compound 9l binding is shown. (B) Backbone trace of JNK3 (gray) complexed with compound 9l overlaid with
the backbone trace of JNK3 (green) from Scapin et al.26 (C) Space filling model of JNK3 with compound 9l bound.
Table 3. P450 Inhibition and Microsomal Stability of Key Amino-
stability of 9l for all three species was reasonable, rivaling
pyrimidine JNK Inhibitors
that of 9j, especially in rat and human (Table 3). We also
P450% Inhibition @ 10 μMa
microsomal stabilityb (min)
determined the plasma protein binding for 9l in mouse, rat,
dog, monkey, and human (92%, 98%, 96%, 96%, and 92%,
respectively).
compd
2C9
2D6
3A4
1A2
mouse/rat/human
Rat Pharmacokinetics and Mouse Brain Penetration for
Key Aminopyrimidines. Table 4 presents the pharmacoki-
netic parameters for compounds dosed at 1 mg/kg iv and
2 mg/kg po in rats and the brain and plasma levels at 2 h of
those same compounds dosed at 10 mg/kg ip in mice. Three
of the four compounds had clearance rates e20 mL/min/kg
with compound 9j, having a very low Clp = 3 mL/min/kg
(Table 4). Compound 9h had the poorest overall pharmaco-
kinetic properties with relatively high clearance (44 mL/min/
9f
9h
9j
9l
43
7
4
10
-4
4
48
15
33
89
39
-1
15
5/17/18
26/11/17
51/47/29
21/41/26
-2
75
70
a P450 inhibition assays examine the inhibition of selective marker
reactions: CYP1A2 phenaceten (40 μM) f acetaminophen; CYP2C9,
tolbutamide (130 μM) f hydroxytolbutamide; CYP2D6, bufuralol
(40 μM) f 40-hydroxybufuralol; CYP3A4, midazolam (5 μM) f
10-hydroxymidazolam. b 2 mg/mL mouse, rat, or human liver micro-
somes were used in the stability studies.
kg), low oral exposure (AUC = 0.24 mM h), and low oral
3
bioavailability (%F = 14) (Table 4). Compound 9l had the
best oral bioavailability with %F = 45. Three of the four
compounds showed brain:plasma ratios g50%, with com-
pound 9h having brain:plasma = 117%. Compound 9l had
the second best brain:plasma ratio at 75%, with compound
9j having the worst brain:plasma ratio at 10% (Table 4).
Inhibition of ROS Generated by STZ Treatment of INS-1
Cells by Compound 9l. To determine if the inhibitors had
impact on JNK-mediated physiological responses, such as
ROS generation, we examined STZ-induced ROS generation
in INS-1 cells to be congruent with our phospho c-jun cell-
based assays. Figure 2a presents the microscopic detection of
ROS generated from INS-1 cells treated with STZ for 4 h in
the absence or presence of seven concentrations of com-
pound 9l. Rotenone (data not shown) was used as a positive
control for ROS generation and showed equivalent levels of
ROS detected by the cell-permeable indicator 5-(and-6)-
chloromethyl-20,70-dichlorofluorescin diacetate, acetyl ester
P450 Inhibition and Microsomal Stability of Key Amino-
pyrimidines. Table 3 presents the cytochrome P450 inhibition
of four human enzymes (2C9, 2D6, 3A4, and 1A2) at 10 μM
compound along with the mouse, rat, and human micro-
somal stability. Compound 9f showed <50% inhibition for
all four P450 enzymes but had relatively low microsomal
stability, especially in mice (Table 3). Addition of the nitrile
group to R3 and the tertrazole group to R4 in compound 9h
improved microsomal stability in mice to 26 min and reduced
P450 inhibition to e15% for all four enzymes (Table 3).
Compound 9j had the best microsomal stability in all species
for the four compounds highlighted and also had a good
P450 inhibition profile with e33% inhibition for all four
enzymes (Table 3). Compound 9l had the worst P450 inhibi-
tion profile of the four compounds presented, with three
of the four enzymes (2C9, 3A4, and 1A2) showing between
70 and 90% inhibition at 10 μM. However, the microsomal

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 423
Table 4. Rat Pharmacokinetics and Mouse Brain Penetration of Key Aminopyrimidine JNK Inhibitors
rata
mouseb [μM]
compd
Clp (mL/min/kg)
t1/2 (h)
Vd (L/kg)
oral AUC (μM h)
oral Cmax (μM)
%F
[plasma]
[brain]
3
9f
9h
9j
9l
10
44
3
0.8
1.9
3.0
2.4
0.4
3.3
0.6
2.5
2.0
0.24
5.9
1.4
0.95
0.07
0.5
25
14
27
45
11.2
2.4
1.0
6.0
6.2
2.8
0.1
4.5
20
0.3
a 1 mg/kg iv; 2 mg/kg po. b 10 mg/kg ip 2 h.
neurodegenerative models. Thus, the SAR described in this
report needed to balance all of these challenges.
Structure-Activity Relationships Affecting Biochemical
and Cell-Based Potency for Aminopyrimidines. The key
SAR element which gave rise to improved potency for both
JNK3 and JNK1 in the biochemical assays was addition of
the methyl sulfonamide at R2 to compounds 9d and 9e
(Table 1). Indeed, compound 9e was 91-fold more potent
versus JNK3 than compound 9c (which had H at R2) despite
the fact that compound 9c had the potency enhancing
addition of the morpholino group at R1. Moreover, numer-
ous analogues to compound 9e were made containing alkyl,
amide, amine, and morpholino substitutions (data not
shown) at position R2 and none of these analogues had
IC50s nearly as potent as compound 9e, suggesting that
methyl sulfonamide substitution at this position was un-
iquely potency enhancing. It is unclear why compound 9e is
more potent than other analogues made. It is interesting to
speculate that one reason compound 9e was more potent
than compound 9l was that 9e could potentially hydrogen
bond to the backbone of Met 149 as seen for many JNK
inhibitors, while compound 9l as seen in Figure 1 did not
have this interaction. A crystal structure of 9e would be
needed to address this hypothesis and see if other interac-
tions such as H-bonding with Lys 93 or even Gln 155 or Asn
152 was possible for the sulfonamide moiety. Despite the
improved biochemical potency of 9e on JNK3 and JNK1,
there was not an improved potency in the cell-based inhibi-
tion of c-jun phosphorylation assay owing to the relatively
poor cell penetration of the methyl sulfonamides. This
liability was further manifested in the brain penetration of
methyl sulfonamide analogues, which showed very poor
brain penetration. Improvements in cell potency came with
addition of morpholino to R1 compared to H and addition
of either morpholino or pyridyl substitutions on the triazole
at R4. It is also likely that morpholino substitution at R1
contributed to enhanced solubility of compounds 9f-9l.
