Design, synthesis, and structure-activity relationship studies of thiophene-3-carboxamide derivatives as dual inhibitors of the c-Jun N-terminal kinase

Article abstract of DOI:10.1016/j.bmc.2011.03.017

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.

Full text of DOI:10.1016/j.bmc.2011.03.017

Bioorganic & Medicinal Chemistry 19 (2011) 2582–2588
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and structure–activity relationship studies
of thiophene-3-carboxamide derivatives as dual inhibitors
of the c-Jun N-terminal kinase
Surya K. De a, Elisa Barile a, Vida Chen a, John L. Stebbins a, Jason F. Cellitti a, Thomas Machleidt b,
Coby B. Carlson b, Li Yang a, Russell Dahl a, Maurizio Pellecchia a,
⇑
a Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
b Invitrogen Corporation (now Life Technologies), Invitrogen Discovery Services, 501 Charmany Drive, Madison, WI 53719, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report comprehensive structure–activity relationship studies on a novel series of c-Jun N-terminal
kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both
ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the
kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 5 January 2011
Revised 1 March 2011
Accepted 7 March 2011
Available online 12 March 2011
Keywords:
JNK
JIP1
Kinase inhibitor
1. Introduction
shown to inhibit JNK activity in vitro, displaying noteworthy selec-
tivity with little inhibition of the closely related Erk and p38 MAP-
The c-Jun N-terminal kinases (JNKs) have been originally iden-
tified in the early 1990s as a family of serine/threonine protein
kinases that are activated by a range of stimuli. The activation of
each of the JNK genes results in the phosphorylation of the
N-terminal transactivation domain of the c-Jun transcription fac-
tor.1–4 Three JNK isoforms (JNK1, 2 and 3) share more than 90%
amino acid sequence identity and the ATP pocket is highly con-
served (>98% identities). These proteins are often activated in
response to a large variety of cellular stresses including irradiation,
hypoxia, peroxides, heat shock, and chemotoxins as well as various
cytokines, thus participating in the onset of apoptosis.5,6 It has
been clearly established that excessive up-regulation of JNK activ-
ity results or is associated with a number of human disorders
including type-2 diabetes and obesity, neurodegeneration and
stroke, cancer and inflammation.1–3 Hence, JNK inhibitors are ex-
pected to be viable agents to devise novel therapies against these
diseases, and there have been large efforts in identifying small
molecule JNK inhibitors targeting its ATP binding site.7–13
Ks.16–19 Furthermore, recent in vivo data, generated for studies
focusing on pepJIP1 fused to the cell permeable HIV-TAT peptide,
show that its administration in various mice models of insulin
resistance and type-2 diabetes restores normoglycemia without
causing hypoglycemia.20 Despite these encouraging data, peptide’s
instability in vivo may hamper the development of novel JNK-
related therapies based on such peptides.16–20
Based on these premises, a drug discovery program in our lab-
oratory was initiated with the aim of identifying and characteriz-
ing small molecule JNK inhibitors as novel chemical entities
targeting its JIP binding site rather than the highly conserved ATP
binding site of the protein. Very recently, we have reported the
identification of 5-(5-nitrothiazol-2-ylthio)-1,3,4-thiadiazol-2-
amine series21 related to compound BI-78D322 (Fig. 1), as initial
JIP mimetic inhibitors. These compounds were discovered using a
displacement assay with
a biotinylated-pepJIP1 peptide and
employing a DELFIA assay platform in a medium size screening
campaign.22 In our continued interest in the development of JNK
inhibitors21–23 we now report further structure–activity relation-
ship studies describing novel small molecules thiophene-
carboxamide derivatives as JNK inhibitors targeting its JIP/
substrate docking site. Intriguingly, we believe that the compounds
are also able to function as ATP mimetics for JNK, which makes
them particularly interesting. The 4,5-dimethyl-2-(2-(naphtha-
len-1-yl)acetamido)thiophene-3-carboxamide (1, Fig. 1) was
qualified as a hit and became the starting point of our medicinal
Peculiar to JNKs substrates and scaffold proteins, is a JNK
interacting conserved consensus sequence R/KXXXXLXL termed
the D-domain.14,15 A short peptide corresponding to the D-domain
of the scaffolding protein JIP1 (aa 153–163; pepJIP1) has been
⇑
Corresponding author.
