Discovery of 2-(5-nitrothiazol-2-ylthio)benzo[d]thiazoles as novel c-Jun N-terminal kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.02.046

A new series of 2-thioether-benzothiazoles has been synthesized and evaluated for JNK inhibition. The SAR studies led to the discovery of potent, allosteric JNK inhibitors with selectivity against p38.

Full text of DOI:10.1016/j.bmc.2009.02.046

Bioorganic & Medicinal Chemistry 17 (2009) 2712–2717
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 2-(5-nitrothiazol-2-ylthio)benzo[d]thiazoles as novel c-Jun
N-terminal kinase inhibitors
Surya K. De a, Li-Hsing Chen a, John L. Stebbins a, Thomas Machleidt b, Megan Riel-Mehan a, Russell Dahl a,
Vida Chen a, Hongbin Yuan a, Elisa Barile a, Aras Emdadi a, Ria Murphy a, Maurizio Pellecchia a,
*
a Burnham Institute for Medical Research, La Jolla, CA, 92037, USA
b Invitrogen Corporation, 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:
A new series of 2-thioether-benzothiazoles has been synthesized and evaluated for JNK inhibition. The
SAR studies led to the discovery of potent, allosteric JNK inhibitors with selectivity against p38.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 10 December 2008
Revised 17 February 2009
Accepted 20 February 2009
Available online 26 February 2009
Keywords:
JNK1
JNK2
JIP1
DJNKI
Allosteric kinase inbhibitors
The c-Jun N-terminal kinases (JNKs) were initially described in
the early 1990s as a family of serine/threonine protein kinases,
activated by a range of stress stimuli and able to phosphorylate
the N-terminal transactivation domain of the c-Jun transcription
factor.1
Three distinct genes encoding JNKs have been identified as JNK-
1, JNK-2, and JNK-3, and at least 10 different splicing isoforms exist
in mammalian cells.2–4 The three JNK isoforms share more than
90% amino acid sequence identity and the ATP pocket is >98%
homologous. These proteins are activated in response to cellular
stresses such as heat shock, irradiation, hypoxia, chemotoxins,
and peroxides. They are also activated in response to various cyto-
kines and participate in the onset of apoptosis.5,6 It is reported that
up-regulation of JNK activity is associated with a number of disease
states such as type-2 diabetes, obesity, cancer, inflammation, and
stroke.1–3 Therefore, JNK inhibitors are expected to be effective
therapeutic agents against a variety of diseases.
JNKs bind to substrates and scaffold proteins, such as JIP-1, that
contain a D-domain, as defined by the consensus sequence R/
KXXXXLXL.7,8 A peptide corresponding to the D-domain of JIP-1
(aa 153-163; pep-JIP1), inhibits JNK activity in vitro and displays
remarkable selectivity with little inhibition of the closely related
Erk and p38 MAPKs.9–12 Recent in vivo data, generated for studies
focusing on pep-JIP1 fused to the cell permeable HIV-TAT peptide,
show that its administration in various mice models of insulin
resistance and type-2 diabetes, it restores normoglycemia without
causing hypoglycemia in lean mice.13 The final sequence of the
most extensively used peptide is GRKKRRQRRRPPRPKRPTTLNL
FPQVPRSQDT. The peptide was further improved by the synthesis
of an all-D retro-inverso peptide, D-JNK1; that is a JIP1-derived
peptide synthesized in the reverse sequence from D rather than
L amino acids. However, the peptide’s instability in vivo, its short
half-life, and the costly and inefficient large-scale manufacturing
and purification processes all hampered the development on novel
therapies for diabetes based on D-JNK1 alone.9–13
There has been considerable effort to identify JNK inhibitors
over the past several years.14–22 A drug discovery program in our
lab was initiated with the aim of identifying and characterizing
N
N
N
NO2
HO
S
S
N
N
N
S
O
NO2
S
S
O
A: BI-78D3
B: 87G3
* Corresponding author. Tel.: +1 858 646 3159; fax: +1 858 713 9925.
E-mail address: mpellecchia@burnham.org (M. Pellecchia).
Figure 1. (A) Chemical structure of the previously reported BI-78D3.23 (B) Chemical
structure of BI-87G3.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.046