The biochemical potency of the compounds described in
Table 1 is similar to those seen for other aminopyrimidines
reported.18,19 The most potent phenyl-substituted pyrazolo-
pyridine analogue of the aminopyrimidines (compound 30 in
their manuscript) reported by Alam et al. had biochemical
IC50s for JNK2 and JNK3 at 5 nM, respectively, and 22 nM
for JNK1.18 This compares favorably to compounds 9e
(Table 1) in our work where IC50s for JNK3 and JNK1 were
4 and 16 nM, respectively. It was suggested by Alam et al., by
preliminary docking studies, that the phenyl group in com-
pound 30 accessed the hydrophobic pocket in JNK3 and this
contributed to the potency.18 Confirmation of this was not
reported by crystal structure work. The most striking differ-
ence between the aminopyrimidines reported in our current
work compared to that of Alam et al.18 is that despite the
single digit nanomolar biochemical potency reported for
compound 30, the cell-based IC50 for inhibition of c-jun
Figure 2. Compound 9l inhibits STZ-induced intracellular ROS
generation in INS-1 cells. (A) Fluorescent micrographs of H2O2
generation detected by H2DCFDA. The absence of STZ treatment
shows no ROS generation and 4 mM STZ treatment in the absence
of compound 9l shows maximum ROS generation. Seven concen-
trations of compound 9l are shown in the presence of 4 mM STZ. (B)
The normalized relative fluorescence units (RFU) for a 12-point
dose response curve for compound 9l is presented. The data are
from four biological replicates with each dose repeated in duplicate.
The IC50 presented is for the average of the four experiments ( the
standard error of the mean (SEM).
(CM-H2DCFDA), as was seen for the STZ only (panel 2
of Figure 2a). Figure 2b presents the IC50 determination of
compound 9l for the inhibition of ROS from the average of
four biological replicates (with each concentration repeated
in duplicate). These results showed a signal-to-background
of three and that compound 9l was a potent inhibitor of ROS
generation in INS-1 cells.
Discussion
Given the strong preclinical validation for JNK as a target
in neurodegenerative disease,1-4 we set a goal of developing
selective JNK inhibitors which were potent in cell-based
assays, had good pharmacokinetic properties, and good brain
penetration to make them viable candidates to probe in vivo

424 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Kamenecka et al.
phosphorylation was only 3.8 μM. This is 18-fold less potent
than compound 9e and 71-fold less potent than compound 9l
reported in our work (Table 1), suggesting better cell pene-
tration of the triazole-substituted aminopyrimidines re-
ported in this work. The 4-substituted-2-aminopyrimidines
reported by Humphries et al. compare a little more favorably
to the compounds reported in this manuscript, where a
4-OH-cycolhexyl analogue (compound 9c from ref 19) had
a biochemical IC50 versus JNK1=12 nM and a cell-based
inhibition of c-jun phosphorylation=98 nM.19
A broader comparison of the biochemical IC50s against
the benzothiazol-2-yl acetonitrile, JNK3 inhibitors, revealed
that compound 9l was equipotent (148 nM for compound 9l
vs 120 nM for AS601245 from Gaillard et al.16) and com-
pound 9e was 30-fold more potent than AS601245. It is
difficult to compare cellular activities of these compounds
because Gaillard et al. did not report cell-based IC50s, but
merely % inhibition at 10 μM compound, where they showed
90% inhibition of inteleukin-2 (IL-2) release in Jurkat cells at
this concentration. In 2006, Szczepankiewicz et al. reported a
series of aminopyridine-based JNK inhibitors which showed
suitability for in vivo use. Of the many compounds reported,
four were highlighted (compounds 6o, 6s, 18b, and 35)
and they had cell-based IC50s values =920, 1300, 650, and
1750 nM, respectively. Compound 9l was 12-fold more
potent in cells than the most potent compound highlighted
by Szczepankiewicz et al. and 32-fold more potent than the
least potent compound highlighted.
While many of these JNK inhibitors have been character-
ized for selectivity against broad panels of kinases, few have
reported the selectivity of these compounds versus p38R.13,16
For the aminopyrimidines presented by Alam et al.,18 the
major focus for selectivity was versus CDK-2, so comparison
of selectivity over p38 in their work cannot be made with the
compounds reported here. Similarly, Humphries et al. did
not report the IC50 for their compounds versus p38R but did
do so for p38β, where they showed that compound 9c was
∼1000-fold selective over p38β.19 If this selectivity holds for
p38R as well, which one can speculate is highly likely, then
the compounds presented by Humphries et al. compare
favorably in their p38 selectivity to those reported in Table 1.
The well described toxicity of p38 inhibitors necessitates this
desired selectivity in any JNK inhibitor program.27
The lack of a shift in cell-based IC50 for compounds 9f, 9g,
9i, 9j, 9k, and 9l (Table 1) compared to biochemical IC50
versus JNK1 is of interest. In contrast to compound 9e,
which showed a 13-fold decrease in potency for the c-jun
phosphorylation cell-based assay compared to the JNK1
biochemical IC50, the aforementioned compounds were es-
sentially equipotent in the biochemical and cell-based assays.
One possible interpretation for the increased potency in the
cells for these compounds could be that they have very slow
off rates from the enzyme and hence have greater cell-based
potency. This has been seen for other ATP competitive
kinase inhibitors, including those for p38 where the bio-
chemical and cell-based IC50s were nearly identical.21,28 It is
less likely that there is another enzyme which phosphorylates
c-jun in the cells, as very few enzymes beyond JNK have been
reported to utilize c-jun as a substrate. Moreover, broad
counterscreening of 3 μM 9l against a panel of 400 kinases
showed this class to be highly selective, with only 11 out of
400 kinases showing greater than 70% biochemical inhibi-
tion at this concentration and only 28 showing >25%
inhibition. In addition, all of the upstream kinases which
activate the JNK pathway were not inhibited at this con-
centration (MKK7, no inhibition; MKK4, no inhibition;
MLK1-3, no inhibition, MAPK3K1-4, no inhibition, and
ASK-1, no inhibition) by 9l, further implicating direct JNK
inhibition as the likely target in cells. Thus, inhibition of
another putative enzyme which phosphorylates c-jun that
was inhibited by the compounds in Table 1 is unlikely but
cannot be strictly ruled out.
Crystallography of JNK3-Compound 9l Complex. The
binding of compound 9l to JNK3 was unique because of
the lack of any hydrogen bonding in the structure, the
significant contributions from hydrophobic interactions to
the binding, the lack of movement of the gatekeeper Met 146,
˚
and the ∼2.5 A drop of the Gly-rich loop to compress the
active site. A drop in the Gly-rich loop was not seen for the
aminopyrimidines reported by Alam et al.,18 where they
showed more traditional type binding having hydrogen
bonding interactions with Met 149 and steric interactions
of a chlorine atom with gatekeeper Met 146 residue. More-
over, it appeared that compound 6 from Alam et al.18 had
key interactions with Gln 155, which were not present in the
JNK3-compound 9l structure presented in Figure 1. It is
likely that the highly planar nature of compound 9l is
responsible for this movement of the Gly-rich loop as well
as for the lack of the traditional interactions reported with
Met 149 and Gln 155. Indeed, the highly planar compound 3
presented by Scapin et al. also showed a large drop in the
position of the Gly-rich loop upon binding where the less
planar compound 1 from that study did not show this drop.26
Swahn et al. showed that the anilino-bipyridines bound to
JNK3 in the ATP pocket and were able to move the gate-
˚
keeper Met 146 by more than 2 A, giving a few of these
compounds selectivity for JNK3 over JNK1.12 This move-
ment of Met 146 was not seen for compound 9l, nor was it
seen for the compounds in the Alam et al. study and is likely
the reason there is no selectivity for JNK3 over JNK1 for the
compounds in Table 1. Attempts to utilize the Met 146
gatekeeper residue for improved potency and selectivity by
substitution of our aminopyrimidines did not prove helpful.