E-mail addresses: mpellecchia@sanfordburnham.org, lslivka@burnham.org
(M. Pellecchia).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.017

S. K. De et al. / Bioorg. Med. Chem. 19 (2011) 2582–2588
2583
N
N
N
NO2
O
HO
S
S
N
NH2
O
O
S
NH2
O
N
H
S
O
S
N
H
O
BI-78D3
1
25
Figure 1. Previously reported key JIP1 site binding JNK inhibitor (BI-78D3) and thiophene 3-carboxamide inhibitors indentified via HTS (1 and 25).
chemistry efforts, with an IC50 value for the displacement of pep-
JIP1 in the DELFIA assay of 15.8 M, inhibiting JNK1 kinase activity
in the Lantha assay platform with an IC50 value of 26.0 M. To
investigate the effects on potency induced by small changes in
the structure of 1, we developed the general synthetic route for
the preparation of this series. A variety of commercially available
2-aryl acetic acids were treated with aryl 2-amino-3-carboxamides
in the presence of EDC at room temperature to give 5a–5g and 11–
74 (Schemes 1–3) in moderate to good yields. Replacement of the
thiophene moiety with a phenyl ring led to compound 3 that
observations, we synthesized additional analogs of compound 7
with a variety of aryl or heteroaryl substitutions (Scheme 3). The
mono fluoro or difluoro substitutions (compounds 29–31, 52–56,
and 71) on the benzene ring were well tolerated (IC50 = 8.3, 9.4,
l
l
5.1, 8.2, 10.2, 9.7, 7.4, 5.8, 4.9
lM, respectively) but the penta-
fluoro benzene, compound 60, was inactive (IC50 >50
lM). Simi-
larly, a chlorine on positions 2 and 3 on the benzene ring resulted
in compounds with good inhibitory activities (compound 26,
IC50 = 1.4
rine (28, IC50 = 18.7
(34, IC50 >25 M), isopropyl (50, IC50 >25
>50 M), and isobutyl (20, IC50 >25 M) in the 4-position on the
l
M and compound 27, IC50 = 2.6
M) or larger substituents such as bromine
M), phenyl (62, IC50
lM). However a chlo-
l
showed a drastic drop in activity (IC50 >100
lM), similarly replac-
l
l
ing the 3-carboxamide group on the thiophene with an acid, result-
ing in compound 5a, or an ester, resulting in compound 5b, or a
cyano group, as in compound 5c, also resulted in a significant loss
of JNK1 inhibitory activity (Table 1). The position of carboxamide is
also important for JNK1 inhibitory activity as the analogue with the
carboxamide at the 5-position on the thiophene (compound 5f)
was completely inactive. The 4-methyl (5d) or 5-methyl (5e) or
4,5-dimethyl substitutions on the thiophene of compound 1 also
l
l
benzene ring were not tolerated, whereas similar substitutions in
positions 2 or 3 resulted in compounds with good inhibitory prop-
erties against JNK1. Furthermore, introducing potential hydrogen-
bonding acceptors such as methoxy (compound 37, IC50 = 11.9
compound 38, IC50 = 2.6 M and compound 39, IC50 = 5.7
methylene dioxy (compound 19, IC50 = 1.8 M), and benzodioxane
(compound 18 IC50 = 2.7 M) groups on the benzene ring resulted
lM,
l
lM),
l
l
resulted in less active compounds (IC50 >25
the un-substituted compound (5g, IC50 = 5.4
retained 4- and 5-positions unsubstituted and carboxamide on
the 3-position on the thiophene, and explored modifications at
the 2-position. We observed that introducing substituents with
one carbon linker did not affect the inhibitory properties of the
l
M), compared to
in compounds with significant JNK1 inhibition while other polar
groups, such as a morpholine or a piperazine (compounds 64–68)
resulted in compounds with poor inhibitory activities (IC50
l
M). Therefore, we
>50
lM), except for compound 66 in which some activity was re-
tained (IC50 = 7.6
l
M). Finally, we found that replacing the benzene
with a benzothiophene (compound 25, Fig. 1) resulted in a signif-
icant improvement with respect to both JNK1 kinase activity inhi-
bition and JNK1/pepJIP displacement.