S. K. De et al. / Bioorg. Med. Chem. 17 (2009) 2712–2717
2713
N
Br
NO2
S
N
R1
R2
N
X
R1
R2
N
X
SH
NO2
S
S
NaOMe, MeOH
rt
X = NH or O or S (see Table 1)
Scheme 1.
Table 1
Inhibition results for benzothiazoles, benzimidazoles, benzoxazoles derivatives against JNK
Compounds
ID BI-
Lantha screen kinase activity assay, IC50
(l
M)
pepJIP1 DELFIA displacement assay, IC50 (lM)
N
NO2
S
N
S
N
87B8
55 21.8
1.0
N
H
H2N
N
H
N
N
N
87C2
87C4
8.1 0.74
4.2 0.51
0.67
0.4
NO2
NO2
S
S
S
N
S
N
S
N
NH2
N
N
N
S
87C7
70 33.9
27 14.2
ND
<1
N
H
N
OH
NH2
O
N
N
NO2
87C10
S
S
N
H
N
N
S
OH
87D4
>100
ND
N
H
O
N
S
N
O2N
NO2
87D5
87D6
6.4 0.43
23 4.60
3.2
1.5
S
S
N
H
N
N
Cl
Cl
NO2
S
N
H
N
N
O
87D7
2.7 0.11
0.24
NO2
S
S
N
N
MeO
MeO
N
NO2
NO2
87D10
87D11
4.7 0.96
1.8 0.04
0.13
0.10
S
S
S
S
N
H
N
S
(continued on next page)

2714
S. K. De et al. / Bioorg. Med. Chem. 17 (2009) 2712–2717
Table 1 (continued)
Compounds
ID BI-
Lantha screen kinase activity assay, IC50
3.8 0.01
(
lM)
pepJIP1 DELFIA displacement assay, IC50
0.02
(lM)
N
N
NO2
S
87D12
S
N
H
N
F3C
N
NO2
S
87E1
87E7
87E9
87F1
87F2
20.2 5.37
0.14
ND
0.11
ND
>1
S
N
H
NO2
N
O
>100
S
NO2
N
Cl
N
27 3.37
NO2
S
S
O
O
S
N
>50
NH2
S
O
O
N
Cl
N
S
>50
NO2
S
S
N
N
87F3
87F4
87F6
>50
50
0.29
0.098
1.7
NO2
S
S
O
N
S
S
S
S
S
N
O
N
S
N
>50
NH2
S
N
MeO
N
N
N
NO2
87F7
87F8
87F11
>50
>50
>50
1.5
S
S
N
N
N
S
0.796
ND
NO2
S
S
S
Cl
Cl
Cl
N
N
S
S
N
N
N
N
N
S
87F12
87G2
>50
>50
ND
ND
S
N
N
N
NO2
S
S
O
S

S. K. De et al. / Bioorg. Med. Chem. 17 (2009) 2712–2717
2715
Table 1 (continued)
Compounds
ID BI-
87G3
Lantha screen kinase activity assay, IC50
1.8 0.09
(lM)
pepJIP1 DELFIA displacement assay, IC50
0.16
(lM)
N
N
S
S
NO2
N
S
N
N
S
N
S
Cl
87G10
>50
ND
O
O
OMe
N
N
N
S
S
N
87G11
>50
ND
S
Cl
Cl
N
N
S
N
87G12
87H1
>50
>50
ND
ND
N
S
Cl
Cl
N
HO3S
N
NO2
S
N
H
ND, no displacement at 100
lM.
small molecule JNK inhibitors as novel chemical entities. Very re-
cently, we have reported the identification of 4-(2,3-dihydro-
benzo[b][1,4]dioxin-6-yl)-5-(5-nitrothiazol-2-ylthio)-4H-1,2,4-
triazol-3-ol (BI-78D3)23 (Figure 1A) inhibitor from our HTS
libraries as the first potent and selective JNK inhibitor of this class
that demonstrates in vivo activity in a diabetes mice model of insulin
resistance. In our continued interest in the development of JNK
inhibitors,22,23 we now report a comprehensive structure–activity
relationship studies describing novel small molecules benzothiazole
derivatives as JNK inhibitors that target the JIP-JNK interaction site.
Initially, we synthesized several analogs of purine-thioether
derivatives (BI-87B8, BI-87C7, and BI-87C10) but none of these
were good JNK inhibitors. We therefore decided to focus on the
benzimidazole series. We synthesized several analogs of this series
(Scheme 1).24 Compounds were synthesized by nucleophilic sub-
stitution of 2-bromo-5-nitrothiazole with the corresponding thiol
of benzimidazole, benzoxazole, and benzothiazole in the presence
of NaOMe in methanol at room temperature.24 We observed that
methoxy (BI-87D10), methyl (BI-87D12), and nitro (BI-87D5)
groups were tolerated on benzimidazole derivatives. Interestingly,
1-position on benzimidazole blocked with methyl (BI-87F7)
showed no activity in kinase assay. The benzoxazole series showed
almost the same activity as the benzimidazole series (BI-87D7).
Benzothiazoles showed much better inhibition potency than other
two series. In the benzothiazole series, the activity was similar
with or without methoxy (BI-87D11, BI-87G3). However, when a
chloro (BI-87F2 or BI-87F8) or an ethoxy (BI-87G2) was present
at the 5 or 6 position, compounds were inactive likely due to steric
hindrance. We also tested several disulfide analogs of benzothia-
zole compounds, they were not active in kinase assay but some
had a decent activity in a DELFIA based pepJIP1 displacement as-
say23 (Table 1).
Figure 2. (A) and (B) Docked structure of compound 87G3 in the JIP site of JNK1.

