Design, synthesis, and biological activity of novel, potent, and selective (benzoylaminomethyl)thiophene sulfonamide inhibitors of c-Jun-N-terminal kinase

Article abstract of DOI:10.1021/jm031112e

Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a critical role in a wide range of disease states including cell death (apoptosis)-related and inflammatory disorders (epilepsy, brain, heart and renal ischemia, ne

Full text of DOI:10.1021/jm031112e

J. Med. Chem. 2004, 47, 6921-6934
6921
Design, Synthesis, and Biological Activity of Novel, Potent, and Selective
(Benzoylaminomethyl)thiophene Sulfonamide Inhibitors of c-Jun-N-Terminal
Kinase
Thomas Ru¨ckle,† Marco Biamonte,†, Tania Grippi-Vallotton,† Steve Arkinstall,X Yves Cambet,§
Montserrat Camps,‡ Christian Chabert,‡ Dennis J. Church,† Serge Halazy,† Xuliang Jiang,| Isabelle Martinou,§
Anthony Nichols,§ Wolfgang Sauer,† and Jean-Pierre Gotteland*,†
Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland, and
Serono Reproductive Biology Institute, 1-Technology Drive, Rockland, Massachusetts 02370
Received November 27, 2003
Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a
critical role in a wide range of disease states including cell death (apoptosis)-related and
inflammatory disorders (epilepsy, brain, heart and renal ischemia, neurodegenerative diseases,
multiple sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome). The screening of
a compound collection led to the identification of a 2-(benzoylaminomethyl)thiophene sulfon-
amide (AS004509, compound I) as a potent and selective JNK inhibitor. Chemistry and
structure-activity relationship (SAR) studies performed around this novel kinase-inhibiting
motif indicated that the left and central parts of the molecule were instrumental to maintaining
potency at the enzyme. Accordingly, we investigated the JNK-inhibiting properties of a number
of variants of the right-hand moiety of the molecule, which led to the identification of
2-(benzoylaminomethyl)thiophene sulfonamide benzotriazole (AS600292, compound 50a), the
first potent and selective JNK inhibitor of this class which demonstrates a protective action
against neuronal cell death induced by growth factor and serum deprivation.
Introduction
splicing isoforms exist in mammalian cells.4 JNK1 and
JNK2 are widely expressed in a variety of tissues, while
in contrast, JNK3 is selectively expressed in the brain,
heart, and testis.4,9 Each JNK isoform binds to its
substrates with a different affinity, suggesting that
substrate specificity is the central regulatory element
of JNK-dependent signaling pathways in vivo.4
In terms of the physiological function of JNKs, mice
lacking jnk1 or jnk2 exhibit deficits in T-helper (CD4+)
cell function.10-12 Double knockout animals are embry-
onic lethal, although fibroblasts from such animals are
viable in vitro and exhibit a remarkable resistance to
radiation-induced apoptosis.13 The jnk3 knockout mouse
exhibits resistance to kainic acid-induced apoptosis in
the hippocampus and to subsequent seizures.14 It there-
fore appears that JNK activity is critical for both the
immune response and for programmed cell death,15 and
that therapeutic inhibition of JNKs may provide clinical
benefits in a wide range of apoptosis-related and
inflammatory disorders such as neurodegenerative dis-
eases, reperfusion injury, multiple sclerosis, and rheu-
matoid arthritis.16 Along the same lines, recent evidence
further suggests that JNK inhibitors may also prove
beneficial in the treatment of vascular, metabolic,17 and
oncological diseases.18
The c-Jun N-terminal kinases (JNKs) (also known as
‘stress-activated protein kinases’) are members of the
mitogen-activated protein kinase (MAPK) family to-
gether with p38 mitogen-activated protein kinases (p38
kinases) and extracellular signal-regulated kinases
(ERKs). MAP kinases are serine/threonine kinases that
are activated by dual phosphorylation of the threonine
and tyrosine residues of the Thr-X-Tyr segment located
on a loop adjacent to the active site.1,2 The activation of
each MAP kinase is carried out by a specific MAP kinase
kinase. Activated MAP kinases phosphorylate various
substrates, including transcription factors such as c-Jun,
ATF-2, Elk1, NFAT, p53, and a cell death domain
protein,5-8 which in turn mediate the response to
stimuli by regulating the expression of specific sets of
genes. Accordingly, members of the JNK family of
kinases are activated by the proinflammatory cytokines
tumor necrosis factor-R (TNF-R) and interleukin-1â (IL-
1â), as well as by environmental stress, such as that
induced by anisomycin, UV irradiation, hypoxia, and
osmotic shock.3
Three distinct genes encoding JNKs have been identi-
fied (jnk1, jnk2, and jnk3), and at least 10 different
* To whom correspondence should be addressed. Phone:
+41227069824. Fax: +41227946965. E-mail: jean-pierre.gotteland@
serono.com.
An increasing number of JNK inhibitors have entered
development pipelines in the past few years,16 such as
the anthrapyrazolone SP600125, which is an ATP-
competitive JNK inhibitor that exhibits moderate se-
lectivity in a range of Ser/Thr- and Tyr- protein kinases
assays.17,19 Another important contribution to the iden-
tification of JNK inhibitors has been made very recently
by Merck researchers, who have published the JNK3
† Department of Chemistry, Serono Pharmaceutical Research In-
stitute.
‡ Department of Signaling, Serono Pharmaceutical Research Insti-
tute.
§ Department of Screening, Serono Pharmaceutical Research Insti-
tute.
| Department of Chemistry, Serono Reproductive Biology Institute.
X Department of Signaling, Serono Reproductive Biology Institute.
Present address: Conforma Therapeutics Corp., 9393 Towne
Centre Dr., Suite 240, San Diego, CA 92121.
10.1021/jm031112e CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/26/2004

6922 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Table 1. Kinase Selectivity Profile of Initial Hit Compound I
Ru¨ckle et al.
kinase
JNK3
0.3
JNK2
1.1
JNK1
1.36
p38
MEK1
ERK1
PKC
PI3K
AKT
GSK3
P56Lck
>10
EGF
IC50 (µM)
>10
>10
>10
>10
>10
>10
>10
>10
Chart 1. SAR Mapping of Screening Hit I (AS604509)
Scheme 2a
crystal structure complex with various JNK small
molecule inhibitors.20
In view of the above, we initiated a drug discovery
program aimed at identifying and characterizing novel,
small molecule JNK inhibitors. Here we report on a
class of (benzoylaminomethyl)thiophene sulfonamide
JNK inhibitors21 that demonstrate high selectivity for
the enzymes in question when tested in a panel of
serine/threonine and tyrosines kinases (Table 1). The
starting point for this work was the identification of
compound I in high throughput screening, which led us
to initiate SAR investigations around the “western”,
“central”, and “eastern” parts of the molecule (Chart 1).
a Reagents: (a) Et3N, MeOH, 100 °C; (b) H2SO4, MeOH, reflux
(59%); (c) hydrazine, MeOH, reflux (76%); (d) 2, 4, or 6, Py, CHCl3,
reflux (40-94%).
Chemistry
yield. This was coupled to the sulfonyl chlorides 2, 4,
and 6 under optimized conditions (pyridine, CHCl3) to
yield the acyl hydrazides I, 12, and 13 in crystalline
form (Scheme 2). Initial SAR studies were focused on
point-modifications of inhibitor I. The N-sulfonyl-N′-
acylhydrazine moiety in I was replaced by a glycine-
amide, considered as a bioisostere, by reacting sulfonyl
chloride 2 with glycine-tert-butyl ester in quantitative
yield, followed by an ester-deprotection using 25% TFA
in dichloromethane. Intermediate 14 was then coupled
to the appropriate ethylenediamine 15 (PyBOP/DIEA),
affording 16 in almost quantitative yield.
The synthesis of compound I was carried out on a
multigram scale, without chromatographic purification
of the intermediates (Scheme 1). Commercial 2-ami-
nomethylthiophene was benzoylated and treated with
chlorosulfonic acid to provide the sulfonyl chloride 2 in
moderate yield.22 This reaction could be applied to the
synthesis of the closely related sulfonyl chlorides 4 and
6. On the other hand, γ-aminobutyric acid was treated
with the 2-chloropyridine (8) at 100 °C in an autoclave
to provide carboxylate 9. Esterification and treatment
with hydrazine provided the acyl hydrazide 11 in 59%
Scheme 1a
a Reagents: (a) 4-chlorobenzoyl chloride, DIEA, DCM, 0 °C (98%); (b) HSO3Cl, rt (43-63%); (c) NaH, DMF, 0 °C (98%); (d) 4-chloroaniline,
Et3N, DCM (86%).

Inhibitors of c-Jun-N-Terminal Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6923
Scheme 3a
a Reagents: (a) Glycine-tert-butyl ester, DIEA, DCM, 1h (95%); (b) 25% TFA in DCM, 1 h (quant.); (c) 15, PyBOP, DIEA, DCM, 15h,
rt (95%); (d) H2SO4/MeOH (2 M), reflux, 2 h (89%); (e) NBS, dibenzoylperoxide, CCl4, reflux, 24 h (98%); (f) NaN3, DMF, 80 °C, 2 h; (g)
H2 (50 psi), Pd/C, EtOH, 15 h (50%); (h) 4-chlorobenzoyl chloride, DIEA, DCM, 3 h (90%); (i) LiOH‚H2O, THF/water, 5 h, 60 °C (98%); (j)
11, DCI, HOBt, DCM, 3 h (60%).
Scheme 4a
a Reagents: (a) 11, pyridine, CHCl3, reflux, 2 h (97%); (b) LiAlH4, THF, 30 min (67-87%); (c) 4-chlorobenzoyl chloride, DCM/Py 30:1,
3 h (35-78%); (d) K2CO3, MeOH, 50 °C, 1 h (97%); (e) SnCl2‚2H2O, DMF, 15 h (36%).
Scheme 5a
a Reagents: (a) HSO3Cl, THF; (b) NaOH, Bu4NOH (97%, two steps); (c) (COCl2)3, DMF, DCM (60%).
The carbonyl analogue 18 was synthesized starting
from commercially available 5-methylthiophene-2-car-
boxylic acid. Intermediate 17 could be accessed over four
steps without purification in 34% yield. The methyl
group on 2-methylthiophene was converted into an
aminomethyl group by a bromination/azide nucleophilic
replacement/hydrogenation sequence23 to give interme-
diate 17. Ester 17 was then saponified and coupled
(DCI, HOBt) to hydrazide 11, affording 18 in moderate
yield (Scheme 3).
In a second stage, modifications on the central
thiophene ring were addressed. Replacement of the
thiophene moiety by a furan heterocycle was performed
using chemistry analogous to the one described in
Schemes 1-3 and is not further discussed here. Bio-
isosteric replacement by a benzene ring, substituted in
either the meta or para position, started from the
corresponding commercially available cyanobenzene-
sulfonyl chlorides. Meta- and para-substituted cyanoben-
zenes were coupled to hydrazide 11 under reflux,
followed by nitrile reduction and ultimately acylation,
leading to compounds 21 and 22 with good overall
yields. First efforts to rigidify the flexible aminomethyl
moiety were undertaken by shortening the linkage to
an anilino derivative and later by replacing the core
structure by a tetrahydro-isoquinoline moiety. The
aniline derivative 26 was accessed by coupling 3-ni-
trobenzenesulfonyl chloride to hydrazide 11, followed
by reduction (SnCl2-dihydrate), and by acylating the
aniline intermediate 25 (overall yield 75%). The tet-
rahydroisoquinoline analogue 24 was synthesized start-
ing from its commercially available trifluoroacetyl de-
rivative, which was coupled quantitatively to hydrazide
11, followed by deprotection using potassium carbonate
in methanol. The free amine intermediate 23 was
acylated as above (Scheme 4).
The route to sulfonyl chlorides 2, 4, and 6 could not
always be successfully applied to analogues carrying
substituents others than p-chloro. For instance, treat-
ment of m-methoxybenzamide 27 (Scheme 5) with
HSO3Cl gave a gummy reaction mixture, and partial
HSO3Cl-catalyzed hydrolysis during workup lowered the
yield. Accordingly, it was preferable to deliberately
induce hydrolysis (1 equiv of aq NaOH) and to extract
the sulfonic acid as its tetrabutylammonium salt (Bu4-
NOH/DCM).24 The nicely tractable sulfonate 28 was