Subsequently, we performed dose–response measurements and
enzyme kinetics assays with compound 25. Compound 25 showed
series (i.e., compound 7, IC50 = 3.6
IC50 = 5.9 M), while longer chains (i.e., compound 9 with a 2-car-
bon linker, IC50 >100 M, or compound 10 with a trans-2-carbon
lM vs compound 8, no linker,
l
l
linker, IC50 >100
lM) are not tolerated (Table 1). Based on these
an IC50 of 1.32
lM in the kinase assay (Fig. 2) and an IC50 of
4.62 M in displacing pepJIP1, as measured by DELFIA assay, that
l
indicates that the compound may bind directly at JIP site (Fig. 2).
However, enzyme kinetics experiments suggested that compound
25 is an ATP competitive inhibitor as well as a mixed substrate
competitive inhibitor (Fig. 2). This is quite intriguing, suggesting
that the compound may bind independently to both the ATP and
the JIP binding sites on JNK. Interestingly, our findings are similar
to what recently reported by Chen et al.24 that have identified a
similar dual JIP site and ATP site JNK inhibitor based on kinetic
experiments.
O
O
NH2
NH2
O
a
NH2
N
H
2
3
Hence, to determine the selectivity, compound 25 was tested
against a panel of 26 kinases. Compound 25 was found to be nearly
inactive (Table 2) against p38a (2% inhibition at 25 lM), a member
R2
R3
R1
NH2
4a-g
R2
R1
a
O
of the MAPK family with high structural similarity to JNK (52% se-
quence identity, considering the kinase domains), and inactive
against other MAP kinases and lipid kinases (Table 2), further cor-
roborating that these compounds may elicit their activity by bind-
ing not only to the highly conserved ATP site but also the JNK
docking site. This compound showed a preferential inhibition of
JNK1 and JNK2 over JNK3 (Table 2), in agreement with our previous
findings with compound BI-78D322 (Fig. 1) and with the reported
data on pepJIP1.16–20
R3
S
S
N
H
5a-g
Scheme 1. Reagents and conditions: (a) 2-(1-Naphthyl) acetic acid, EDC, HOBt,
DIEA, DMF, rt. a: R1 = carboxylic acid, R2 = R3 = H; b: R1 = methyl ester, R2 = R3 = H;
c: R1 = cyano, R2 = R3 = H; d: R1 = carboxamide, R2 = Me, R3 = H; e: R1 = carboxamide,
2 = H, R3 = Me; f: R1 = H, R2 = H, R3 = carboxamide; g: R1 = carboxamide,
2 = R3 = H.
R
R

2584
S. K. De et al. / Bioorg. Med. Chem. 19 (2011) 2582–2588
O
NH2
O
S
N
H
10
c
O
O
O
NH2
O
NH2
O
NH2
a
d
S
NH2
S
N
S
N
H
6
H
9
7
b
O
NH2
O
S
N
H
8
Scheme 2. Reagents and conditions: (a) phenyl acetic acid, EDC, HOBt, DIEA, DMF, rt; (b) benzoic acid, EDC, HOBt, DIEA, DMF, rt; (c) 3-phenylpropanoic acid, EDC, HOBt, DIEA,
DMF, rt; (d) trans-cinnamic acid, EDC, HOBt, DIEA, DMF, rt.
O
O
the ATP and JIP binding pockets derived from the X-ray crystal
structure of the ternary complex including JNK1, pepJIP1, and the
ATP mimic SP600125 (PDB ID 1UKI). From the docked binding
pose, compounds of this series, represented by compound 25
(Fig.3) appeared to be deeply inserted in the ATP binding site
and further stabilized by hydrogen-bonding interactions involving
its carboxamide NH2 and side-chain atoms of the residue Gln 37.
The docked poses of compounds 18, 19 and 38 suggest the pres-
ence of additional hydrogen-bonding interactions with the back-
bone amide of Met 111 in the ATP binding site, providing a
justification for the increased inhibitory properties for these com-
pounds (Supplementary data).
Furthermore, the JIP1 docking site seemed also able to
potentially accommodate compounds of this series, as shown for
compound 25 (Fig. 3) through hydrophobic contacts and hydro-
gen-bonding interactions between the carboxamide –CO– and –
NH2 groups with the backbone NH of Val 118 and the backbone
carbonyl of Asn 114, respectively. Hence, both binding modes
would reflect the observed SAR including the direct ability of the
compounds to displace the pepJIP1/JNK interactions in the DELFIA
assay, suggesting that the dual inhibition is possible also on a the-
oretical basis.