P450 Inhibition and Microsomal Stability of Key Amino-
pyrimidines. Compounds 9h and 9j had the least P450
inhibition of any of the four compounds presented in Table 3.
Addition of the CN group at R3 of compound 9h was likely
the major contributing factor to improving the inhibition
profile versus 2C9, 3A4, and 1A2 compared to compound 9f.
Like 9h, compound 9j also contained CN at R3 and likely
was the reason for low P450 inhibition of this compound too.
Addition of the 3-pyridyl substitution to the triazole in 9j
further improved the P450 inhibition profile over a 3-mor-
pholino substitution as seen in compound 9l. These obser-
vations suggest a relatively strong interaction of the
3-morpholino substitution in compound 9l with the P450s
2C9, 3A4, and 1A2, which was not found when pyridyl was
at that position. While the 3-morpholino substitution was
detrimental for P450 inhibition, this substitution enhanced
microsomal stability in all species compared to simple methyl
substitution of the triazole (compound 9f). The same en-
hancement in microsomal stability was true for the pyridyl
substitution. Both of these observations are consistent with
the well-known ability of these groups to provide stability
against liver microsomal metabolism of xenobiotics. The
major liability of compound 9l was the high P450 inhibition
seen with the three enzymes noted in Table 3. Future SAR
will be focused on improving this inhibition profile while still

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 425
maintaining all of the potency, selectivity, and PK properties
of this series.
from the significant role JNK plays in pathways which affect
mitochondrial ROS generation. The major source of ROS in
cells is the mitochondria.33 Translocation of JNK to the
mitochondria has been shown, and a possible role for the
kinase in the organelle bioenergetics has been suggested.34,35
Phosphorylation of mitochondrial proteins like Bcl-2, Bcl-
XL, and Bax by JNK has been demonstrated, connecting
JNK pathway directly with mitochondrial dependent apo-
ptosis.34-37 Hence, it is possible that inhibition of JNK not
only decreases activation of the most well-known target of the
kinase (c-jun) but it also inhibits a number of events that
would irreversibly lead to disruption of the mitochondrial
functions and cell death. Finally, despite the good selectivity
profile of compound 9l, it cannot be ruled out that off-target
inhibition may contribute to some of the decreased ROS.
Rat Pharmacokinetics and Mouse Brain Penetration for
Key Aminopyrimidines. Despite the favorable P450 profile
and good microsomal stability of compound 9j, it was found
that this compound did not have good brain penetration
compared to compounds 9f, 9h, and 9l (Table 4). The poor
brain penetration (brain:plasma ratio = 0.1) prohibited this
compound from being considered for further assessment as a
possible in vivo candidate in neurodegenerative models
despite the good overall PK properties of the compound
(Table 4). Brain penetration has been shown to be directly
correlated with the polar surface area (PSA) of a compound
(i.e., the lower the PSA, the greater the brain penetration).29
Comparison of compound 9j, which had a calculated PSA =
118 to compound 9l which had a calculated PSA = 93,
supported the experimental findings where compound 9l had
a brain:plasma ratio = 0.75. Compound 9l also showed good
rat pharmacokinetic parameters with good oral exposure
Summary and Conclusion
As in any medicinal chemistry program, multiple parameters
need to be balanced to obtain a compound with the desired
properties. CNS drugs in particular present a greater challenge,
as one must incorporate brain penetration into the desired
properties while still maintaining potency of the target of
interest, selectivity, good microsomal stability, and good phar-
macokinetic properties. In this report, we have managed to
strike a balance of these properties by designing potent ATP-
competitive JNK inhibitors which had cell-based IC50 values
near 50 nM (compound 9l) while maintaining a 200-fold selec-
tivity over p38 and a broad set of kinases. This broad and high
level of selectivity should help minimize potential side effects of
these compounds. Moreover, these compounds have been
shown to be highly brain penetrant while showing functional
efficacy in reducing ROS production in cells. Given ROS pro-
uction is a central mechanism for cell death in many neurode-
generative disorders, we feel that this class of aminopyrimidines
represents good candidates for testing in vivo models of
neurodegeneration. The major liability of compound 9l is the
high level of inhibition for some of the CYP450 enzymes.
Future work will be focused on improving the CYP450 inhibi-
tion profile and trying to improve cell-based potency.
(AUC = 1.4 mM h at 2 mg/kg dose), good oral bioavail-
3
ability (45%), and reasonable half-life (Table 4). It is likely
that the combination of the fluoro substitution at R3, along
with the 3-morpholino-1,2,4-triazole at R4 provided the
balance needed for achieving good pharmacokinetic proper-
ties while maintaining the brain penetration needed to test
neurodegenerative animal models. Indeed, it has been shown
that polar surface area is positively correlated with both CNS
exposure and oral bioavailability30 and hence it is likely that
compound 9l strikes the balance needed for good perme-
ability to cross the gut and blood-brain barrier, be stable
against liver metabolism, as well as having potent cell
inhibition of JNK. It should be mentioned that despite the
very potent biochemical inhibition of JNK1 and JNK3 by
compound 9e, this compound was not pursued further
because all of the methyl-sulfonamide substituted analogues
we made had very poor brain penetration, which was likely
associated with the very high polar surface area of these
compounds (calculated PSA = 123 for compound 9e). This
compound also had %F = 5, in keeping with the high PSA
value and poor microsomal stability in all species.
Because none of the other JNK inhibitors reported were
designed for CNS therapeutic indications, it is difficult to
compare our compounds to the aminopyrimidines and other
classes of compounds in the literature. It can be inferred,
however, from the stated peripheral indications for these
compounds that it is unlikely those compounds were opti-
mized for brain penetration given the diabetes and inflam-
mation indications cited for those programs.
Inhibition of ROS Generated by STZ Treatment of INS-1
Cells by Compound 9l. The potent inhibition of ROS genera-
tion (IC50 = 0.81 ( 0.32 nM) by compound 9l suggests that
JNK has significant contribution to ROS generation in INS-
1 cells. One potential explanation for this potency is that
JNK activation (by STZ) induces ROS generation and there-
fore the inhibition of JNK prevents ROS generation. Thus
even low levels of JNK inhibition may go a long way in
preventing ROS generation. This possibility is supported by
the findings of Davis and colleagues, who showed that
TNFR-induced ROS production was JNK-dependent and
that almost no ROS production, as measured by CM-
H2DCFDA detection, was seen in jnk-/- fibroblasts.31
Similar findings were reported by Karin and colleagues,
who also showed that TNFR-induced ROS production had
a significant JNK dependence.32 A second possibility comes
Experimental Section
Commercially available reagents and anhydrous solvents were
used without further purification unless otherwise specified.