6924 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Ru¨ckle et al.
Chart 2. Focused Set of Western End Replacements
Scheme 6a
The latter was treated with a slight deficit of an acyl
chloride (Scheme 6, route e), sulfonyl chloride (route f),
or isocyanate (route g), respectively, to provide the
amides I, 34a-w, 35a-g, 36a-f, the sulfonamides (e.g.
37), or the ureas 38a-c in typically 82-100% optical
purity (254 nm). 32b could also be reductively alkylated
using the corresponding aldehydes in the presence of
NaBH(OAc)3 (route h) to afford secondary amines (e.g.
39).
The N-acyl-N′-sulfonylhydrazine moiety was per-
ceived undesirable as far as ‘druglike properties’ are
concerned (high MW, low permeability, N-N-linkage,
high number of rotatable bonds).28 Efforts were there-
fore directed at replacing this portion of the molecule.
The sulfonyl chlorides 2 and 29 were reacted in parallel
with a number of amines. These syntheses were carried
out in solution using polymer-supported reagents af-
fording the corresponding sulfonamides without further
purification. Typically, an equimolar mixture of sulfonyl
chlorides with the corresponding amines in the presence
of polymer-bound morpholine were stirred overnight in
DCM/DMF at room temperature using a Quest210
parallel synthesizer. Polymer-bound sulfonyl chloride
and/or polymer-bound aminomethyl-PS were added to
scavenge eventual excess of starting materials as de-
tected by HPLC. The corresponding sulfonamides were
obtained in high yield (60-98%) and purity (90-99%)
(Scheme 7). At first, amines were selected based on their
diversity and then in an increasingly focused way to
culminate in 4-acyl- and alkylpiperidines, 4 arylpipera-
zines, and finally 4-benzotriazolopiperidines (Chart 2).
a Reagents: (a) allyl bromide, DIEA (80%); (b) t-BuLi, THF, -78
°C; SO2, -78 °C to rt; NCS (53%); (c) 11, Py, CHCl3, reflux (98%);
(d) N-dimethylbarbituric acid, Pd(PPh3)4, DCM (67%); (e) RCOCl,
pyridine:THF 1:5, -40 °C (50-80%); (f) RSO2Cl, Py:THF 1:5, rt
(50%); (g) RNCO, Py:THF 1:5, rt (70%); (h) RCHO, NaBH(OAc)3,
DCE, rt (97%).
Scheme 7a
a Reagents: (a) polymer-bound morpholine (4 equiv), amine (1
equiv), DCM/DMF, 15 h, rt; (b) polymer-bound aminomethylben-
zene (2 equiv), polymer-bound isocyanate (2 equiv), 5 h, rt (60-
98%).
readily converted to the sulfonyl chloride 29 with
triphosgene in the presence of catalytic amounts of
DMF.25
The route to hit compound I was modified again for
the exploration of the SAR around the 4-chlorobenz-
amido motif. 2-Aminomethylthiophene was protected as
its bisallyl derivative 30 (allyl-Br, Et3N) and converted
into the thien-2-ylsulfonyl chloride 31 via metalation
(i. t-BuLi, -78C. ii. SO2. iii. NCS).26 It is important to
note that while chlorosulfonylation of 27 under ‘stan-
dard’ conditions with HSO3Cl provided 2% of the
unwanted thien-3-yl regioisomer, treatment of 30 with
t-BuLi/SO2/NCS afforded in contrast less than 0.5% of
the thien-3-yl isomer. 31 was then coupled to the
corresponding acyl hydrazide 11 to yield the bis-allyl
aduct 32a, which after deprotection (Pd(PPh3)4, N,N′-
dimethylbarbituric acid)27 gave the primary amine 32b.
The latter led to the very promising benzotriazole
subseries as highly active JNK inhibitors, which caused
us to synthesize additional analogues. Because only two
4-benzotriazolopiperidines are commercially available,
we elaborated a synthetic scheme to access such motifs
(Scheme 8); the most straightforward way turned out
to be a Mitsunobu-type reaction.29 Benzotriazoles, ben-
zimidazoles, or azabenzotriazoles were reacted with
N-Boc-4-hydroxypiperidine in the presence of PPh3 and
DEAD in anhydrous THF, affording the corresponding
Scheme 8a,b
a Reagents: (a) N-Boc-4-hydroxypiperidine, DEAD, PPh3, THF (20-80%), 3 h; (b) 20% TFA/DCM, 2 h (100%), (c) i. 2 or 29, polymer-
bound morpholine, DCM/DMF, ii polymer-bound aminomethylbenzene, polymer bound isocyanate. bIntermediates 44a-k are deprotected
to give intermediates 47a-k, which are coupled ultimately to 2 or 29 to generate 50a-k. Intermediates 45a-e are deprotected to give
intermediates 48a-e, which are coupled to 2 or 29 to yield 51a-e. Intermediates 46a-d are deprotected to give intermediates 49a-d,
which are coupled ultimately to 2 or 29 to produce 52a-d.

Inhibitors of c-Jun-N-Terminal Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6925
Chart 3. Assignment of Regioisomers of
1-(4-Piperidyl)-1-Boc-triazoles 44a-k, 45a-e, 46a-d
Using 2D-NMR NOESY Experiments (cross-peaks
between H7arom with H4piperdine and H3piperidine were
studied)
Scheme 9a
a Reagents: (a) 2-nitro-1-fluorobenzene, DMF, 80 °C, 15 h (78%);
(b) H2, Pd/C, EtOH, 3 h (75%); (c) TFA, DCM, 3 h (45%); (d) 2,
DIEA, DCM/DMF (60%).
1-(4-piperidyl)-1-Boc-benzotriazoles, -benzimidazoles, or
-azabenzotriazoles. In the case of unsubstituted benzo-
triazoles, two regioisomers could be expected, while for
substituted benzotriazoles or heteroaromatic benzazoles,
three different isomers were expected.
In all cases, all possible isomers were formed, but
could be separated on gram scale using silica gel
chromatography. The structures were determined using
2D-NMR experiments (Chart 3). Removal of Boc using
standard techniques led to the novel building blocks of
N-(4-piperidinyl)triazoles and imidazoles, which were
ultimately coupled to sulfonyl chlorides 2 and 29.
Derivative 50m could be accessed via a different route
starting from 2-nitro-fluoro-benzene, which was coupled
to N-Boc-4-aminopiperidine in an SNAr type reaction.
Reduction of the nitro-group led to the key intermediate,
which was cyclized in one step to the desired 2-trifluo-
romethylbenzimidazole, after the Boc group had been
removed (see Scheme 9).
Results and Discussion
Already at the beginning of this work it was evident
that the western benzoylaminomethyl moiety had an
instrumental contribution for JNK activity, as pointed
out in Table 2. Removal of this group led to a significant
loss of activity (as exemplified by compound 32b),
Table 2. In Vitro Activity of Key Compounds
a All values in triplicate. b Method A: prepared according to Scheme 2. Method B: prepared according to Scheme 6. Method C: prepared
according to Scheme 3. Method D: prepared according to Scheme 4. c Analytical data. a: CHN, NMR, LC-MS; b: NMR, HPLC, LC-MS.