To further corroborate this hypothesis, we conducted isothermal
titration calorimetry experiments to monitor the thermodynamics
of bindings of compound 25 to JNK2 in presence and absence of sat-
urating concentrations of either a non-hydrolizable ATP or of pep-
JIP1. These studies revealed indeed that compound 25 binds to
JNK2 with a Kd value of 640 nM with a 2:1 ligand to protein stoichi-
ometry, and that the binding decreases significantly in presence of
either the non-hydrolizable ATP or pepJIP1. The measured Kd values
for compound 25 to JNK1 under these saturating conditions were
NH2
NH2
O
a
R
S
NH2
S
N
H
6
11-74
Scheme 3. Reagents and conditions: (a) 2-aryl-acetic acids, EDC, HOBT, DIEA, DMF,
rt. Compound 11: R = 2-bromo-6-methoxy-1-naphthyl; 12: R = 2-methoxy-6-
bromo-1-naphthyl; 13: R = 2-naphthyl; 14: R = thiophene-2-yl; 15: R = 3,5-dimeth-
ylphenyl; 16: R = pyrene-1-yl; 17: R = 4-thiopheny-2-yl-thiazol-2-yl; 18: R =
3,4-benzodioxane; 19: R = 3.4-methylenedioxyphenyl; 20: R = 4-isobutylphenyl;
21: R = 2-pheny-thiazol-4-yl; 22: R = 2-methoxy-5-methylphenyl; 23: R = benzofu-
ran-3-yl; 24: R = benzoimidazol-2-yl; 25: R = benzothiophen-3-yl; 26: R = 2-chloro-
phenyl; 27: R = 3-chlorophenyl; 28: R = 4-chlorophenyl; 29: R = 2-fluorophenyl; 30:
R = 3-fluorophenyl; 31: R = 4-flurophenyl; 32: R = 2-bromophenyl; 33: R = 3-bromo-
phenyl; 34: R = 4-bromophenyl; 35: R = 2-iodophenyl; 36: R = 3-iodophenyl; 37:
R = 2-methoxyphenyl; 38: R = 3-methoxyphenyl; 39: R = 4-methoxyphenyl, 40:
R = 2-methylphenyl; 41: R = 3-methylphenyl; 42: R = 4-methylphenyl; 43: R =
3-nitrophenyl; 44: R = 4-nitrophenyl; 45: R = 3-trifluoromethylphenyl; 46: R =
4-trifluorophenyl; 47: R = 4-trifluoromethoxyphenyl; 48: R = 3-cyanophenyl; 49:
R = 4-cyanophenyl; 50: R = 4-isopropyl; 51: R = 4-ethoxyphenyl; 52: R = 3,5-difluo-
rophenyl; 53: R = 3,4-difluorophenyl; 54: R = 2,4-difluorophenyl; 55: R = 2,6-difluo-
rophenyl; 56: R = 2,5-difluorophenyl; 57: R = 2-chloro-6-fluorophenyl; 58: R =
3,4-dimethoxyphenyl; 59: R = 3,5-dimethoxyphenyl; 60: R = 2,3,4,5,6-pentafluor-
ophenyl; 61: R = 3-pyridyl; 62: R = 4,4-biphenyl; 63: R = indol-3-yl; 64: R = mor-
pholino; 65: R = 2,6-dimethylmorpholino; 66: R = 4-furan-2-yl-piperazin-1-yl; 67:
R = 4-cyclopropane-carbonyl-piperazin-1-yl; 68: R = (4-(2-(trifluoromethyl)phenyl-
sulfonyl)piperazin-1-yl); 69: R = 5,6,7,8-tetrahydronaphthalen-2yl; 70: R = 2,3-
dihydro-1H-inden-5-yl; 71: R = 6-fluoro-4H-benzo[d][1,3]dioxin-8-yl; 72: R =
(2-(6-(thiophen-2-yl)imidazo[2,1-b]thiazol-3-yl); 73: R = (2-(9-chloro-3,4-dihydro-
2H-benzo[b][1,4]dioxepin-7-yl); 74: R = (2-(5-methylimidazo[2,1-b]thiazol-2-yl).