Reactions were monitored by HPLC with YMC Pack-Pro C18
column (4.6 mm ꢀ 75 mm). Analytical HPLC data were gener-
ated by injecting 5 μL of very dilute sample solution in methanol
or acetonitrile to a reverse phase HPLC system run over 14 min
(5-95% acetonitrile/water with 0.1% TFA in each solvent). The
products were detected by UV in the detection range of 215-
310 nm. All compounds were determined to be >95% pure by
this method. HRMS (electrospray ionization) experiments were
performed with a Thermo Finnigan orbitrap mass analyzer. Data
were acquired in the positive ion mode at a resolving power of
100000 at m/z 400. Calibration was performed with an external
calibration mixture immediately prior to analysis. Thin layer
chromatography (TLC) analyses were performed with precoated
silica gel 60 F254. Flash chromatography was performed on
prepacked columns of silica gel (230-400 mesh, 40-63 μm) by
CombiFlash with EtOAc/hexane or MeOH/CH2Cl2 as eluent.
The purification by preparative HPLC was performed on YMC
Pack-Pro C18 column with CH3CN þ 50%MeOH/H2O þ 0.1%
TFA as eluent. The mass spectra were recorded by LCMS with
the Finnigan LCQ Advantage MAX spectrometer of Thermo
Electron. NMR spectra were recorded with a Bruker 400 MHz
spectrometer at ambient temperature with the residual solvent

426 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Kamenecka et al.
peaks as internal standards. The line positions of multiplets are
given in ppm (δ).
4-(4-Phenylpyrimidin-2-ylamino)benzamide (9a).
7.65-7.62 (m, 1H), 7.52 (d, 1H), 7.42 (t, 1H), 7.16 (d, 1H),
3.81-3.79 (m, 4H), 3.25-3.23 (m, 4H). MS (ESI) 400 (M þ H).
N-(4-(2-(4-(1H-1,2,3-Triazol-1-yl)phenylamino)pyrimidin-4-yl)-
phenyl)methanesulfonamide (9e).
A mixture of 2-chloro-4-phenylpyrimidine (0.10 g, 0.52 mmol)
(Combi-Blocks) and 4-aminobenzamide (0.21 g, 1.6 mmol) in
2-ethoxyethanol (3.0 mL) was heated in a sealed tube at 190 °C
for 1 h (or at 120 °C overnight if aqueous 2-ethoxyethanol was
used). The reaction mixture was cooled to room temperature
and diluted with water. The precipitate was filtered, washed with
water, and dried under air to provide the desired product as a
tan solid >95% pure as judged by analytical HPLC analysis.
q1H NMR (DMSO-d6, 400 MHz) δ 9.9 (s, 1H), 8.6 (d, 1H),
8.2 (br s, 2H), 7-7-8.0 (m, 5H), 7.6 (br s, 3H), 7.5 (d, 1H), 7.2
(br s, 1H). MS (ESI) 291.2 (M þ H).
A mixture of N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)methanesulfonamide (0.85 g, 2.9 mmol), 2,4-dichlor-
opyrimidine (0.84 g, 5.7 mmol), Pd(PPh3)4 (0.33 g, 0.29 mmol),
Na2CO3 (5 mL of 2 M aq solution), and CH3CN (15 mL) was
purged with Ar for 10 min and then heated in a sealed tube at
90 °C overnight. The reaction mixture was cooled to room
temperature and diluted with water and EtOAc. The layers were
separated, and the aqueous layer was extracted with EtOAc (2ꢀ).
The combined organics were dried (MgSO4) and concentrated in
vacuo to give a pale-yellow solid. Trituration of this solid with
Et2O provided N-(4-(2-chloropyrimidin-4-yl)phenyl)methane-
sulfonamide (0.60 g, 37%) which was used without further
purification. 1H NMR (DMSO-d6, 400 MHz) δ 10.3 (br s, 1H),
8.8 (d, 1H), 8.2 (d, 2H), 8.1 (d, 1H), 7.3 (d, 2H), 3.1 (s, 3H).
The title compound was made following the general proce-
dures described for compound 9a using 4-(1H-1,2,3-triazol-1-
yl)aniline. 1H NMR (CDCl3, 400 MHz) δ 1H NMR (DMSO-d6,
400 MHz) δ 10.18 (s, 1H), 9.96 (s, 1H), 8.74 (d, 1H), 8.58 (d, 1H),
8.19 (d, 2H), 8.07 (d, 2H), 7.95 (d, 1H), 7.85 (d, 2H), 7.43 (d, 1H),
7.37 (d, 2H), 3.32 (s, 6H), 3.12 (s, 3H). MS (ESI) 408.0 (M þ H).
4-(4-(4-(Methylsulfonamido)phenyl)pyrimidin-2-ylamino)benzene-
sulfonamide (9d).
N-(4-(1H-1,2,4-Triazol-1-yl)phenyl)-4-phenylpyrimidin-2-amine
(9b).
Compound 9b was obtained following the same general
protocol used in the preparation of compound 9a using
2-chloro-4-phenylpyrimidine and 4-(1H-1,2,4-triazol-1-yl)ani-
line. 1H NMR (DMSO-d6, 400 MHz) δ 9.77 (s, 1H), 8.52
(dd, 1H), 8.34 (t, 1H), 8.14-8.10 (m, 2H), 7.92-7.88 (m, 2H),
7.73-7.70 (m, 2H), 7.64 (d, 1H), 7.53-7.48 (m, 2H), 7.37
(d, 1H), 6.45 (dd, 1H). MS (ESI) 315 (M þ H).
N-(4-(1H-1,2,3-Triazol-1-yl)phenyl)-4-(3-morpholinophenyl)-
pyrimidin-2-amine (9c).
The title compound was made following the general proce-
dures described for compound 9a using N-(4-(2-chloropyri-
midin-4-yl)phenyl)methanesulfonamide and 4-aminobenzene-
sulfonamide. 1H NMR (DMSO-d6, 400 MHz) δ 10.00 (s, 1H),
8.53 (d, 1H), 8.10 (d, 2H), 8.01 (d, 2H), 7.70 (d, 2H), 7.69-7.54
(m, 2H), 7.41 (d, 1H), 7.24 (d, 2H), 7.16 (s, 1H), 2.98 (s, 3H). MS
(ESI) 420 (M þ H).
A mixture of 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)morpholine (2.38 g, 8.23 mmol), 2,4-dichloropyrimi-
dine (1.35 g, 9.05 mmol), Pd(PPh3)4 (0.48 g, 0.41 mmol), K2CO3
(12 mL of 2 M aq solution), and DME (24 mL) was purged with
Ar for 10 min, then heated in a sealed tube at 90 °C overnight. The
reaction mixture was cooled to room temperature and extracted
with EtOAc (2ꢀ). The combined organics were dried (MgSO4)
and concentrated in vacuo to give an oil. Purification of this
material by column chromatography on silica gel (25% EtOAc/
hexane) provided 4-(3-(2-chloropyrimidin-4-yl)phenyl)morpho-
line as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ 8.64 (d, 1H),
7.70 (s, 1H), 7.64 (d, 1H), 7.51-7.47 (m, 1H), 7.41 (t, 1H),
7.10-7.08 (m, 1H), 3.92-3.90 (m, 4H), 3.29-3.26 (m, 4H).
A mixture of 4-(3-(2-chloropyrimidin-4-yl)phenyl)morpho-
line was then coupled with 4-(1H-1,2,3-triazol-1-yl)aniline to
give the title compound as described for compound 9a. 1H NMR
(DMSO-d6, 400 MHz) δ 9.98 (s, 1H), 8.74 (d, 1H), 8.59 (d, 1H),
8.09-8.05 (m, 2H), 7.86 (d, 1H), 7.86-7.82 (m, 2H), 7.76 (s, 1H),
N-(4-(3-Methyl-1H-1,2,4-triazol-1-yl)phenyl)-4-(3-morpholino-
phenyl)pyrimidin-2-amine (9f).