6926 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Ru¨ckle et al.
Table 3. SAR Table of Western End Modifications
whereas modifications of the benzoylaminomethyl func-
tionality offered only minor freedom to operate. Indeed,
alkylation of the amide moiety resulted in compounds
of reduced potency (12 and 24, Table 2), and only the
inverse amide (cpd 13, Table 2) retained activity to a
certain degree. Replacement of the thiophene central
core by a benzene ring (21 and 22, Table 2) was
tolerated, and gave similar activity as hit I. However,
removal of the methylene group (resulting in an aniline)
lowered activity as shown by 26 (Table 2). At this point
the decision was made to keep the central thiophene
ring for further SAR evaluation, being a well-balanced
compromise between ease of synthesis and ability to
confer JNK activity.
Additional exploration of the key binding elements in
I revealed that the thiophene-sulfonamide linkage was
crucial for activity, since loss of potency was observed
when replacing it by an amide moiety (18, Table 2).
Surprisingly, the effect was less pronounced when the
SO2 functionality was replaced by a methylene group,
leading to only a 4-fold loss in activity. This may suggest
that none of the oxygen atoms of the sulfonamide are
directly implicated in binding, but the sulfonamide
rather serves as a scaffold.
IC50 (µM)b
r-JNK3 r-JNK2 methoda anal.c
prep
compd
R
Benzamides, X ) R-Ph-CONH
34a
I
H
0.45
0.30
0.33
0.46
0.52
0.64
5.2
0.51
0.98
0.62
1.6
1.01
1.8
1.5
0.79
1.6
n.d.
1.58
1.7
2
n.d.
7.5
3.5
2.7
2.1
1.7
3.6
1.2
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
b
a
b
b
b
b
a
a
b
b
b
b
b
b
a
b
b
a
b
a
b
b
b
4-Cl
34b
34c
34d
34e
34f
34g
34h
34i
34j
34k
34l
4-F
4-Br
4-CH3
4-CH2CH3
4-C(CH3)3
4-CF3
4-NO2
4-OCH3
4-N(CH3)2
4-OH
>5
0.62
0.54
0.64
0.43
0.20
0.21
0.26
4.2
4-NH2
34m 3-Cl
34n
34o
34p
34r
34s
34t
3-Br
3-OH
3-OCH3
3-NO2
2-Cl
2-Br
2-OH
>5
>5
4.3
n.d.
10
3.0
34u
34v
0.25
Finally, switching NH-NH-CO-CH2 to NH-CH2-
CO-NH (16, Table 2) resulted in a 4-fold loss of
inhibitory potency, suggesting that the eastern part of
compound I could potentially be tailored.
2,6-dichloro
34w 3,5-dichloro
>5
1.1
Heteroaromatic Amides X ) R-CONH
35a
35b
35c
35d
35e
35f
fur-2-yl
1.09
0.38
0.68
0.78
0.40
0.23
7.9
8.6
A
A
A
A
A
A
A
b
a
a
b
b
a
a
Further profiling of the key western region of com-
pound I established the preferred substitution of the
aromatic ring (Table 3). The p-chlorine atom is not
necessary for activity (34a) and can be replaced by many
other substituents provided they keep the section flat
(34b-e, 34g-i and 34k,l). However introduction of
bulky substituents led to a drop in activity (34f, 34j).
Single meta substituents were acceptable (34m-r), but
not meta disubstitution (34w), while ortho substituents
tended to be deleterious (34s,t and 34v) with the notable
exception of OH (34u). We hypothesized here that the
o-hydroxy group stabilizes the planarity of the benz-
amide moiety of these molecules.
We further demonstrated the critical importance of
the aromatic amide group. Other aryl amides retain the
activity (35a-f), but alkyl amides do not (36a,f) recon-
firming that JNK inhibitory activity cannot be explained
by a distinct hydrogen bond acceptor/donor interaction
but rather by nonspecific hydrophobic (aromatic) inter-
actions. Spacers between the aromatic ring and the
amido group decrease the activity (36b-e). Further-
more, we found that the amide linkage could not be
replaced by sulfonamide (37), urea (38a-c), or amine
linkages (entry 39) without loss of activity. For further
optimization on the eastern end, we maintained the
4-chloro substitution, also because of its capability to
prevent oxidative metabolism on the aromatic ring. We
found that the (arylamino)butyrylhydrazino moiety on
the eastern end was not strictly necessary for activity
(Table 2) and that the isomeric glycine 16 was also
active. However, when the γ-aminobutyric spacer was
replaced by a more rigid linker, such as 4-carboxypip-
eridine, significant drop in activitity was observed (33).
Alternatively, this moiety could be truncated altogether
(40a,b, Table 4) or replaced by a piperazine or piperidine
(40c,d) with a 3-10-fold loss of activity (Table 4).
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
3-pyridiyl-2-OH
3-pyridiyl-2-SH
3-pyridyl-2-NH2
n.d.
8.7
7.8
n.d.
n.d.
n.d.
35g
Alkyl Amides X ) R-CONH
36a
36b
36c
36d
36e
36f
CH3
>5
>5
>5
>5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
A
A
A
A
A
A
a
b
b
b
a
b
Ph-CH2
Ph-OCH2
Ph-CH2CH2
Ph-CHdCH
cyclohexyl
6.5
>5
Sulfonamides, Ureas, and Amines X ) R
37
Ph-SO2NH
>5
2.0
1.3
>5
> 10
n.d.
>5
B
C
C
C
D
a
b
a
b
b
38a
38b
38c
39
Ph-CH2NHCONH
4-Cl-Ph-NHCONH
2-Cl-Ph-NHCONH
Ph-CH2NH
n.d.
n.d.
n.d.
a Method A: prepared according to Scheme 6 route e. Method
B: prepared according to Scheme 6 route f. Method C: prepared
according to Scheme 6 route g. Method D: prepared according to
Scheme 6 route h. b Purity ) 87 ( 6%; all values in triplicate.
c Analytical data. a: CHN, NMR, LC-MS; b: HPLC, LC-MS.
Activity could be regained by proper substitution of the
piperazine or piperidine scaffold. In the piperazine
series, N-aryl (41a-d) and N-alkyl (41e-g) derivatives
proved to be poorly active, while N-acyl derivatives
(42b-e) showed IC50’s < 1 µM. In the piperidines series,
substitution was implemented via -O- (43a-c), -NH-
(43d,e), -CH- (43f,g), or -CO- (entry 43h), linking
groups. Compounds with IC50’s e 1 µM were found in
all of the piperidine subseries, with the 1-hydroxyben-
zotriazolo- and the anilinopiperidines being the most
interesting ones (43c,d). According to the results de-
scribed above and the marked differences in activity
depending on the carbocycle and its substitution pat-

Inhibitors of c-Jun-N-Terminal Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6927
Table 5. SAR-table of Focused Eastern End Modifications
Table 4. SAR Table of Eastern End Modifications
a All values in triplicate, standard deviations are below 15%.
b Analytical data a: CHN, NMR, LC-MS; b: NMR, LC-MS; typical
purity >90%.
Table 6. SAR Table of Focused Eastern End Modifications.
Heteroaromatic Benzazoles
a All values in triplicate, standard deviations are below 15%.
b Analytical data. a: CHN, NMR, LC-MS; b: NMR, LC-MS; typical
purity >90%.
tern, it appears that a kink between the carbocyclic unit
and an aromatic center is beneficial for JNK activity.
Compounds 41b, 41c, and 42a were the first com-
pounds synthesized in this SAR program that exhibited
activity in a functional JNK3 cell assay of neuronal
death, demonstrating that JNK inhibitors of this type
block neuronal apoptosis in primary cultures of superior
cervical ganglia (SCG) cells subjected to neuronal
growth factor (NGF) deprivation. Compounds possessing
a heteroatom in the kink or in the aromatic ring (e.g.
41b,c) not only show improved inhibitory activity, but
were also interesting at the cellular level. From a
medicinal chemistry point of view, 43c was considered
as an attractive starting point for further investigations
in this direction. Therefore, series of benzotriazolo-,
benzimidazolo- (see Table 5) and azabenzotriazolopip-
eridines (see Table 6) were synthesized. The benzotri-
azole derivatives (Table 5, 1-benzazoles) confirmed our
a All values in triplicate, standard deviations are below 15%.
b Analytical data. a: CHN, NMR, LC-MS; b: NMR, LC-MS; typical
purity >90%.
previous observations: compounds 50a, 50c-f, 50k
improved JNK3-activity by a factor of 2-3 as compared
to I. Benzimidazoles were equally accepted as shown
by 50i-k, whereas benzimidazolones (e.g. 50l) were less
active. The fact that the benzene moiety points toward
a hydrophobic pocket is underlined by the reduced
activity of the 5-carboxy- and 6-carboxybenzotriazole
derivatives (50g-h). Overall, the SAR is rather flat in
this pocket, and a variety of different substituents
appear to be equally acceptable. It is important to notice,
however, that the isomeric 2-benzazole analogues (51a-
d) rather disfavor JNK-inhibitory activity. Only the
smallest unit, the 3,4H-triazole, retained some activity
(51e).

6928 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Ru¨ckle et al.
Figure 1. Effect of compound 50a on neuronal survival. A: Control (-NGF). B: BAF treated, C: +NGF, D: 50a @ 10 µM.
Table 7. Cell Activity of JNK Inhibitors in Neuronal Apoptosis Assay in SCG Cells
rescued neurons (%)
visual estimation
compd
concn (µM)
24 h
48 h
of shape of neuronsa
SB-23906330
I
10
10
10
10
10
10
10
3
18.1 ( 1.8 (2)
10.9 ( 1.8
-
<10
<10
41c
41b
22
22.9 ( 12.1 (4)
43.3 ( 3.5 (2)
n.d.
13.7 ( 0.3 (2)
27.3 ( 8.5 (10)
22.9 (1)
29.9 (1)
++
n.d.
2.1
16
33.4 (1)
+-
+++
++
50a
50a
50a
34u
34u
42a
42d
40c
50e
50i
56.0 ( 9.7 (20)
25.2 ( 8.0 (2)
2.2 ( 0.5 (2)
63.8 ( 22 (5)
1
10
3
10
10
10
10
10
36.3 ( 19.6 (2)
<10
+++
<10
<10
<10
<10
<10
<10
<10
n.d.
n.d.
67.3 ( 25.6 (2)
36.1 ( 10.6 (2)
+++
++
a Toxicity: visual estimation of shape of neurons; from - ) bad shape (toxicity of cpd) to +++ perfect shape of neurons.
Having completed the SAR around this series, we
wondered if the benzazole unit mimicked the adenine
ring of ATP. Therefore, adenine and close analogues
were selected and reacted according to Scheme 8. The
fact that no gain in activity was obtained (Table 6)
suggests that this is not the case. 52a as the smallest
aza analogue of 50i displays a loss of activity by a factor
of 2. Other analogues containing additional nitrogens
(52b) and amino groups (52c,d) on the benzo moiety,
which could potentially mimic adenine, are weak inhibi-
tors in the micromolar range.
The most striking property of benzotriazole deriva-
tives is a clearly improved cellular activity (Table 7).
While I demonstrated activity below 5%, compounds 41c
and 41d showed inhibitory activity in the range of 30%.
However, the real breakthrough was achieved with 50a
and 50e in particular, showing an excellent inhibition
of neuronal cell death induced by in NGF deprived SCG
cells at 10 µM (56.0% and 67%, respectively), which was
still observed at 3 µM for 50a. Full dose-response
determination allowed to evaluate an IC50 value of
1.7 µM.
As shown in Figure 1, neurons are kept alive by
treatment with 50a, a significant protective effect
comparable with that of the caspase inhibitor BAF (@
100 µM). The antiapoptotic potential of this compound
was further evaluated in a neuronal cell apoptosis model
using human teratocarcinoma cells differentiated into
neurons. In this model, apoptosis induced by serum
deprivation was almost completely blocked by compound
50a at 10µM, as well as by the caspase inhibitor BAF
(100 µM).
Another important feature of this class of compounds
is their excellent selectivity profile. 50a was screened
against 80 different Ser/Thr and Tyr-kinases, and did
not significantly inhibit any of them at a concentration
of 10 µM as shown in Table 8.
Nevertheless, the antiapoptotic potential of this new
series of JNK inhibitor and especially compound 50a
did not correlate with in vivo potency in models of
ischemia reperfusion injury due to a very poor biophar-
maceutical profile. This was mainly due to very poor
solubility of the compound (1 µg/mL in PBS) and to a
PK profile characterized by low plasma exposure by both