Finally, to assess the activity of the compounds in cell we em-
ployed a cell-based LanthaScreen™ kinase assay. In this assay,
compound 25 is able to inhibit TNF-
a
stimulated phosphorylation
1.1 lM (in presence of ATP) and 3.1 lM (in presence of pepJIP1),
of c-Jun with an IC50 value of 7.5 M (Supplementary data), which
l
both with a 1:1 ligand to protein stoichiometry.
parallels well the in vitro activity and previous findings with this
Finally, to assess basic pharmacological properties of the pro-
posed thiophene carboxamide derivatives, we determined
in vitro plasma and microsomal stability for selected compounds.
Compounds 25, 33, and 26 remained basically unaltered after
type of assay and other JNK inhibitors.22
To shed light into the possible binding mode of these com-
pounds, we performed molecular modeling studies,25–27 using

S. K. De et al. / Bioorg. Med. Chem. 19 (2011) 2582–2588
2585
Table 1
after 60 min of incubation in rat microsomal preparations, data
suggesting that the molecules might be suitable for further optimi-
zations and in vivo studies. Thiophenes are considered potential
toxicophores due to their metabolites that can cause hepatotoxic-
ity, immunotoxicity and nephrotoxicity.28–30 However, di-substitu-
tions or ring deactivation by addition of adjacent functionalities,
have been proven to minimize metabolic oxidation hence reducing
toxicity.28,30 Hence, the observed relatively long microsomal half-
lives of these compounds can be attributed to the presence of di-
substitutions on the thiophene ring. Finally, two very recent re-
ports, describing the pharmacology of other thiophene-based JNK
inhibitors, albeit with rather different structures form those re-
ported herein, corroborated the present observations.31,32
In summary, we report data on a series of novel JNK inhibitors
that resulted in the selection of compound 25, that apparently
inhibits JNK by possibly binding to both the ATP and JIP binding
site, independently. Because the compound possesses a good bal-
ance of potency, selectivity, solubility, metabolic stability, and cel-
lular activity, we anticipate that this series may hold promise for
further development of novel JNK inhibitors as chemical probes
and eventually for therapeutic purposes.
Inhibition results for thiophene carboxamide series against JNK-1
Compd Kinase
activity assay
Compd Kinase
activity assay
Compd Kinase
activity assay
a
a
a
IC50
(lM)
IC50
(lM)
IC50 (lM)
1
3
26.0
>100
>100
>100
>100
>25
>25
>100
5.4
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
>25
1.3
1.4
2.6
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
>25
1.9
8.2
10.2
9.7
5a
5b
5c
5d
5e
5f
5g
7
18.7
8.3
9.4
5.1
2.9
1.8
>25
3.4
1.8
11.9
2.6
5.7
6.0
5.4
4.3
>25
2.9
7.6
6.4
5.8
20.9
3.4
7.4
5.8
12.5
>25
3.1
>50
8.1
>50
9.4
>50
65.3
7.6
83.5
75.1
3.6
2.5
4.9
3.6
5.9
8
9
>100
>100
>25
>25
>25
3.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2.7
>50
>25
2.7
1.8
2. Methods
>25
>25
2.30
>25
2.5
1.9
2.5
2.1. Synthetic chemistry
Unless otherwise indicated, all anhydrous solvents were com-
mercially obtained and stored in Sure-seal bottles under nitrogen.
All other reagents and solvents were purchased as the highest
grade available and used without further purification. Thin-layer
chromatography (TLC) analysis of reaction mixtures was per-
formed using Merck Silica Gel 60 F254 TLC plates, and visualized
a
Values are means of at least three or more experiments. Standard deviation
generally within 25%.
60 min of incubation in rat plasma while were degraded 85% (T1/2
18 min), 74% (T1/2 24 min), and 70% (T1/2 27 min), respectively,
100
100
A
B
80
60
40
80
60
40
HillSlope
IC50
0.9070
4.618
HillSlope
IC50
1.136
1.325
20
20
-2
-1
0
1
2
-3
-2
-1
0
1
2
3
Log compound 25 [µM]
Log compound 25 [µM]
3.62 µM
1.32 µM
0.66 µM
0 µM
3.62 µM
1.32 µM
0.66 µM
0 µM
2.0
1.5
1.0
0.5
C
D
2.5
2.0
1.5
1.0
0.5
-10
-5
5
10
-0.5
1/ATP Concentration [µM]
-20
-10
0
10
20
30
1/ATF2 Concentration [µM]
Figure 2. Dose–response curves for the displacement of pepJIP1 from JNK1 by compound 25 in the DELFIA assay (A), and for the inhibition of JNK1 kinase activity in the
LANTHA assay by compound 25 (B). Lineweaver–Burk analyses for the inhibition of JNK1 kinase activity (LANTHA assay) by compound 25 with increasing substrate ATF2 (C)
or ATP (D) concentrations. Data were collected in triplicates.