A mixture of 4-fluoro-1-nitrobenzene (0.77 g, 5.5 mmol),
3-methyl-1H-1,2,4-triazole (0.50 g, 6.0 mmol), K2CO3 (0.83 g,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 427
6.0 mmol), and DMF (25 mL) was heated at 75 °C overnight.
The reaction mixture was cooled to room temperature, diluted
with water, and extracted with EtOAc (3ꢀ). The organic layer
was dried (MgSO4) and concentrated to provide a solid. Pur-
ification of this material by column chromatography on silica
gel (40% EtOAc/hexane) provided 3-methyl-1-(4-nitrophenyl)-
1H-1,2,4-triazole as the major product. 1H NMR (CDCl3, 400
MHz) δ 8.59 (s, 1H), 8.39 (d, 2H), 7.88 (d, 2H), 2.53 (s, 3H). MS
(ESI) 205.1 (M þ H). HRMS, MHþ: calculated for C23H23N7O
414.20366; obtained 414.2038.
Compound 9g was prepared from this crude 2-chloropyrimi-
dine and 4-(3-methyl-1H-1,2,4-triazol-1-yl)aniline as previously
described for compound 9a. 1H NMR (DMSO-d6, 400 MHz) δ
9.95(s, 1H), 9.05 (s, 1H), 8.64 (d, 1H), 8.02-7.92 (m, 3H), 7.74
(d, 2H), 7.60-7.55 (m, 2H), 3.81-3.78 (m, 4H), 3.34-3.29
(m, 4H). MS (ESI) 439 (M þ H).
3-(2-(4-(2-Methyl-2H-tetrazol-5-yl)phenylamino)pyrimidin-4-
yl)-5-morpholinobenzonitrile (9h).
A mixture of 3-methyl-1-(4-nitrophenyl)-1H-1,2,4-triazole
(0.24 g, 1.2 mmol), 5% Pt/C, and MeOH (12 mL) was stirred
at room temperature under H2 balloon overnight. The reaction
mixture was filtered through celite and concentrated in vacuo to
provide the desired product as a beige solid which was used
1
without further purification. H NMR (CDCl3, 400 MHz) δ
8.31 (s, 1H), 7.40 (d, 2H), 6.77 (d, 2H), 2.51 (s, 3H).
The title compound was made following the general proce-
dure described for compound 9a using 4-(3-(2-chloropyrimidin-
4-yl)phenyl)morpholine and 4-(3-methyl-1H-1,2,4-triazol-1-
A mixture of 4-(2H-tetrazol-5-yl)aniline (1.13 g, 7.0 mmol),
MeI (1.09 g, 7.7 mmol), and K2CO3 (1.93 g, 14.0 mmol) in
acetone (14 mL) was stirred at 40 °C for 1 h. The reaction mixture
was filtered, and the filtrate was concentrated in vacuo to give a
solid. Purification of this material bycolumnchromatographyon
silica gel (40% EtOAc/hexane) provided 4-(2-methyl-2H-tetra-
1
yl)aniline. H NMR (CDCl3, 400 MHz) δ 8.42 (d, 1H), 8.31
(s, 1H), 7.80 (d, 2H), 7.67 (s, 1H), 7.59 (d, 2H), 7.46 (d, 1H), 7.34
(t, 1H), 7.13 (d, 1H), 7.01 (d, 1H), 3.86-3.84 (m, 4H), 3.21-3.19
(m, 4H), 2.44 (s, 3H). MS (ESI) 414.25 (M þ H).
3-(2-(4-(3-Methyl-1H-1,2,4-triazol-1-yl)phenylamino)pyrimidin-
4-yl)-5-morpholinobenzonitrile (9g).
1
zol-5-yl)aniline as a white solid. H NMR (CDCl3, 400 MHz)
δ 7.94 (d, 2H), 6.77 (d, 2H), 4.37 (s, 3H), 3.90 (brs, 2H).
A mixture of 3-(2-chloropyrimidin-4-yl)-5-morpholinoben-
zonitrile (0.040 g, 0.13 mmol) and 4-(2-methyl-2H-tetrazol-5-
yl)aniline (0.024 g, 0.13 mmol) in 2-ethoxyethanol (0.2 mL) was
heated in a sealed tube at 190 °C for 1 h. The reaction mixture
was cooled to room temperature and diluted with water. The
precipitate was filtered, washed with water, and dried under air
to provide 3-(2-(4-(2-methyl-2H-tetrazol-5-yl)phenylami-
no)pyrimidin-4-yl)-5-morpholinobenzonitrile as a brown solid.
1H NMR (CDCl3, 400 MHz) δ 8.49 (d, 1H), 8.05 (d, 2H), 7.86
(s, 1H), 7.76 (d, 2H), 7.65 (s, 1H), 7.36 (s, 1H), 7.16 (d, 1H),
7.10 (d, 1H), 4.33 (s, 3H), 3.86-3.84 (m, 4H), 3.24-3.22 (m, 4H).
MS (ESI) 440.11 (M þ H). HRMS, MHþ: calculated for
C23H21N9O, 440.19414; obtained, 440.1941.
A mixture of 3-bromo-5-fluorobenzonitrile (5.4 g, 27.0 mmol)
and morpholine (100 mL) was heated in a sealed tube at 120 °C
overnight. When the reaction was done as judged by analytical
HPLC analysis, the reaction was cooled to room temperature
and quenched with water (400 mL). The resulting colorless
precipitate was collected by filtration, dried in vacuo, and used
4-(3-Morpholinophenyl)-N-(4-(3-(pyridin-3-yl)-1H-1,2,4-tria-
zol-1-yl)phenyl)pyrimidin-2-amine (9i).
1
without further purification. H NMR (CDCl3, 400 MHz) δ
7.25 (s, 1H), 7.24 (s, 1H), 7.07 (t, 1H), 3.88 (t, 4H), 3.22 (t, 4H).
MS (ESI) 267.1, 269.1 (Br isotope) (M þ H).
A mixture of 3-bromo-5-morpholinobenzonitrile (4 g, 15.0
mmol), bis(pinacolato)diboron (5.0 g, 19.5 mmol), Pd(dppf)Cl2-
(CH2Cl2) (0.68 g, 0.75 mmol), KOAc (4.4 g, 45.0 mmol), and
DMSO (30 mL) was degassed with argon and then heated in a
sealed tube at 100 °C for 6 h. The reaction mixture was cooled to
room temperature, diluted with water, and extracted with ethyl
acetate (2ꢀ). The combined organics were dried (MgSO4),
filtered through a short pad of silica gel, and concentrated to
give the borate as a tan solid, which was used without further
purification. 1H NMR (CDCl3, 400 MHz) δ 7.49 (s, 1H), 7.45 (d,
1H), 7.10-7.09 (m, 1H), 3.79 (t, 4H), 3.14 (t, 4H), 1.19 (s, 12H).
MS (ESI) 315.2 (M þ H).
3-Bromo-1-(4-nitrophenyl)-1H-1,2,4-triazole was obtained
from 3-bromo-1H-1,2,4-triazole and 4-fluoro-1-nitrobenzene
as described for compound 9g. 1H NMR (CDCl3, 400 MHz) δ
8.60 (s, 1H), 8.44 (d, 2H), 7.91 (d, 2H).