Inhibitors of c-Jun-N-Terminal Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6929
Table 8. Selectivity Profile of Compound 50a for 80 Protein
by NGF-deprived superior cervical ganglia cells (SCG
cells) with an IC50 value of 1.7 µM. Its high enzymatic
and cellular potency, associated with the high selectivity
profile, allows 50a to be a valuable tool to study JNK
in biological systems.
Kinasesa
protein
kinase
activity
(% of control)
protein
kinase
activity
(% of control)
JNK1R1(h)
JNK2R2(h)
JNK3(h)
Abl(m)
AMPK(r)
Arg(m)
Aurora-A(h)
Axl(h)
Blk(m)
20
16
6
80
90
91
79
134
78
88
93
93
102
99
94
MEK1(h)
MKK4(m)
MKK6(h)
MKK7â(h)
MSK1(h)
p70S6K(h)
PAK2(h)
PDGFRR(h)
PDGFRâ(h)
PDK1(h)
PKA(h)
PKBR(h)
PKBâ(h)
PKBγ(h)
PKCR(h)-His
PKCâII(h)-His
PKCγ(h)-His
PKCδ(h)
PKCꢀ(h)
98
112
106
121
86
77
88
103
91
98
78
51
118
76
101
88
79
95
95
100
99
Experimental Section
General Experimental Methods. Procedures. All chemi-
cals were purchased from Fluka-Aldrich, Buchs (CH), unless
otherwise stated. All polymer-bound reagents were purchased
from Argonaut Technologies. Parallel syntheses were carried
out in a Quest210 parallel synthesizer from Argonaut Tech-
nologies. Parallel evaporations were performed in a HT-4 Atlas
Evaporator from GeneVac. Melting points were measured with
an apparatus Bu¨chi Melting Point B-545 and were uncor-
rected. NMR spectra were recorded on a Brucker DPX-300
MHz spectrometer. Data were reported as follows: chemical
shift in ppm using either residual DMSO (2.49 ppm) or CHCl3
(7.19 ppm) as internal standards on the δ scale, multiplicity
(s ) singlet, d ) doublet, t ) triplet, q ) quadruplet, m )
multiplet), coupling constants (s) in hertz, and integration. MS
data provided were obtained using a mass spectrometer
Perkin-Elmer API 150 EX (APCI). The analytical HPLC was
performed using a HPLC column Waters Symmetry C8 50 ×
4.6 mm, conditions: a- MeCN/H2O 0.09% TFA, 0 to 100% (10
min); b- MeCN/H2O 0.09% TFA, 0 to 100% (20 min); c- MeCN/
H2O 0.09% TFA, 5 to 100% (10 min), max plot 230-400 nm;
d- MeCN/H2O, 5 to 100% (10 min), max plot 230-400 nm.
Elemental analyses were performed on an Erba Science 11108
CHN analyzer.
Bmx(h)
CaMKII(r)
CaMKIV(h)
CDK1/cyclinB(h)
CDK2/cyclinA(h)
CDK2/cyclinE(h)
CDK3/cyclinE(h)
CDK5/p35(h)
CDK6/cyclinD3(h)
CDK7/cyclinH/MAT1(h)
CHK1(h)
97
103
102
94
130
75
PKCη(h)
PKCι(h)
CHK2(h)
CK1(y)
CK2(h)
c-RAF(h)
CSK(h)
cSRC(h)
Fes(h)
FGFR3(h)
Flt3(h)
85
99
93
130
67
134
76
78
PKCµ(h)
PKCθ(h)
PKD2(h)
PRAK(h)
PRK2(h)
ROCK-II(h)
Rsk1(h)
93
113
95
74
92
101
93
77
Rsk2(h)
4-Chloro-N-thiophen-2-ylmethylbenzamide (1). A solu-
tion of 4-chlorobenzoyl chloride (14.52 mL, 114 mmol) in 50
mL of dry DCM was added over 30 min to a stirred solution of
2-aminomethylthiophene (14.1 mL, 137 mmol) and i-Pr2NEt
(43.0 mL, 251 mmol) in DCM (200 mL) at 0 °C. A white solid
was formed, and the reaction was allowed to warm to room
temperature over 1 h. The mixture was diluted with 200 mL
of DCM, washed twice with HCl aq (0.1 N), and dried over
MgSO4. Evaporation gave 28 g (98%) of the title benzamide
as a white solid: mp 153-54 °C, 1H NMR (CDCl3) δ 7.90 (d, J
) 8.67 Hz, 2H), 7.58 (d, J ) 8.67 Hz, 2H), 7.44 (dd, J ) 3.77,
1.13 Ηz, 1H), 7.22 (d, J ) 5.27 Hz, 1H), 7.16 (dd, J ) 3.39,
5.27 Hz, 1H), 6.62 (br d, 1H), 4.98 (d, J ) 5.65 Hz, 2H).
5-({[1-(4-Chlorophenyl)methanoyl]-amino}methyl)-
thiophene-2-sulfonyl Chloride (2). A solution of 1 (10 g,
40 mmol) in DCM (500 mL) was treated with a solution of
chlorosulfonic acid (20.1 mL, 198 mmol) in DCM (80 mL) at
-80 °C. The reaction mixture was allowed to reach rt over 5
h. The mixture was poured on ice and quickly extracted with
DCM. The organic layer was dried over MgSO4, and the solvent
was evaporated to dryness to yield 8.8 g (63%) of 2 as a white
powder which was used without further purification: mp 133-
35 °C, 1H NMR (DMSO-d6) δ 9.21 (t, J ) 6.4 Hz, 1H), 7.87 (d,
J ) 8.7 Hz, 2H), 7.53 (d, J ) 8.7 Hz, 2H), 6.91 (d, J ) 3.4 Hz,
1H), 6.77 (d, J ) 3.4 Hz, 1H), 4.53 (d, J ) 3.8 Hz, 2H).
4-Chloro-N-methyl-N-thiophen-2-ylmethylbenz-
amide (3). To a slurry of 318 mg of NaH (60% purity, 7.94
mmol) in dry THF (50 mL) was added 1 (1 g, 3.97 mmol) in
THF at 0 °C under N2. The slurry was stirred for 15 min at rt
prior to the addition of 1 mL of MeI (15.9 mmol). The reaction
was stirred for 1 h, water was slowly added, and the reaction
mixture was evaporated to dryness. EtOAc was added, and
the organic layer was washed with water, dried over MgSO4,
and evaporated to dryness to yield 1 g (98%) of 3 as a
transparent oil. 1H NMR (DMSO-d6 60°C) δ 7.49 (d, J ) 8.66
Hz, 2H), 7.45-7.39 (m, 3H), 7.03 (br s, 1H), 7.01-6.90 (m, 1H),
4.72 (br s, 2H), 2.88 (s, 3H), MS m/z 266.5 (M + H).
Fyn(h)
IGF-1R(h)
IKKR(h)
IKKâ(h)
IR(h)
Lck(h)
Lyn(h)
MAPK1(h)
MAPK2(h)
MAPKAP-K2(h)
46
108
92
96
103
85
89
97
106
74
Rsk3(h)
97
77
100
106
101
67
75
83
61
121
SAPK2a(h)
SAPK2b(h)
SAPK3(h)
SAPK4(h)
SGK(h)
Syk(h)
TrkB(h)
Yes(h)
ZAP-70(h)
a Protein kinases were assayed with 10 µM of 50a in the
presence of 10 µM of ATP.
iv (AUCrat ) 240 h‚ng/mL @ 10 mg/kg), ip (AUCrat ) 85
h‚ng/mL @ 10 mg/kg) and oral administration. It is
therefore our next medicinal chemistry challenge to
improve the ‘druglike properties’ of this series of JNK
inhibitors.
Conclusion
We have identified a novel class of (benzoylamino-
methyl)thiophene sulfonamide JNK inhibitors. Exten-
sive SAR studies around the scaffold suggest that the
western end and the central core appear to be essential
features for inhibitory activity, allowing relatively little
freedom to operate. In contrast, the eastern end of the
molecule can accommodate a much larger range of
substitutions, allowing for an improvement of potency
in cell-based apoptosis assays. Taken together, these
aspects have led to the identification of potent benzazole
analogues. Compound 50a as one representative of this
subclass that inhibits the enzymatic activity of h-JNK3
with an IC50 value of 150nM and exhibits 4-fold selec-
tivity against h-JNK2. 50a displays an excellent selec-
tivity profile toward a large range of receptors and
enzymes, and more particularly, a stunning profile
toward 80 Ser/Thr and Tyr-kinases. Moreover, 50a
shows excellent inhibition of neuronal cell death induced
5-{[(4-chlorobenzoyl)methyl-amino]methyl}thiophene-
2-sulfonyl Chloride (4). 4 was synthesized according to the
chlorosulfonylation procedure of 2. Isolated yield: 650 mg
(48%). 1H NMR (DMSO-d6) δ 7.86 (d, J ) 8.7 Hz, 2H), 7.51 (d,
J ) 8.7 Hz, 2H), 6.90 (d, J ) 3.4 Hz, 1H), 6.78 (d, J ) 3.4 Hz,
1H), 4.76 (s, 2H), 2.95 (s, 3H).