2586
S. K. De et al. / Bioorg. Med. Chem. 19 (2011) 2582–2588
Table 2
Selectivity profile for compound 25a,b
Kinase
% inhibition at 25
lM
Kinase
% inhibition at 25
l
M
Kinase
% inhibition at 25 lM
JNK1
JNK2
JNK3
MAPK14 (p-38
MAPK3 (ERK1)
MAPK1 (ERK2)
AKT1
AMPK A1
AURK A1
79%c
72%c
18%c
2%
3%
4%
5%
0%
0%
BRAF
CAMK1
CDK1
CHEK1 (CHK1)
CHUK (IIK
FRAP1 (mTOR)
GSK3
MAK2K1 (MEK1)
MAPK3K9 (MLK1)
2%
2%
3%
0%
0%
0%
16%
0%
0%
MAPK14 (p-38
NEK1
PDK1 direct
PI3Ka
a
)
11%
0%
0%
4%
0%
0%
0%
1%
1%
a, direct)
a
)
PIM1
PRKCA (PKC
a)
a
ROCK1
SPHK1
MAP4K2 (GCK)
a
b
c
From Invitrogen (Life Technologies) kinase profiling service.
Values are means of at least two independent experiments.
Z’-LYTE assay.
Figure 3. Docking studies of compound 25 within the JIP (A) and ATP (B) binding sites of JNK1 (PDB ID 1UKI). The surface was generated with MOLCAD27 and color coded
according to cavity depth (blue, shallow; yellow, deep). Predicted hydrogen-bonding interactions are highlighted with yellow dashed lines.
using ultraviolet light. NMR spectra were recorded on Varian 300
or 500 MHz instruments. Chemical shifts (d) are reported in parts
per million (ppm) referenced to Me4Si. Coupling constants (J) are
reported in Hertz (Hz) throughout. Mass spectral data were ac-
quired on Shimadzu LC/MS-2010EV for low resolution, and on an
Agilent ESI-TOF for either high or low resolution. Purity of key
compounds was obtained in a HPLC Breeze from Waters Co. using
2.3. Microsomal stability assay (RLM assay)
Solutions of three compounds were incubated with RAT liver
microsomes (RLM) for 60 min at 37.5 °C. The final incubation solu-
tions contained 4 lM test compound, 2 mM NADPH, 1 mg/ml (total
protein) microsomes, and 50 mM phosphate (pH 7.2). Compound
solutions, protein, and phosphate were pre-incubated at 37.5 °C
for 5 min and the reactions were initiated by the addition of NADPH
and incubated for 1 h at 37.5 °C. Aliquots were taken at 15 min time-
points and quenched with the addition of methanol containing
internal standard. Following protein precipitation and centrifuga-
tion, the samples were analyzed by LC–MS. Test compounds were
run in duplicate with two control compounds of known half life.
an Atlantis T3 3
lm 4.6 Â 150 mm reverse phase column. The elu-
ant was a linear gradient with a flow rate of 1 ml/min from 95% A
and 5% B to 5% A and 95% B in 15 min followed by 5 min at 100% B
(Solvent A: H2O with 0.1% TFA; Solvent B: ACN with 0.1% TFA). The
compounds were detected at k = 254 nm. Purity of key compounds
was established by elemental analysis as performed on a Perkin
Elmer series II-2400. Combustion analysis was performed by
NuMega Resonance labs, Inc., San Diego, CA. Details on the
experimental procedures and key analytical data for each of the
reported compound are available as Supplementary data.