A mixture of 3-bromo-1-(4-nitrophenyl)-1H-1,2,4-triazole
(0.51 g, 1.9 mmol), SnCl2-2H2O (2.14 g, 9.5 mmol), and EtOH
(4 mL) was heated at reflux for 4 h. The reaction mixture was
cooled to room temperature and basified with NaOH (2 M aq)
until pH 7-9. The resulting precipitate was filtered through
celite and washed with EtOH. The EtOH filtrate was concen-
trated in vacuo, and the crude residue was dissolved in water and
extracted with EtOAc (3ꢀ). The combined organics were dried
(MgSO4) and concentrated in vacuo to provide the desired
A mixture of this borate (3.6 g, 11.5 mmol), 2,4-dichloropyr-
imidine (2.2 g, 14.9 mmol), Pd(PPh3)4 (0.66 g, 0.6 mmol),
K2CO3 (20 mL of 2 M aq. solution), and DME (40 mL) was
purged with Ar for 10 min and then heated in a sealed tube at
95 °C for 12 h. The reaction mixture was cooled to room
temperature, diluted with water and EtOAc, and the layers were
separated. The aqueous layer was extracted with EtOAc (2ꢀ).
The combined organics were dried (MgSO4) and concentrated
in vacuo to give a pale-yellow solid that was used without
further purification. 1H NMR (CDCl3, 400 MHz) δ 8.73
(d, 1H), 7.91-7.90 (m, 1H), 7.73-7.72 (m, 1H), 7.65 (d, 1H),
7.27-7.26 (m, 1H), 3.93-3.91 (m, 4H), 3.34-3.31 (m, 4H). MS
(ESI) 301.1 (M þ H).
1
product as a white solid. H NMR (CDCl3, 400 MHz) δ 8.28
(s, 1H), 7.40 (d, 2H), 6.79 (d, 2H), 3.90 (br s, 2H).
A mixture of 4-(3-(2-chloropyrimidin-4-yl)phenyl)mor-
pholine (0.11 g, 0.40 mmol) and 4-(3-bromo-1H-1,2,4-triazol-
1-yl)aniline (0.12 g, 0.48 mmol) in 2-ethoxyethanol (2 mL)/water

428 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Kamenecka et al.
(1 mL) was heated in a sealed tube at 120 °C overnight. The
reaction mixture was cooled to room temperature and diluted
with water. The precipitate was filtered, washed with water,
and dried under air to provide N-(4-(3-bromo-1H-1,2,4-triazol-
4-(3-Fluoro-5-morpholinophenyl)-N-(4-(3-(4-methylpiperazin-
1-yl)-1H-1,2,4-triazol-1-yl)phenyl)pyrimidin-2-amine (9k).
1-yl)phenyl)-4-(3-morpholinophenyl)pyrimidin-2-amine as
a
brown solid. 1H NMR (CDCl3, 400 MHz) δ 8.53 (d, 1H),
8.40 (s, 1H), 7.92 (d, 2H), 7.69 (s, 1H), 7.62 (d, 2H), 7.55
(d, 1H), 7.44 (t, 1H), 7.37 (s, 1H), 7.24 (s, 1H), 7.12 (d, 1H),
3.95-3.93 (m, 4H), 3.31-3.29 (m, 4H). MS (ESI) 478.17 and
480.20 (M þ H).
A mixture of N-(4-(3-bromo-1H-1,2,4-triazol-1-yl)phenyl)-
4-(3-morpholinophenyl)pyrimidin-2-amine (0.034 g, 0.07 mmol),
pyridin-3-ylboronic acid (0.017 g, 0.14 mmol), K2CO3 (0.058 g,
0.42 mmol), Pd(PPh3)4 (0.008 g, 0.007 mmol), toluene (0.40 mL),
and MeOH (0.10 mL) was heated in a sealed tube at 120 °C with
microwave irradiation for 1 h. The reaction mixture was cooled
down to room temperature and filtered. The filtrate was
concentrated to a yellow solid. Purification of this material by
column chromatography on silica gel (80% EtOAc/hexane)
provided 4-(3-morpholinophenyl)-N-(4-(3-(pyridin-3-yl)-1H-
1,2,4-triazol-1-yl)phenyl)pyrimidin-2-amine as a white yellow
solid. 1H NMR (CDCl3, 400 MHz) δ 9.37 (br s, 1H), 8.60 (brs,
1H), 8.48 (s, 1H), 8.44 (d, 1H), 8.39 (d, 1H), 7.83 (d, 2H),
7.64-7.60 (m, 3H), 7.50 (s, 1H), 7.46 (d, 1H), 7.35-7.34 (m, 2H),
7.14 (d, 1H), 7.00 (dd, 1H), 3.84-3.82 (m, 4H), 3.20-3.18
(m, 4H). MS (ESI) 477.34 (M þ H).
A mixture of 1-bromo-3,5-difluorobenzene (8.0 g, 41.5 mmol) in
morpholine (100 mL) was heated in a sealed tube at 120 °C for 30 h
and then concentrated in vacuo. The resulting residue was dissolved
in EtOAc and washed with saturated aqueous NaHCO3 (2ꢀ),
brine (1ꢀ), dried (MgSO4), and concentrated. The resulting pale-
yellow oil slowly crystallized and was judged to be >90% pure by
analytical HPLC analysis. It was used without further purification.
A mixture of 4-(3-bromo-5-fluorophenyl)morpholine (4.38 g,
16.8 mmol), bis(pinacolato)diboron (5.56 g, 21.9 mmol), Pd-
(dppf)Cl2(CH2Cl2) (0.69 g, 0.84 mmol), KOAc (4.96 g,
50.5 mmol), and DMSO (17 mL) was purged with Ar for 3 min
and then heated in a sealed tube at 100 °C overnight. The reaction
mixture was cooled to room temperature, diluted with water, and
extracted with ethyl acetate (2ꢀ). The combined organics were
dried (MgSO4), filtered through a short pad of silica gel, and
concentrated to provide an oil. Purification of this material by
column chromatography on silica gel (10% EtOAc/hexane)
provided 4-(3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)morpholine as a white solid. 1H NMR (CDCl3,
400 MHz) δ 7.14 (s, 1H), 7.06-6.99 (m, 1H), 6.71-6.66
(m, 1H), 3.88-3.86 (m, 4H), 3.22-3.20 (m, 4H), 1.37 (s, 12H).
4-(3-(2-Chloropyrimidin-4-yl)-5-fluorophenyl)morpholine
was prepared from 2,4-dicholorpyrimidine and 4-(3-fluoro-
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpho-
line as described for compound 9g. Purification by chromatog-
raphy on silica gel (20% EtOAc/hexanes) provided the coupled
product as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ 8.67 (d,
1H), 7.60 (d, 1H), 7.52-7.51 (m, 1H), 7.24-7.21 (m, 1H),
6.81-6.77 (m, 1H), 3.93-3.86 (m, 4H), 3.31-3.28 (m, 4H).