6930 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Ru¨ckle et al.
N-(4-Chlorophenyl)-2-thiophen-2-ylacetamide (5). 5
was synthesized starting from 5 g of thiophen-2-yl-acetyl
chloride according to the synthesis of 1. Isolated yield: 6.7 g
(86%) of a beige solid. 1H NMR (CDCl3) δ 7.65 (d, J ) 9.0 Hz,
2H), 7.58 (dd, J ) 1.5 Hz, 4.9 Hz, 1H), 7.51 (d, J ) 9.0 Hz,
2H), 7.35-7.25 (m, 2H), 4.20 (s, 2H), MS m/z 252.0 (M + H);
250.0 (M - H).
5-[(4-Chlorophenylcarbamoyl)methyl]thiophene-2-sul-
fonyl Chloride (6). 6 was synthesized according to the
chlorosulfonylation procedure for 2. Isolated yield: 330 mg
(47%) of white crystals. 1H NMR (CDCl3) δ 7.76 (d, J ) 3.7
Hz, 1H), 7.44 (d, J ) 8.6 Hz, 2H), 7.29 (d, J ) 8.6 Hz, 2H),
7.24 (s, 1H), 7.0 (d, J ) 3.7 Hz, 1H), 3.98 (s, 2H).
s, 3H), 2.23 (t, J ) 6.2 Hz, 2H), 1.85 (quin, J ) 6.6 Hz, 2H),
MS m/z 624.9 (M + H); 622.2 (M - H); Anal. (C23H22-
Cl2F3N5O4S2): C, H, N.
N-(4-Chlorophenyl)-2-(5-{[2-(4-{[3-chloro-5-(trifluoro-
methyl)pyridin-2-yl]amino}butanoyl)hydrazino]sulfon-
yl}-2-thienyl)acetamide (13). 13 was synthesized using 6
as sulfonyl chloride according to the synthesis of I. Isolated
yield after silica gel chromatography: 40%. 1H NMR (DMSO-
d6) δ 10.3 (s, 1H), 10.0 (d, J ) 2.6 Hz, 1H), 9.86 (d, J ) 3.4 Hz,
1H), 8.29 (s, 1H), 7.91 (d, J ) 2.3 Hz, 1H), 7.59 (d, J ) 9.0 Hz,
2H), 7.44 (d, J ) 3.7 Hz, 1H), 7.33 (d, J ) 9.0 Hz, 2H), 7.24 (t,
J ) 5.4 Hz, 1H), 7.00 (d, J ) 3.7 Hz, 1H), 4.06 (s, 2H), 3.4-3.3
(m, 2H), 2.05 (t, J ) 7.5 Hz, 2H), 1.66 (quint, J ) 6.9 Hz, 2H),
MS m/z 610.1 (M + H); 608.1 (M - H); Anal. (C22H20Cl2F3-
N5O4S2): C, H, N.
4-(3-Chloro-5-trifluoromethylpyridin-2-ylamino)bu-
tyric Acid Methyl Ester (10). A mixture of γ-aminobutyric
acid (8.18 g, 79.3 mmol), 2,3-dichloro-5-(trifluoromethyl)-
pyridine (11.0 mL, 79.3 mmol), triethylamine (27.6 mL, 198.3
mmol), and methanol (270 mL) was heated at 100-04 °C in a
Parr autoclave (450 mL vessel) with mechanical agitation for
3 h. Evaporation, addition of DCM (200 mL), and filtration
removed the unreacted, insoluble γ-aminobutyric acid (2.5 g).
Evaporation, addition of t-BuOMe (200 mL), and filtration
removed most of the Et3N‚HCl salt (4.4 g). The ether solution
was filtered through a silica gel plug and concentrated to afford
the crude N-substituted γ-aminobutyric acid 9 as its triethyl-
ammonium salt. The crude was esterified to the corresponding
methyl γ-aminobutyrate 10 by heating to reflux for 1.5 h in
methanolic H2SO4 (1.9 M H2SO4 in MeOH, 50 mL). Concentra-
tion, addition of EtOAc (100 mL) and cyclohexane (100 mL),
washing (NaHCO3 sat.; H2O; brine), drying (Na2SO4), and
evaporation afforded 13.8 g (59%) 10 as a colorless oil: 1H
NMR (CDCl3) δ 8.18 (d, J ) 0.9 Hz, 1H), 7.53 (d, J ) 2.2 Hz,
1H), 5.54-5.42 (br. t, J ) 6 Hz, 1H), 3.61 (s, 3H), 3.51 (q, J )
6.8 Hz, 2H), 2.35 (t, J ) 7.2 Hz, 2H), 1.92 (quint, J ) 7.0 Hz,
2H).
4-(3-Chloro-5-trifluoromethylpyridin-2-ylamino)bu-
tyric Acid Hydrazide (11). A solution of 10 (5.61 g, 19.0
mmol) in 80% aqueous hydrazine (7 mL) and MeOH (14 mL)
was heated to reflux for 2 h. The reaction mixture was diluted
with EtOAc (250 mL). The unreacted hydrazine was extracted
with a minimum amount of water. The organic layer was dried
(Na2SO4), concentrated to 50 mL, and poured into a crystallizer
containing 150 mL of cyclohexane. The desired hydrazide
rapidly crystallized, and filtration after 2 h afforded 4.24 g
(76%) of 11 as pale yellow needles: 1H NMR (DMSO-d6) δ 8.96
(br s, 1H), 8.32 (br s, 1H), 7.94 (d, J ) 2.1 Hz, 1H), 7.25 (t, J
) 5.5 Hz, 1H), 4.51 (s, 2H), 3.40 (q, J ) 6.6 Hz, 2H), 2.07 (t, J
) 7.6 Hz, 2H), 1.88 (quint, J ) 7.2, 2H); MS m/z 297 (M + H).
{5-[(4-Chlorobenzoylamino)methyl]thiophene-2-
sulfonylamino}acetic Acid (14). Glycine tert-butyl ester
(263 mg, 1.57 mmol) and DIEA (537 µL) were dissolved in 20
mL of DCM. To this solution was added 2 (500 mg, 1.43 mmol)
in 10 mL of DMF. The reaction was stirred overnight. After
aqueous workup, the corresponding tert-butyl ester was iso-
lated as a white solid (400 mg, 63%) and deprotected without
any further purification using DCM/TFA 1:1. The reaction
mixture was stirred for 1 h. The solvents were evaporated to
1
dryness to yield 14 as a white solid (330 mg, 95%). H NMR
(DMSO-d6) δ 9.34 (t, J ) 5.8 Hz, 1H), 8.20 (t, J ) 6.0 Hz, 1H),
7.88 (d, J ) 8.7 Hz, 2H), 7.55 (d, J ) 8.7 Hz, 2H), 7.42 (d, J )
3.7 Hz, 1H), 7.04 (d, J ) 3.7 Hz, 1H), 4.62 (d, J ) 5.6 Hz, 2H),
3.58 (d, J ) 6.0 Hz, 2H), MS m/z 387.6 (M - H).
4-Chloro-N-[5-({[2-(3-chloro-5-trifluoromethyl-pyridin-
2-ylamino)ethylcarbamoyl]methyl}sulfamoyl)thiophen-
2-ylmethyl]benzamide (16). The intermediate 14 (320 mg,
0.82 mmol), DIEA (2.46 mmol), and PyBOP (860 mg, 1.7 mmol)
were dissolved in 30 mL of DCM. To this solution was added
the amine 15 (296 mg, 1.25 mmol) in 20 mL of DCM. The
reaction was stirred for 15 h, the solvent was evaporated to
dryness, and the crude material was purified on silica gel
(cyclohexane/EtOAc 1:1) affording 480 mg (95%) of 16 as a
1
white solid. H NMR (DMSO-d6) δ 9.34 (t, J ) 5.8 Hz, 1H),
8.31 (s, 1H), 8.09 (q, J ) 6.5 Hz, 2H), 7.95 (d, J ) 2.3 Hz, 1H),
7.87 (d, J ) 8.7 Hz, 2H), 7.54 (d, J ) 8.7 Hz, 2H), 7.42 (d, J )
3.7 Hz, 1H), 7.28 (t, J ) 5.5 Hz, 1H), 7.05 (d, J ) 3.7 Hz, 1H),
4.62 (d, J ) 5.6 Hz, 2H), 3.56-3.07 (m, 6H). MS m/z 610.3 (M
+ H); 608.1 (M - H); Anal. (C22H20Cl2F3N5O4S2): C, H, N.
5-Aminomethylthiophene-2-carboxylic Acid Methyl
Ester (17). 5-Methylthiophene-2-carboxylic acid (5 g, 35 mmol)
was refluxed in H2SO4 in MeOH (2 M) for 6 h. The reaction
was neutralized with NaOH (10 N) at 0 °C, and the methyl
ester was extracted with DCM affording 4.85 g (89%) of
5-methylthiophene-2-carboxylic acid methyl ester. Without
further purification, the methyl ester (4.8 g, 31 mmol) was
refluxed in CCl4 in the presence of NBS (6 g, 34 mmol) and
benzoyl peroxide (242 mg, 0.03 equiv) for 20 h. The reaction
is cooled to 0 °C and filtered. The filtrate is concentrated to
dryness affording an oily orange liquid representing 5-bro-
momethylthiophene-2-carboxylic acid methyl ester (7 g, 98%).
NMR shows 80% of the monobromo derivative, which was used
for the next step without further purification. Seven grams
(24 mmol, 80%) of the obtained mixture was heated at 70 °C
in DMF in the presence of 2.2 g (33 mmol) of NaN3 for 3 h.
EtOAc was added, and the organic layer was washed with
brine several times affording 5-azidomethylthiophene-2-car-
boxylic acid methyl ester (5.5 g). Mass spectrometry indicated
the absence of the bromo derivative. The azido intermediate
(5.5 g, 80% purity) was reduced in the presence of Pd/C with
H2 at 2 bar in 10% HCl (2 M) in EtOH for 20 h. The acidic
solution was then washed with DCM and further basified to
pH 9.5. The aqueous basic solution was then extracted several
times with DCM to yield 2 g of 17 (50%) as a yellow liquid,
which spontaneously crystallized after solvent evaporation. 1H
NMR (CDCl3) δ 7.64 (d, J ) 3.9 Hz, 1H), 6.89 (d, J ) 3.9 Hz,
1H), 4.05 (s, 2H), 3.84 (s, 3H), 1.54 (s, 2H), MS m/z 171 (M +
H).
4-Chloro-N-[(5-{[2-(4-{[3-chloro-5-(trifluoromethyl)py-
ridin-2-yl]amino}butanoyl)hydrazino]sulfonyl}-2-thienyl)-
methyl]benzamide (I). A solution of 2 (211 mg, 0.60 mmol),
the acyl hydrazide 11 (179 mg, 0.60 mmol), and pyridine (71
mg, 0.90 mmol) in CHCl3 (10 mL) was heated to reflux for 3 h
and cooled to rt. The precipitate was collected by filtration and
washed with CHCl3 to give 321 mg (88%) of the title compound
1
I as a white powder. H NMR (DMSO-d6) δ 10.07 (d, J ) 3.2
Hz, 1H), 9.88 (s, 1H), 9.25 (t, J ) 5.3 Hz, 1H), 8.32 (s, 1H),
7.95 (d, J ) 2.0 Hz, 1H), 7.78 (d, J ) 8.6 Hz, 2H), 7.54 (d, J )
8.6 Hz, 2H), 7.42 (d, J ) 3.8 Hz, 1H), 7.25 (br. t, J ) 5.5 Hz,
1H), 7.03 (d, J ) 3.8 Hz, 1H), 4.61 (d, J ) 5.9 Hz, 2H), 3.32
(buried q, 2H), 2.04 (t, J ) 7.4 Hz, 2H), 1.65 (quint, J ) 7.1
Hz, 2H). MS m/z 610.2(M + H); 608.1 (M - H); Anal. (C22H20-
Cl2F3N5O4S2): C, H, N.
4-Chloro-N-[(5-{[2-(4-{[3-chloro-5-(trifluoromethyl)py-
ridin-2-yl]amino}butanoyl)hydrazino]sulfonyl}-2-thienyl)-
methyl]-N-methylbenzamide (12). 12 was synthesized us-
ing 4 as sulfonyl chloride according to the synthesis of I.
Isolated yield after silica gel chromatography (DCM/MeOH 20:
1
1): 20 mg (83%) of a white solid. H NMR (CDCl3) δ 9.98 (br
s, 1H), 8.33 (s, 1H), 7.62 (d, J ) 1.9 Hz, 1H), 7.53 (d, J ) 3.7
Hz, 1H), 7.36 (s, 4H), 7.24 (s, 1H), 6.99 (br s, 1H), 5.70 (t, J )
5.8 Hz, 1H), 4.76 (br s, 2H), 3.48 (q, J ) 6.5 Hz, 2H), 2.95 (br