2.4. DELFIA assay (dissociation enhanced lanthanide fluoro-
immuno assay)
To each well of 96-well streptavidin-coated plates (Perkin-El-
2.2. Plasma stability assay
mer) 100 lL of a 100 ng/ml solution of biotin-labeled pepJIP11
(Biotin-lc-KRPKRPTTLNLF, where lc indicates a hydrocarbon chain
Test compound solution was incubated (1 lM, 2.5% final DMSO
of six methylene groups) was added. After 1 h incubation and elim-
concentration) with fresh rat plasma at 37 °C. The reactions were
terminated at 0, 30, and 60 min by the addition of two volumes
of methanol containing internal standard. Following protein
precipitation and centrifugation, the samples were analyzed by
LC–MS. The percentage of parent compound remaining at each
time point relative to the 0 min sample is calculated from peak
area ratios in relation to the internal standard. Compounds were
run in duplicate with a positive control known to be degraded in
plasma.
ination of unbound biotin-pepJIP11 by three washing steps, 87
of Eu-labeled anti-GST antibody solution (300 ng/ml; 1.9 nM),
2.5 L DMSO solution containing test compound, and 10 L solu-
tion of GST-JNK1 for a final protein concentration of 10 nM was
added. After 1 h incubation at 0 °C, each well was washed five
times to eliminate unbound protein and the Eu-antibody if dis-
placed by a test compound. Subsequently, 200 lL of enhancement
solution (Perkin-Elmer) was added to each well and fluorescence
measured after 10 min incubation (excitation wavelength,
lL
l
l

S. K. De et al. / Bioorg. Med. Chem. 19 (2011) 2582–2588
2587
340 nm; emission wavelength, 615 nm). Controls include unla-
beled peptide and blanks receiving no compounds. Protein and
peptide solutions were prepared in DELFIA buffer (Perkin-Elmer).
compound (indicated concentration) followed by 30 min of stimu-
lation with 2 ng/ml of TNF-alpha which stimulates both JNK and
p38. The medium was then removed by aspiration and the cells
were lysed by adding 20 lL of lysis buffer (20 mM Tris–HCl pH
2.5. In vitro kinase assay
7.6, 5 mM EDTA, 1% NP-40 substitute, 5 mM NaF, 150 mM NaCl,
1:100 protease and phosphatase inhibitor mix, SIGMA P8340 and
P2850, respectively). The lysis buffer included 2 nM of the terbium
labeled anti-pc-Jun (pSer73) detection antibodies (Invitrogen).
After allowing the assay to equilibrate for 1 h at room temperature,
TR-FRET emission ratios were determined on a BMG Pherastar
fluorescence plate reader (excitation at 340 nm, emission 520
The LanthaScreen™ assay platform from Invitrogen was uti-
lized. The time-resolved fluorescence resonance energy transfer as-
say (TR-FRET) was performed in 384 well plates. Each well received
JNK1 (35 ng/mL), ATF2 (400 nM), and ATP (0.2 lM) in 50 mM
HEPES, 10 mM MgCl2, 1 mM EGTA and 0.01% Brij-35, pH 7.5 and
test compounds. The kinase reaction was performed at room tem-
perature for 1 h. After which, the terbium labeled antibody and
EDTA were added into each well. After an additional hour incuba-
tion, the signal was measured at 520/495 nm emission ratio on a
fluorescence plate reader (Victor 2, Perkin-Elmer).
and 490 nm; 100 ls lag time, 200 ls integration time, emission ra-
tio = Em520/Em 490).
Acknowledgment
We gratefully acknowledge financial support from the NIH
(grant # DK080263 to M.P.).
2.6. Isothermal titration calorimetry
Titrations were done using a VP-ITC calorimeter from Microcal
(Northampton, MA). Depending upon the solubility of the titrants,
Supplementary data
JNK2 was used at 25–50
(pH 7.4), 5% DMSO, and 0.01% triton X-100. Titrants were used at
15Â protein concentration (375–750 M) in the same buffer. For
competition titrations, 350 M 25 was titrated to 25 M JNK2 in
the presence of 250 M ATP S (Biomol International, Plymouth
Meeting, PA) or pepJIP1 (i.e., 250 M ATP S or pepJIP1 was present
in both the syringe and cell of the ITC). Titrations of compound
alone into buffer were done to determine the heats of dilution of
compounds and were negligible compared to compound to protein
titrations. Compound heats of dilution were subtracted from com-
pound/protein titrations. Titrations were carried out at 25 °C. Data
were analyzed using Microcal Origin software provided by the ITC
manufacturer.
lM in 20 mM sodium phosphate buffer
Supplementary data (detailed synthetic procedures and analyt-
ical data are available for the reported compounds. A dose response
curve for the inhibition of JNK in cell is also reported. Docking stud-
ies with compounds 19 and 39 are also included) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.03.017.
l
l
l
l
c
l
c
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