2716
S. K. De et al. / Bioorg. Med. Chem. 17 (2009) 2712–2717
100
80
60
40
20
0
0.45
0.40
0.35
0.30
IC50 = 1.804 µM
IC50~15 µM
0.25
0.20
0.1
1
10
100
0.001
0.01
0.1
1
10
100
Concentration, µM
Concentration, µM
Figure 3A. Kinase inhibition assay for 87G3.
Figure 4. TR-FRET analysis of c-Jun phosphorylation upon TNF-alpha stimulation of
HeLa cells in the presence of increasing 87G3.
Delfia Assay
150
100
50
be at least 50 times less active (Table 2) against p38
the MAPK family with high structural similarity to JNK and inactive
against Akt kinase (no significant inhibition at 100 M). These
a, a member of
l
compounds were also inactive against other unrelated proteins un-
der investigation in our laboratory, including proteases such as
lethal factor and furin (Table 2), further corroborating that this
compound may selectively interfere with the JNK docking site. This
selectivity is in agreement with our previous findings with com-
pound BI-78D323 and with the reported data on pepJIP1.9–13 Liquid
chromatography/mass spectrometry analysis demonstrated
that compound BI-87G3 has favorable microsome stability
(t1/2 = 70 min) and plasma stability (80% remaining after 1 h in
plasma stability analysis).31
0
-4
-3
-2
-1
0
Log Concentrations (µM)
In an attempt to further profile the properties of compound BI-
87G3 in the context of a complex cellular milieu, we employed the
Figure 3B. Dose dependent displacement of biotinylated pepJIP1 from GST-JNK1.
cell-based LanthaScreen kinase assay.32 In this assay compound
TM
BI-87G3 is able to inhibit TNF-a stimulated phosphorylation of c-
Jun in cell (IC50 = 15 M; Figure 4). It should be noted that the
l
Table 2
Selectivity profile
cell-based system employed makes use of a GFP-c-Jun stable
expression system. As a result, the levels of GFP-c-Jun in these cells
are higher than endogenous levels. This could have an inflationary
effect on the IC50 values obtained with this assay when testing
substrate competitive compounds. Nonetheless, this finding
establishes that compound BI-87G3 is able to function in a cellular
context.
Compound JNK, IC50
M)
p-38
a
, IC50
Akt, IC50
(
l
M)
Furin, IC50
M)
LF, IC50
M)
(l
(l
M)
(l
(l
BI-87D11
BI-87G3
1.8
>100
5% inhibition at
100
2% inhibition at
100
>50
>100
l
M
1.8
>100
>50
>100
l
M
In conclusion, we successfully developed a new series of JNK
inhibitors, many of which are potent in vitro. Our results indicate
that targeting the protein–protein interaction between JNK and
JIP with a small molecule is a new and promising avenue for JNK
related therapeutics.
Modeling studies25–30 suggest that compound BI-87G3 may
bind at the JIP site with the nitrothiazole group crossing the ridge
close to residues Arg127 and Cys163 (Fig. 2 A and 2B). Its benzo-
thiazole group could then form a hydrogen bond with Arg127 and
could bind into a sub-pocket nearby, which based on this hypoth-
esis, could tolerate different groups with the similar size to sub-
stitute the benzothiazole group of BI-87G3. We have previously
recognized that the Cys residue plays a major role in the binding
of these compounds and it is entirely possible that the observed
SAR is function of the ability of the various substituents to either
bring the thiols from the compound and the Cys in close proxim-
ity for reactivity or to directly increase the reactivity of the com-
pound itself.23 While we have not completely ruled out these
possibilities, we could not observe clear evidence for covalent
binding.
Acknowledgments
We gratefully acknowledge financial support from the NIH
(Grants # DK073274 and DK080263) and Syndexa pharmaceuticals
(to M.P.).
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kinase activity and the pepJIP1 DELFIA displacement assays.23
Compound BI-87G3 showed an IC50 of 1.8 lM in the kinase assay
(Fig. 3A) in a substrate competitive manner. In addition, compound
BI-87G3 also showed an IC50 of 160 nM in displacing pepJIP 1
(Fig. 3B). Compounds BI-87G3 and BI-87D11 were also found to