Inhibitors of c-Jun-N-Terminal Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6931
4-Chloro-N-(5-{N′-[4-(3-chloro-5-trifluoromethylpyri-
din-2-ylamino)butyryl]hydrazinocarbonyl}thiophen-2-
ylmethyl)benzamide (18). Intermediate 17 (500 mg, 2.92
mmol) was capped with 4-chlorobenzoyl chloride (355 µL, 2.8
mmol) in the presence of DIEA (750 µL, 4.4 mmol) in DCM.
The reaction was stirred for 2 h at rt. Aqueous workup
produced 890 mg (90%) of the corresponding methyl ester,
which was saponified directly. The methyl ester (770 mg, 2.48
mmol) was dissolved in 18 mL of THF/water (5:1), LiOH‚H2O
(208 mg, 4.98 mmol) was added, and the reaction was heated
at 60 °C for 5 h. pH was decreased to 1, and the precipitate
was filtered off and washed with a small amount of water. The
corresponding acid was isolated as a white powder (725 mg,
98%) (NMR indicated absence of the methyl group). The acid
(30 mg, 0.1 mmol) was coupled directly to 11 (33 mg, 0.11
mmol) using DCI (14 mg, 0.11 mmol) and HOBt (15 mg, 0.11
mmol) as coupling reagents in DCM/DMF 1:1. The reaction
mixture was stirred for 3 h at rt, while 18 precipitated out as
4-{[3-Chloro-5-(trifluoromethyl)pyridin-2-yl]amino}-
N′-(1,2,3,4-tetrahydroisoquinolin-7-ylsulfonyl)butanohy-
drazide (23). 23 was produced following the synthetic pro-
cedure for I using 2-acetyl-1,2,3,4-tetrahydroisoquinoline-7-
sulfonyl chloride (357 mg, 1.09 mmol). The corresponding
trifluoroacetamide of 23 was isolated in quantitative yield and
was directly deprotected using 1 mL of sat. K2CO3 in 10 mL
of MeOH at 50 °C for 1 h. Filtration over silica gel using DCM/
MeOH/NH4OH 80:20:5 as eluents produced 280 mg (94%) of
1
23 as a white powder. H NMR (DMSO-d6) δ 9.99 (br s, 1H),
8.30 (s, 1H), 7.93 (s, 1H), 7.60 (s, 1H), 7.57 (s, 1H), 7.29 (d, J
) 7.2 Hz, 2H), 4.10 (s, 2H), 3.30 (q, J ) 6.3 Hz, 2H), 3.16 (t, J
) 5.8 Hz, 2H), 2.89 (t, J ) 5.6 Hz, 2H), 2.01 (t, J ) 7.7 Hz,
2H), 1.62 (quint, J ) 7.2 Hz, 2H), MS m/z 492.1 (M + H); 490.5
(M - H).
N′-{[2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-
7-yl]sulfonyl}-4-{[3-chloro-5-(trifluoromethyl)pyridin-2-
yl]amino}butanohydrazide (24). Compound 24 was syn-
thesized following the procedure for the synthesis of 21.
Isolated yield: 19.7 mg (51%). 1H NMR (DMSO-d6) δ 9.99 (br
s, 1H), 8.30 (s, 1H), 7.93 (s, 1H), 7.88 (d, J ) 8.7 Hz, 2H), 7.60
(s, 1H), 7.55 (d, J ) 8.7 Hz, 2H), 7.57 (s, 1H), 7.29 (d, J ) 7.2
Hz, 2H), 4.10 (s, 2H), 3.30 (q, J ) 6.3 Hz, 2H), 3.16 (t, J ) 5.8
Hz, 2H), 2.89 (t, J ) 5.6 Hz, 2H), 2.01 (t, J ) 7.7 Hz, 2H), 1.62
(quint, J ) 7.2 Hz, 2H), MS m/z 630.3 (M + H); 628.5 (M -
H); Anal. (C26H24Cl2F3N5O4S): C, H, N.
1
a colorless solid (34 mg, 60%). H NMR (DMSO-d6) δ 10.2 (s,
1H), 9.84 (s, 1H), 9.29 (t, J ) 5.8 Hz, 1H), 8.32 (s, 1H), 7.94 (d,
J ) 2.3 Hz, 1H), 7.88 (d, J ) 8.7 Hz, 2H), 7.64 (d, J ) 3.7 Hz,
1H), 7.54 (d, J ) 8.7 Hz, 2H), 7.36 (t, J ) 5.5 Hz, 1H), 7.04 (d,
J ) 3.7 Hz, 1H), 4.61 (d, J ) 5.7 Hz, 2H), 3.45 (q, J ) 6.4 Hz,
2H), 2.20 (t, J ) 7.5 Hz, 2H), 1.81 (quint, J ) 7.3 Hz, 2H), MS
m/z 574.3 (M + H); 572.1 (M - H); Anal. (C23H20Cl2F3N5O2S):
C, H, N.
N′-[(3-Aminophenyl)sulfonyl]-4-{[3-chloro-5-(trifluo-
romethyl)pyridin-2-yl]amino}butanohydrazide (25). To
a solution of 3-nitrobenzenesulfonyl chloride (89 mg, 0.40
mmol) and DMAP (72 mg, 0.59 mmol) in DMF was added 11
(120 mg, 0.40 mmol). The reaction was stirred at rt for 5 h.
Aqueous workup produced a brown solid, which was purified
on silica gel using CHCl3/EtOAc 4:1 to 1:1. 92 mg (47%) of the
corresponding nitro intermediate were isolated as a white
solid. The nitro intermediate (89 mg, 0.185 mmol) was dis-
solved in 2 mL of DMF followed by the addition of 52 mg (0.23
mmol) of SnCl2‚2H2O. The reaction was stirred overnight and
was driven to completion by adding additional 53 mg of the
reducing agent. The crude product was filtered over silica gel
using EtOAc/MeOH 98:2 as eluent to yield 63 mg (75%) of 25
as a pale yellow solid. MS m/z 452.8 (M + H); 450.7 (M - H).
N′-{[3-(Aminomethyl)phenyl]sulfonyl}-4-{[3-chloro-5-
(trifluoromethyl)pyridin-2-yl]amino}butanohydrazide
(19). 19 was synthesized from 3-cyanobenzenesulfonyl chloride
(372 mg, 1.86 mmol) according to the synthesis of I. Isolated
yield of the corresponding nitrile after silica gel chromatog-
raphy: 750 mg (87%). The nitrile (360 mg, 0.78 mmol) was
reduced using LiAlH4 (1.9 mL (1 M in THF), 1.95 mmol) in
anhydrous THF at rt. The reaction mixture was stirred for 30
min at rt before quenching with H2O. Aqueous workup
1
afforded 363 mg (97%) of 19. H NMR (DMSO-d6) δ 8.30 (s,
1H), 7.92 (d, J ) 2.3 Hz, 1H), 7.78 (s, 1H), 7.64 (d, J ) 7.9 Hz,
1H), 7.55 (d, J ) 7.9 Hz, 1H), 7.44 (t, J ) 7.7 Hz, 1H), 7.27 (t,
J ) 5.8 Hz, 1H), 3.8 (s, 2H), 3.30 (q, J ) 6.5 Hz, 2H), 2.01 (t,
J ) 7.3 Hz, 2H), 1.61 (quint, J ) 7.2 Hz, 2H), MS m/z 466.2
(M + H); 464.1 (M - H).
4-Chloro-N-(3-{[2-(4-{[3-chloro-5-(trifluoromethyl)py-
ridin-2-yl]amino}butanoyl)hydrazino]sulfonyl}phenyl)-
benzamide (26). Compound 26 was synthesized using the
protocol of I and could be isolated as a pale yellow powder after
silica gel chromatography in 35% yield. MS m/z 591.4 (M +
H); 589.5 (M - H); Anal. (C23H20Cl2F3N5O4S): C, H, N.
N′-{[4-(Aminomethyl)phenyl]sulfonyl}-4-{[3-chloro-5-
(trifluoromethyl)pyridin-2-yl]amino}butanohydrazide
(20). 20 was synthesized according to the synthesis of 19 to
yield 260 mg (67%) of 20. 1H NMR (CD3OD) δ 8.22 (s, 1H),
7.94 (d, J ) 8.3 Hz, 2H), 7.81 (d, J ) 1.9 Hz, 1H), 7.66 (d, J )
8.3 Hz, 2H), 4.21 (s, 2H), 3.44 (t, J ) 6.9 Hz, 2H), 2.16 (t, J )
7.3 Hz, 2H), 1.78 (quint, J ) 7.2 Hz, 2H), MS m/z 466.2 (M +
H); 464.1 (M - H).
5-[(3-Methoxybenzoylamino)methyl]thiophene-2-sul-
fonyl Chloride (29). To a solution of 2-aminomethylthiophene
(10.6 mL, 103 mmol) and pyridine (9.1 mL, 104 mmol) in 100
mL of chloroform was added at 0 °C a solution of 3-methoxy-
benzoyl chloride (19.2 g, 103 mmol) in DCM. The reaction
mixture was allowed to warm to rt during 1 h and stirred for
additional 3 h. Water was added while 3-methoxy-N-(thien-
2-ylmethyl)benzamide 27 (10.1 g) precipitated. The solid was
filtered off and washed with water. The remaining organic
layer was washed with brine, dried over MgSO4, and evapo-
rated to dryness to afford additional 15.2 g of 27. The overall
yield was 25.3 g (99.9%). 27 was used for the next step without
further purification. Chlorosulfonic acid (5.62 mL, 84 mmol)
was dissolved in 20 mL DCM and added to a solution of 27
(11.0 g, 42 mmol) in 100 mL of DCM under vigorous stirring.
A gummy solid was formed, and the reaction mixture was
stirred for 3 h. The reaction was quenched with ice, and ice
cold NaHCO3 solution was added to reach pH 8.5. The aqueous
layer was washed twice with DCM. Tetrabutylammonium
hydroxide (40% in water) (32 mL, 50 mmol) was added to the
aqueous layer, while a solid was formed. The precipitate was
extracted with DCM, and the aqueous layer was washed 3×
with DCM. The combined organic layers were dried over
MgSO4 and evaporated to dryness to afford a slightly colored
foam of tetrabutylammonium 5-{[(3-methoxybenzoyl)-amino]-
methyl}thiophene-2-sulfonate 28 (24 g, 97%). NMR spectra
indicated pure compound, which was used for the following
4-Chloro-N-(3-{[2-(4-{[3-chloro-5-(trifluoromethyl)py-
ridin-2-yl]amino}butanoyl)hydrazino]sulfonyl}benzyl)-
benzamide (21). To a solution of 20 (15.7 mg, 34 µmol) in
DCM/pyridine 30:1 was added 4-chlorobenzoyl chloride (0.7
equiv) at rt. The reaction mixture was stirred for 2 h at rt.
The crude product was filtered through silica gel affording pure
1
21 (15.9 mg, 78%) as a colorless solid. H NMR (DMSO-d6) δ
10.5 (s, 1H), 9.98 (s, 1H), 9.79 (br s, 1H), 8.28 (br s, 1H), 8.03
(d, J ) 8.7 Hz, 2H), 7.90 (d, J ) 1.9 Hz, 1H), 7.64 (d, J ) 8.7
Hz, 2H), 7.52-73.4 (m, 3H), 7.27-7.13 (m, 1H), 1.99 (t, J )
6.4 Hz, 2H), 1.62 (quint, J ) 6.9 Hz, 2H), 1.22 (s, 2H), MS m/z
604.1 (M + H); 602.0 (M - H); Anal. (C24H22Cl2F3N5O4S): C,
H, N.
4-Chloro-N-(4-{[2-(4-{[3-chloro-5-(trifluoromethyl)py-
ridin-2-yl]amino}butanoyl)hydrazino]sulfonyl}benzyl)-
benzamide (22). 22 was synthesized according to synthesis
of 21. Isolated yield: 15 mg (73%). 1H NMR (DMSO-d6) δ 9.95
(d, J ) 3.0 Hz, 1H), 9.73 (d, J ) 3.4 Hz, 1H), 9.20 (t, J ) 5.6
Hz, 1H), 8.29 (s, 1H), 7.92 (d, J ) 1.9 Hz, 1H), 7.89 (d, J ) 8.7
Hz, 2H), 7.74 (d, J ) 8.7 Hz, 2H), 7.53 (d, J ) 8.7 Hz, 2H),
7.44 (d, J ) 8.7 Hz, 2H), 7.23 (t, J ) 5.6 Hz, 1H), 4.51 (d, J )
5.6 Hz, 2H), 3.28 (q, J ) 6.4 Hz, 2H), 1.99 (t, J ) 6.9 Hz, 2H),
1.61 (quint, J ) 6.9 Hz, 2H), MS m/z 604.1 (M + H); 602.0 (M
- H); Anal. (C24H22Cl2F3N5O4S): C, H, N.