A mixture of 4-(3-(2-chloropyrimidin-4-yl)-5-fluorophenyl)-
morpholine was prepared from 2,4-dicholorpyrimidine was
treated with 4-(3-bromo-1H-1,2,4-triazol-1-yl)aniline as des-
cribed for compound 9g to give N-(4-(3-bromo-1H-1,2,4-tria-
zol-1-yl)phenyl)-4-(3-fluoro-5-morpholinophenyl)pyrimidin-2-
amine after precipitation from the reaction mixture. 1H NMR
(DMSO-d6, 400 MHz) δ 10.00 (s, 1H), 9.23 (s, 1H), 8.62 (d, 1H),
8.01 (d, 2H), 7.75 (d, 2H), 7.59 (s, 1H), 7.55 (d, 1H), 7.40-7.39 (m,
1H), 7.01-7.00 (m, 1H). MS (ESI) 496.17 and 498.17 (M þ H).
This crude residue was treated with 1-methylpiperazine in the
presence of K2CO3 with DMSO as the solvent at 190 °C over-
night. The crude reaction was subjected to reverse-phase prep-
3-Morpholino-5-(2-(4-(3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-
phenylamino)pyrimidin-4-yl)benzonitrile (9j).
A mixture of 3-bromo-1-(4-nitrophenyl)-1H-1,2,4-triazole
(2.15 g, 8.0 mmol), 2-(tributylstannyl)pyridine (4.42 g, 12.0
mmol), Pd(PPh3)4 (0.46 g, 0.40 mmol), and toluene (32 mL)
was heated in a sealed tube at 130 °C overnight. The reaction
mixture was cooled to room temperature, diluted with saturated
aqueous NaHCO3 and EtOAc, and the layers were separated.
The aqueous layer was extracted with EtOAc (2ꢀ). The com-
bined organics were dried (MgSO4) and concentrated in vacuo
to give 2-(1-(4-nitrophenyl)-1H-1,2,4-triazol-3-yl)pyridine as a
brown solid that was used without further purification. 1H
NMR (CDCl3, 400 MHz) δ 8.83-8.81 (m, 1H), 8.79 (s, 1H),
8.43 (d, 2H), 8.27 (d, 1H), 8.06 (d, 2H), 7.87 (t, 1H), 7.43-7.39
(m, 1H).
4-(3-(Pyridin-2-yl)-1H-1,2,4-triazol-1-yl)aniline was ob-
tained following hydrogenation of 2-(1-(4-nitrophenyl)-1H-
1,2,4-triazol-3-yl)pyridine as described for compound 9f. 1H
NMR (CDCl3, 400 MHz) δ 8.78-8.76 (m, 1H), 8.49 (s, 1H), 8.22
(d, 1H), 7.84-7.82 (m, 1H), 7.55 (d, 2H), 7.36-7.32 (m, 1H),
6.78 (d, 2H), 3.86 (brs, 2H).
3-Morpholino-5-(2-(4-(3-(pyridin-2-yl)-1H-1,2,4-triazol-1-
yl)phenylamino)pyrimidin-4-yl)benzonitrile was prepared
following the same general protocol as described for com-
pound 9a using 3-(2-chloropyrimidin-4-yl)-5-morpholinoben-
zonitrile (0.060 g, 0.20 mmol) and 4-(3-(pyridin-2-yl)-1H-
1,2,4-triazol-1-yl)aniline (0.047 g, 0.20 mmol). 1H NMR
(DMSO-d6, 400 MHz) δ 10.03 (s, 1H), 9.33 (s, 1H),
8.73-8.72 (m, 1H), 8.67 (d, 1H), 8.17 (d, 1H), 8.03-7.97 (m,
5H), 7.89 (d, 2H), 7.62 (d, 1H), 7.57 (s, 1H), 7.51-7.50 (m, 1H),
3.81-3.79 (m, 4H), 3.34-3.32 (m, 4H). MS (ESI) 502.38 (M þ
H). HRMS, MHþ: calculated for C28H23N9O, 502.20979;
obtained, 502.2102.
1
HPLC purification to give compound 9k as a yellow solid. H
NMR (CDCl3, 400 MHz) δ 10.70 (brs, 1H), 8.28 (d, 1H), 8.19 (s,
1H), 7.78 (d, 2H), 7.52 (d, 2H), 7.34 (s, 1H), 7.17 (d, 1H), 7.13 (d,
1H), 6.73 (dd, 1H), 4.18-4.17 (m, 2H), 3.82-3.80 (m, 4H),
3.57-3.55 (m, 2H), 3.50-3.48 (m, 2H), 3.19-3.17 (m, 4H),
2.89-2.87 (m, 2H), 2.79 (s, 3H). MS (ESI) 516.34 (M þ H).
4-(3-Fluoro-5-morpholinophenyl)-N-(4-(3-morpholino-1H-
1,2,4-triazol-1-yl)phenyl)pyrimidin-2-amine (9l).

Article
A mixture of 3-bromo-1-(4-nitrophenyl)-1H-1,2,4-triazole
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 429
duplicate. The data were fitted by nonlinear regression of the
four-parameter equation.
Crystallization of JNK3. JNK3 was crystallized as described
by Kamenecka et al.11 and compound 9l was soaked into the
crystals over 36 h.
(1.35 g, 5.0 mmol) and morpholine (8.71 g, 100 mmol) was heated
in a sealed tube at 120 °C overnight. The reaction mixture was
cooled down to room temperature and diluted with water. The
precipitate was filtered, washed with water, and dried under air to
provide the desired product as a brown solid. 1H NMR (CDCl3,
400 MHz) δ 8.40 (s, 1H), 8.34 (d, 2H), 7.80 (d, 2H), 3.85-3.83
(m, 4H), 3.55-3.53 (m, 4H). MS (ESI) 276.13 (M þ H).
Data Collection and Structure Solution of JNK3-9l. Liquid
nitrogen cooled crystals were loaded into an SSRL cassette for
shipping. Data was collected remotely at SSRK on beamline
9-1.39 Data sets were indexed, integrated, and scaled to the
orthorhombic spacegroup C2221 using HKL2000.40 Initial
phases were determined via molecular replacement, with PDB
ID 1JNK as the search model, using PHASER.41 The initial
model was refined by alternating cycles of restrained refinement
using REFMAC542 and manual model building using COOT.43
PHASER and REFMAC5 are part of the CCP444 suite of
software. Model geometry, bond lengths, and steric clashes were
monitored using MOLPROBITY.45 The ligand was placed into
positive density in Fo - Fc electron density map. Figures and
rmsd values were generated using PYMOL.46
P450 Inhibition and Microsomal Stability Assays. P450 in-
hibition for the four major human isoforms was evaluated
by following the metabolism of specific marker substrates
(CYP1A2 phenaceten demethylation to acetaminophen;
CYP2C9, tolbutamide hydroxylation to hydroxytolbutamide;
CYP2D6, bufuralol hydroxylation to 40-hydroxybufuralol; and
CYP3A4, midazolam hydroxylation to 10-hydroxymidazolam)
in the presence or absence of 10 μM test compound for 15 min at
37 °C. The concentration of each marker substrate was approxi-
mately its Km. Specific inhibitors for each isoform were included
in each run to validate the system. Microsome (mouse, rat,
human; Xenotech, Lenexa, Kansas) stability was evaluated by
incubating 1 μM test compound with 2 mg/mL hepatic micro-
somes in 100 mM KPi, pH 7.4. The reaction was initiated at
37 °C by adding NADPH (1 mM final concentration). Aliquots
were removed at 0, 5, 10, 20, 40, and 60 min and added to
acetonitrile (5ꢀ v:v) to stop the reaction and precipitate the
protein. At the end of the assay, the samples were centrifuged
through a Millipore Multiscreen Solvinter 0.45 μm low binding
polytetrafluoroethylene (PTFE) hydrophilic filter plate and
analyzed by LC-MS/MS. Data was log transformed and regres-
sion analysis was used to calculate half-life.