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31. I. Microsomal Stability Assay (RLM assay). Test compound solutions were
incubated with RAT liver microsomes (RLM) for 60 min at 37.5 °C. The final
incubation solutions contained 4 lM test compound, 2 mM NADPH, 1 mg/mL
16. Gaillard, P.; Jeanclaude-Etter, I.; Ardissone, V.; Arkinstall, S.; Cambet, Y.;
Camps, M.; Chabert, C.; Church, D.; Cirillo, R.; Gretener, D.; Halazy, S.; Nichols,
A.; Szyndralewiez, C.; Vitte, P.-A.; Gotteland, J.-P. J. Med. Chem. 2005, 48, 4596.
17. Szczepankiewicz, B. G.; Kosogof, C.; Nelson, L. T. J.; Liu, G.; Liu, B.; Zhao, H.;
Serby, M. D.; Xin, Z.; Liu, M.; Gum, R. J.; Haasch, D. L.; Wang, S.; Clampit, J. E.;
Johnson, E. F.; Lubben, T. H.; Stashko, M. A.; Olejniczak, E. T.; Sun, C.; Dorwin, S.
A.; Haskins, K.; Abad-Zapatero, C.; Fry, E. H.; Hutchins, C. W.; Sham, H. L.;
Rondinone, C. M.; Trevillyan, J. M. J. Med. Chem. 2006, 49, 3563.
(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 centrifugation, the samples were analyzed by LC–MS. Test
compounds were run in duplicate with 2 control compounds of known half life.
HLM t1/2 > 30 min is generally classified as good microsomal stability. II. Plasma
18. Swahn, B.-M.; Huerta, F.; Kallin, E.; Malmström, J.; Weigelt, T.; Viklund, J.;
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stability assay: Test compound solution was incubated (1 lM, 2.5% final DMSO
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.
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24. Synthesis of 2-(5-nitrothiazol-2-ylthio)benzo[d]thiazole (87G3): To a solution
of benzothiazol-2-thiol (167 mg, 1 mmol) in methanol (5 mL) was added
MeONa (2.4 mL, 0.5 M solution in methanol, 1.2 mmol) at room temperature.
After stirring for 5 min, 2-bromo-5-nitro thiazole (229 mg, 1.1 mmol) was
added to the reaction mixture and stirred until deemed complete by TLC (16 h).
The reaction mixture was acidified with 1 N HCl and the resulting precipitate
was collected by filtration and washed with water (2 Â 30 mL), haxanes
(2 Â 30 mL), and 10% ethyl acetate in hexanes (2 Â 30 mL) to give a white solid.
The residue was chromatographed over silica gel (40% ethyl acetate in hexane)
to afford the 87G3 (217 mg, 74%). 1H NMR (300 MHz, DMSO-d6) d 7.54 (t,
J = 7.5 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.20 (d, J = 7.8 Hz,
1H), 8.88 (s, 1H); HRMS calcd for C10H5N3O2S3: 294.9544, found 294.9542.All
compounds were made following the same procedure. Compound 87F4 was
commercially available from Aldrich.
32. Cell based assays for c-Jun phosphorylation: assays for c-Jun and ATF2
phosphorylation were carried out using the LanthaScreen c-Jun (1-79) Hela
(Invitrogen, Carlsbad, CA) which stably express GFP-c-Jun 1-79.
Phosphorylation was determined by measuring the time resolved FRET (TR-
FRET) between a terbium labeled phospho-specific antibody and the GFP-
fusion protein.12 The cells were plated in white tissue culture treated 384 well
plates at a density of 10,000 cell per well in 32
supplemented with 1% charcoal/dextran-treated FBS, 100 U/mL penicillin and
100 g/mL streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium
l ,
l assay medium (Opti-MEMÒ
l
pyruvate, 25 mM HEPES pH 7.3, and lacking phenol red). After overnight
incubation, cells were pretreated for 60 min with compound (indicated
concentration) followed by 30 min of stimulation 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 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
25. Zhao, H.; Serby, M. D.; Xin, Z.; Szczepankiewicz, B. G.; Liu, M.; Kosogof, C.; Liu,
B.; Nelson, L. T. J.; Johnson, E. F.; Wang, S.; Pederson, T.; Gum, R. J.; Clampit, J. E.;
340 nm, emission 520 nm and 490 nm; 100
time, emission ratio = Em520/Em 490).
ls lag time, 200 ls integration