6932 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Ru¨ckle et al.
chlorination step. To a solution of 28 (2.0 g, 3.4 mmol) in 50
mL of DCM was added triphosgene (800 mg, 2.7 mmol, 2.3
equiv), dissolved in 10 mL of DCM. To this reaction mixture
was added DMF (0.1 mL, 1.4 mmol) dropwise during 10 min,
while gas evolution could be observed. The reaction mixture
was stirred for 3 h, and the crude product was directly filtered
through silica gel using EtOAc/hexane 1:2 as eluent. An orange
solid could be isolated which was further recrystallized from
cyclohexane/DCM. 29 (730 mg, 60%) was obtained as colorless
needles. 1H NMR (CDCl3) δ 8.83 (t, J ) 1.5 Hz, 1H), 8.35 (t, J
) 7.5 Hz, 1H), 7.76 (t, J ) 4.1 Hz, 1H), 7.70-7.58 (m, 3H),
7.52-7.40 (m, 2H), 7.05 (t, J ) 3.8 Hz, 1H).
Diallylthiophen-2-ylmethylamine (30). Allyl bromide (55
mL, 65.4 mmol) was added to a solution of 2-aminomethyl-
thiophene (24 mL, 23.3 mmol) and i-Pr2NEt (120 mL, 70.1
mmol) in DCM (270 mL). The moderately exothermic reaction
spontaneously reached the reflux temperature after 1 h. The
reaction was cooled by means of an ice bath and stirred for 14
h at rt whereupon a precipitate appeared. The organic layer
was concentrated, and the precipitates were filtered off. The
EtOAc solution was filtered over SiO2 and concentrated to give
36.1 g (80%) of 30 as a pale yellow oil: 1H NMR (CDCl3) δ 7.25
(br d, J ) 5.9 Hz, 1H), 6.98 (br dd, J ) 5.1, 2.8 Hz, 1H), 6.94-
6.92 (m, 1H), 5.99-5.86 (m, 2H), 5.29-5.18 (m, 4H), 3.85 (s,
2H), 3.16 (dd, J ) 6.3, 0.9 Hz, 4H).), MS m/z 194.2 (M + H).
5-Diallylaminomethylthiophene-2-sulfonyl Chloride
(31). A solution of the allyl-protected thiophene 30 (6.2 g, 32.1
mmol) in Et2O was cooled to - 70 °C by means of an acetone/
dry ice bath. A solution of t-BuLi in pentane (21.38 mL, 1.5
M, 32.1 mmol) was added over 2 min whereupon the internal
temperature rose to -50 °C and the mixture turned orange.
After 10 min, SO2 gas was bubbled for 2 min, which led to the
immediate formation of a thick precipitate. The reaction was
allowed to reach 0 °C, and a suspension of NCS (4.63 g, 32.1
mmol) in THF (20 mL) was added, whereupon the slurry
turned purple. After 45 min at rt, the mixture was filtered
over SiO2, eluting with EtOAc. Evaporation, dilution with
EtOAc:hexane 1:5 and filtration over SiO2 gave the 5.0 g (53%)
of 31 as a pale brown oil, which was used without further
purification. 1H NMR (CDCl3) δ 7.73 (d, J ) 4.1 Hz, 1H), 6.92
(d, J ) 4.1 Hz, 1H), 5.93-5.75 (m, 2H), 5.25 (q, J ) 1.5 Hz,
1H), 5.23-5.13 (m, 3H), 3.82 (s, 2H), 3.16 (d, J ) 6.4 Hz, 4H).
5-Aminomethylthiophene-2-sulfonic Acid, N′-[4-(3-
Chloro-5-trifluoromethyl-pyridin-2-ylamino)butanoyl]-
hydrazide (32b). The bisallylsulfonylhydrazide 32a was
synthesized according to the synthesis of I using sulfonyl
chloride 31. Isolated yield of 32a: 4.0 g (98%). 1H NMR
(DMSO-d6) δ 10.0 (d, J ) 3.7 Hz, 1H), 9.85 (d, J ) 3.7 Hz,
1H), 8.30 (s, 1H), 7.93 (d, J ) 2 0.3 Hz, 1H), 7.42 (d, J ) 3.7
Hz, 1H), 7.29 (t, J ) 5.4 Hz, 1H), 6.94 (d, J ) 3.7 Hz, 1H),
5.9-5.67 (m, 2H), 5.26-5.02 (m, 4H), 3.72 (s, 2H), 3.32 (q, J
) 7.3 Hz, 2H), 3.03 (d, J ) 6.0 Hz, 4H), 2.04 (t, J ) 7.7 Hz,
2H), 1.66 (q, J ) 7.3 Hz, 2H), MS m/z 552.6 (M + H); 550.4 (M
- H). A solution of 32a (4.0 g, 7.25 mmol), N,N′-dimethylbar-
bituric acid (NDMBA 2.8 g, 18.1 mmol), and Pd(PPh3)4 (148.8
mg, 0.13 mmol) in DCM was degassed with argon. The reaction
mixture was stirred for 3 h at rt after which the desired amine
32b precipitated as its NDMBA salt. The mixture was diluted
with EtOAc (200 mL) and hexane (200 mL) and washed with
water (3 × 50 mL). The combined aqueous phases were freeze-
dried, dissolved in a minimal amount of MeOH, and purified
by chromatography (SiO2, DCM/EtOAc/NH4OH.aq 80:20:5).
The chromatography was repeated twice and gave 2.3 g (67%)
of the free amine 32b, which was dissolved in refluxing EtOAc
(80 mL) and cooled to -18 °C to afford 1.7 g (50%) of 32b as
a white powder: 1H NMR (DMSO-d6) δ 10.02-9.85 (br. s, 1H),
8.24-8.19 (br. s, 1H), 7.85 (d, J ) 2.0 Hz, 1H), 7.32 (d, J ) 3.8
Hz, 1H), 7.20 (t, J ) 5.7 Hz, 1H), 6.82 (d, J ) 3.8 Hz, 1H),
5.3-4.3 (br. s, 2H), 3.80 (s, 2H), 3.23 (q, J ) 6.7 Hz, 2H), 1.96
(t, J ) 7.5 Hz, 2H), 1.57 (quint, J ) 7.2 Hz, 2H); MS m/z 472
(M + H); Anal. (C15H17ClF3N5O3S2): C, H, N.
2 as sulfonyl chloride and 3′-chloro-5′-trifluoromethyl-3,4,5,6-
tetrahydro-2H-[1,2′]bipyridinyl-4-carboxylic acid hydrazide ac-
cording to the synthesis of I. Isolated yield after silica gel
chromatography (DCM/MeOH 20:1): 97 mg (76%) of a white
solid. 1H NMR (DMSO-d6) δ 10.1 (d, J ) 3.7 Hz, 1H), 9.90 (d,
J ) 3.7 Hz, 1H), 9.35 (t, J ) 6.0 Hz, 1H), 8.50 (s, 1H), 8.13 (d,
J ) 1.9 Hz, 1H), 7.86 (d, J ) 8.7 Hz, 2H), 7.47 (d, J ) 8.3 Hz,
2H), 7.42 (d, J ) 3.7 Hz, 1H), 7.06 (d, J ) 3.7 Hz, 1H), 4.61 (d,
J ) 5.6 Hz, 2H), 3.76 (d, J ) 12.8 Hz, 2H), 2.75 (t, J ) 11.3
Hz, 2H), 2.39-2.22 (m, 1H), 1.65-1.36 (m, 4H), MS m/z 636.2
(M + H); 634.0 (M - H).
General Procedure for Acylation of 32b. Synthesis of
34a-w, 35a-g, 36a-f. A 20 mg/mL solution of 32b in
pyridine/DCM 1:4 was cooled to -40 °C and treated for 1 h
with 0.8 equiv of the desired acyl chloride RCOCl. The reaction
mixture was brought to room temperature over 30 min. The
desired amide was purified by evaporation, dilution in CH3-
CN, and filtration over a SiO2 pad. The final evaporation
afforded the desired amide in typically 50-80% yield and
purity > 90%.
General Procedure for Sulfonylation, Carbamoylation
of 32b. Synthesis of 37 and 38a-c. A 19 mg/mL solution of
32b in pyridine/DCM 1:4 was mixed for 10 min with 0.9 equiv
of either a sulfonyl chloride RSO2Cl or an isocyanate RNCO.
Evaporation, dilution in CH3CN, filtration over a SiO2 pad,
and evaporation afforded the desired sulfonamide or urea in
ca. 50% yield for the sulfonamides, and ca. 70% for the ureas.
Typical purity: 90-95%.
General Procedure for reductive alkylation of 32b.
Synthesis of N′-({5-[(benzylamino)methyl]-2-thienyl}-
sulfonyl)-4-{[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-
amino}butanohydrazide (39). A solution of 32b (20 mg,
0.043 mmol), benzaldehyde (4.5 µL, 0.043 mmol), and AcOH
(9.0 µL, 0.16 mmol) in MeOH (1.5 mL) was stirred at rt for 20
min before adding NaBH(OAc)3 (24 mg, 0.113 mmol). After 2
h, dilution with EtOAc (5 mL) and filtration over a silica gel
pad afforded 23.4 mg (97%) of the desired amine as a colorless
oil. 1H NMR (DMSO-d6) δ 9.84-9.80 (br. s, 1H), 8.33-8.28 (br.
s, 1H), 7.59 (d, J ) 2.0 Hz, 1H), 7.46 (d, J ) 3.8 Hz, 1H), 7.28-
7.15 (m, 5H), 6.83 (d, J ) 3.9 Hz, 1H), 5.53 (t, J ) 6.0 Hz,
1H), 5.10-4.40 (br. s, 2H), 3.9 (s, 2H), 3.7 (s, 2H), 3.43 (q, J )
6.6 Hz, 2H), 2.17 (t, J ) 7.4 Hz, 2H), 1.79 (quint, J ) 7.1 Hz,
2H). MS m/z 562 (M + H); Anal. (C22H23ClF3N5O3S2): C, H,
N.
General Procedure for Solid-Supported Parallel Syn-
thesis of 40a-d, 41a-g, 42a-e, 43a-h, 50l. In a Quest 210
parallel synthesizer 5 mL reaction vessels were placed 2 mL
of DCM, 0.5 mL of DMF, 0.165 mmol of amine and polymer-
bound morpholine (4 equiv of PS-NMM 2.2 mmol/g). The
mixture (in each vessel) was stirred for 30 min at rt after which
1 mL of a stock solution of 2 or 29 in DCM/DMF 1:1
(0.15molar) was added and the mixture was further stirred
for 15 h at rt. Polymer-bound aminomethyl polystyrene (2
equiv of PS-NH2 1.3 mmol/g) and polymer-bound isocyanate
(2 equiv of PS-NCO 1.5 mmol/g) were added. The mixture
was stirred for 5 h at rt and drained through the lower luer
manifold. The remaining resins were washed three times with
DCM, and the collected solutions were concentrated using a
GeneVac parallel concentrator affording 40a-d, 41a-g, 42a-
e, 43a-h typically as solid (yields: 60-98%). Purities ranged
from 90 to 99%.
General Procedure for the Synthesis of 44a-k, 45a-
e, 46a-d in Case of Two Isomers (as exemplified by the
synthesis of N-Boc-4-benzotriazolylpiperidines (44a, 45a)). To
a solution of 1-Boc-4-hydroxypiperidine (5.2 g, 25 mmol),
benzotriazole (5.95 g, 50 mmol), and triphenylphosphine (13.7
g, 50 mmol) in 375 mL of THF anhydrous was slowly added
DEAD (8.15 mL, 50 mmol) in 250 mL of THF over 3 h. The
yellow solution was stirred overnight, THF was evaporated
to dryness, and the residue was purified by flash chromatog-
raphy using cyclohexane/EtOAc 7:3 as eluent. Two major
fractions were isolated, of which the first eluting fraction
contained 4.5 g (60%) of the 2-isomer N-Boc-4-benzotriazol-2-
4-Chloro-N-[(5-{[2-({1-[3-chloro-5-trifluoromethylpyri-
din-2-yl]piperidin-4-yl}carbonyl)hydrazino]sulfonyl}-2-
thienyl)methyl]benzamide (33). 33 was synthesized using
1
ylpiperidine 45a H NMR (CDCl3) δ 7.87 (d, J ) 3.0 Hz, 1H),