4-(3-Morpholino-1H-1,2,4-triazol-1-yl)aniline was obtained
following hydrogenation of 4-(1-(4-nitrophenyl)-1H-1,2,4-tria-
zol-3-yl)morpholine as described for compound 9f. H NMR
1
(CDCl3, 400 MHz) δ 8.10 (s, 1H), 7.38 (d, 2H), 6.75 (d, 2H),
3.87-3.85 (m, 4H), 3.80 (br s, 2H), 3.52-3.50 (m, 4H). MS (ESI)
246.17 (M þ H).
A
mixture of 4-(3-(2-chloropyrimidin-4-yl)-5-fluorophe-
nyl)morpholine (0.103 g, 0.35 mmol) and 4-(3-morpholino-
1H-1,2,4-triazol-1-yl)aniline (0.086 g, 0.35 mmol) in 2-ethox-
yethanol (0.5 mL) was heated in a sealed tube at 190 °C for 1 h.
The reaction mixture was cooled to room temperature and
diluted with water. The precipitate was filtered, washed with
water, and dried under air to provide compound 9l as a brown
solid. 1H NMR (CDCl3, 400 MHz) δ 8.45 (d, 1H), 8.13 (s, 1H),
7.72 (d, 2H), 7.49 (d, 2H), 7.37 (s, 1H), 7.36 (s, 1H), 7.14 (d, 1H),
7.07 (d, 1H), 6.65 (d, 1H), 3.83-3.81 (m, 4H), 3.79-3.77 (m,
4H), 3.45-3.43 (m, 4H), 3.19-3.17 (m, 4H). MS (ESI) 503.31
(M þ H). HRMS, MHþ: calculated for C26H27FN8O2,
503.23134; obtained, 503.2317.
Expression and Purification of JNK3 and ATF2. JNK3 and
ATF2 were expressed and purified as described by Kamenecka
et al.11 JNK1 and p38 were purchased from Millipore.
Homogeneous Time Resolved Fluoresence Assay for JNK1,
JNK3, and p38 Biochemical Assays. The assays were run as
described by Kamenecka et al.11 JNK3 expression in human, and
rat pancreas, and insulin secreting INS-1 cells has been reported.38
Cell-Based Assay Measuring JNK Activity. Inhibition of c-jun
phosphorylation in INS-1 β-pancreatic cells was run as de-
scribed by Kamenecka et al.11
Cell-Based Assay Measuring Reactive Oxygen Species Gen-
eration in INS-1 β-Pancreatic Cells. Rat insulinoma cells (INS-1
823/13) were plated in 6-well plates (5 ꢀ 105-1 ꢀ 106 cells/well)
in RPMI 1640 containing 10% fetal bovine serum (FBS). Cells
were incubated overnight at 37 °C in 5% CO2 and 95%
humidity. Cells were treated with compound 9l dissolved in
dimethyl sulfoxide (DMSO) for 30 min prior to the administra-
tion of 4 mM streptozotocin (STZ) solubilized in medium.
Inhibitors were present during STZ stimulation at the same
concentrations used during the 30 min pre-STZ step. Cells were
incubated with STZ for 4 h at 37 °C. Cells were washed twice
with red phenol free RPMI 1640 containing 10% FBS. Detec-
tion of ROS generated in INS1 cells during treatments was done
by oxidation (by H2O2) of the cell-permeable indicator 5-(and-
6)-chloromethyl-20,70-dichlorofluorescin diacetate, acetyl ester
(CM-H2DCFDA; Invitrogen, Carlsbad, CA). The ROS indica-
tor was prepared in red phenol free RPMI 1640 at 5 μM, added
to cells and incubated for 1 h at 37 °C. Stained cells were
resuspended and washed twice by centrifugation (5 min at room
temperature) using red phenol free RPMI 1640 medium. Quan-
tification of generated ROS was done in a microplate reader
Spectramax M5e (Molecular Devices, Sunnyvale, CA) using a
96-well black plate with clear bottom (Perkin-Elmer, Whal-
tham, MA); 105 cells were transferred to each well in a 200 μL
volume of red phenol free medium. Excitation of the fluorescein
probe was done at 495 nm and emission was recorded at 525 nm.
Control treatments included DMSO treated cells (no ROS
control) and 10 μM rotenone for 1 h (ROS positive control).
The normalized relative fluorescence units (RFU) were calcu-
lated for each point by dividing the obtained RFU by the RFU
value corresponding to the STZ-treated INS-1 cells in the
absence of inhibitor. The IC50 determined was from the averages
of four biological replicates with each concentration repeated in
Rat Pharmacokinetics and Mouse Brain Penetration. Pharma-
cokinetics of test compounds was assessed in Sprague-Dawley
rats (n = 3). Compounds were dosed intravenously at 1 mg/kg
and orally by gavage at 2 mg/kg. Blood was taken at eight time
points (5, 15, 30 min, 1, 2, 4, 6, 8 h) and collected into EDTA
containing tubes and plasma was generated using standard
centrifugation techniques. Plasma proteins were precipitated
with acetonitrile and drug concentrations were determined by
LC-MS/MS. Data was fit by WinNonLin using a noncompart-
mental model and basic pharmacokinetic parameters including
peak plasma concentration (Cmax), oral bioavailability, expo-
sure (AUC), half-life (t1/2), clearance (CL), and volume of
distribution (Vd) were calculated.
CNS exposure was evaluated in C57Bl6 mice (n=3). Com-
pounds were dosed at 10 mg/kg intraperitoneally and after 2 h
blood and brain were collected. Plasma was generated and the
samples were frozen at -80 °C. The plasma and brain were
mixed with acetonitrile (1:5 v:v or 1:5 w:v, respectively). The
brain sample was sonicated with a probe tip sonicator to break
up the tissue, and samples were analyzed for drug levels by LC-
MS/MS. Plasma drug levels were determined against standards
made in plasma and brain levels against standards made in
blank brain matrix. All procedures were approved by the
Scripps Florida IACUC.
Acknowledgment. This work was supported by NIH grant
U01-NS057153 awarded toP.L. Portions of this research were
carried out at the Stanford Synchrotron Radiation Labora-
tory, a national user facility operated by Stanford University

430 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Kamenecka et al.
on behalf of the U.S. Department of Energy, Office of Basic
Energy Sciences. The SSRL Structural Molecular Biology
Program is supported by the Department of Energy, Office of
Biological and Environmental Research, and by the National
Institutes of Health, National Center for Research Resources,
Biomedical Technology Program, and the National Institute
of General Medical Sciences. We are grateful to Dr. Mike
Chalmers for HRMS determination and Yamille Del Rosario
for administrative assistance in preparing the manuscript.
INS-1 cells were a kind gift from Dr. Christopher Newgard
at Duke University.
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