Inhibitors of c-Jun-N-Terminal Kinase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6933
7.85 (d, J ) 3.0 Hz, 1H), 7.39 (d, J ) 3.0 Hz, 1H), 7.37 (d, J )
3.0 Hz, 1H), 4.92 (quint, J ) 7.3 Hz, 1H), 4.24 (br d, J ) 11.3
Hz, 2H), 3.04 (quint, J ) 6.7 Hz, 2H), 1.48 (s, 9H), MS m/z
247 (M-56+H); 203 (M - Boc + H).
Later eluting fractions contained 2.25 g (30%) of the
1-isomer N-Boc-4-benzotriazol-1-yl-piperidine 44a. 1H NMR
(DMSO-d6) δ 8.12 (br s, 1H), 8.01 (d, J ) 8.3 Hz, 1H), 7.47 (d,
J ) 9.0 Hz, 1H), 5.29-5.15 (m, 1H), 3.44 (br d, J ) 13.1 Hz,
2H), 3.32 (s, 9H), 3.19 (br t, J ) 12.2 Hz, 2H), 2.46-2.22 (m,
4H), MS m/z 203 (M - Boc + H).
General Procedure for the Synthesis of 44a-k, 45a-
e, 46a-d in Case of Three Isomers (as exemplified by the
synthesis of N-Boc-4-chlorobenzotriazolylpiperidines (44e, 44f,
45b). The synthesis was carried out as described in the protocol
for 44a and 45a. The crude product was purified by flash
chromatography using petroleum ether/EtOAc 7:1. Three
major fractions were collected, in which first eluting fractions
(Rf)0.5) contained 350 mg (60%) of the 2-isomer N-Boc-4-(5-
chlorobenzotriazol-2-yl)-piperidine 45b. 1H NMR (CDCl3) δ
7.84 (d, J ) 1.9 Hz, 1H), 7.79 (d, J ) 9.0 Hz, 1H), 7.33 (dd, J
) 9.0, 1.8 Hz, 1H), 4.89 (quint, J ) 7.4 Hz, 1H), 4.23 (br d, J
) 11.3 Hz, 2H), 3.04 (quint, J ) 6.6 Hz, 2H), 2.34-2.2 (m,
4H), 1.48 (s, 9H), MS m/z 337 (M - Boc + H).
The second eluting fraction (Rf)0.3) contained 114 mg (17%)
of N-Boc-4-(6-Chlorobenzotriazol-1-yl)-piperidine 44f. 1H NMR
(CDCl3) δ 7.99 (d, J ) 8.7 Hz, 1H), 7.56 (d, J ) 1.1 Hz, 1H),
7.33 (dd, J ) 9.0, 1.88 Hz, 1H), 3.02 (t, J ) 12.0 Hz, 2H), 4.84-
4.69 (m, 1H), 4.40-4.24 (m, 4H), 2.31 (q, J ) 11.9 Hz, 2H),
2.15 (d, J ) 2.8 Hz, 2H), 1.50 (s, 9H). Structure was assigned
using 2D-NOE experiments. A cross-peak between H7arïm and
H3piperidine was observed. MS m/z 337 (M - Boc + H).
8.3 Hz, 1H), 7.91 (d, J ) 8.3 Hz, 2H), 7.87 (d, J ) 8.3 Hz, 1H),
7.61-7.55 (m, 3H), 7.52 (t, J ) 8.3 Hz, 1H), 7.38 (t, J ) 7.9
Hz, 1H), 7.23 (d, J ) 3.7 Hz, 1H), 5.00 (quint, J ) 7.34 Hz,
1H), 4.70 (d, J ) 5.6 Hz, 2H), 3.78 (d, J ) 12.0 Hz, 2H), 2.79-
2.64 (m, 2H), 2.3-2.16 (m, 4H), MS m/z 516.0 (M + H); 514.0
(M - H). Anal. (C23H22ClN5O3S2) C, H, N.
Similarly, N-[5-(4-benzotriazol-2-ylpiperidine-1-sulfonyl)-
thiophen-2-ylmethyl]-4-chlorobenzamide (51a) was prepared
in 42% yield. 1H NMR (DMSO) δ 9.38 (t, J ) 5.6 Hz, 1H), 7.94-
7.83 (m, 4H), 7.56 (d, J ) 8.6 Hz, 2H), 7.53 (d, J ) 3.7 Hz,
1H), 7.5-7.37 (m, 2H), 7.20 (d, J ) 3.7 Hz, 1H), 5.04-4.9 (m,
1H), 4.68 (d, J ) 5.6 Hz, 2H), 3.68 (br d, J ) 12.4 Hz, 2H),
2.76 (br t, J ) 12.6 Hz, 3H), 2.39 (br d, J ) 13.1 Hz, 2H), 2.22
(br t, J ) 11.1 Hz, 2H), MS m/z 516.0 (M + H); 514.0 (M - H);
Anal. (C23H22ClN5O3S2) C, H, N.
4-Chloro-N-{5-[4-(2-trifluoromethylbenzoimidazol-1-
yl)piperidine-1-sulfonyl]thiophen-2-ylmethyl}benz-
amide (50m). N-Boc-4-aminopiperidine (500 mg, 2.5 mmol)
and 1-fluoro-2-nitrobenzene (210 µL, 2 mmol) where heated
in the presence of DIEA (1 mL, 6.25 mmol) in DMF at 70 °C
for 15 h. After aqueous workup a crude yellow solid was
purified on silica gel using petroleum ether/EtOAc 5:1 as
eluent to yield 500 mg (78%) of N-Boc-4-(2-nitrophenylamino)-
piperidine. MS m/z 222.2 (M - Boc + H). 250 mg (0.78 mmol)
of the secondary nitroaniline derivative was exposed to
hydrogen-gas flow in EtOH in the presence of palladium on
charcoal. After 1 h the yellow color completely disappeared.
The solution was filtered through a microfilter (0.45 µm)
yielding a reddish solution which after solvent evaporation
gave 170 mg (75%) of N-Boc-4-(2-aminophenylamino)-piperi-
dine. MS m/z 194.2 (M - Boc + H). The 2-aminoaniline
derivative (120 mg, 0.41 mmol) was dissolved in 10 mL of
DCM, to which 2 mL of TFA was slowly added. The solution
was stirred for 3 h at rt until the deprotection/cyclization of
the benzimidazole was completed as monitored by LC-MS. The
reaction was evaporated to dryness, and the oily residue was
treated with diethyl ether affording 70 mg (45%) of a pink
precipitate which was identified as 1-piperidin-4-yl-2-triflu-
oromethyl-1H-benzimidazole trifluoroacetate. 1H NMR (DMSO-
d6) δ 8.70 (br s, 1H), 8.51 (br s, 1H), 8.05 (d, J ) 8.3 Hz, 1H),
7.87 (d, J ) 8.3 Hz, 1H), 7.50 (t, J ) 7.3 Hz, 1H), 7.40 (t, J )
7.5 Hz, 1H), 4.94-4.77 (m, 1H), 3.59-3.13 (m, 7H), 2.78-2.59
(m, 2H), 2.09 (br d, J ) 11.6 Hz, 2H), MS m/z 269.8 (M - Boc
+ H).
The third eluting fraction (Rf ) 0.2) contained 23 mg (4%)
of N-Boc-4-(5-chlorobenzotriazol-1-yl)piperidine (44e). 1H NMR
(CDCl3) δ 8.07 (s, 1H), 7.50 (t, J ) 8.8 Hz, 2H), 4.89-4.75 (m,
1), 3.05 (t, J ) 12.4 Hz, 2H), 2.33 (q, J ) 11.3 Hz, 2H), 2.17 (d,
J ) 13.1 Hz, 2H), 1.51 (s, 9H), 1.27 (d, J ) 6.8 Hz, 2H).
Structure was assigned using 2D-NOE experiments. A cross-
peak between H7ar m and H3piperidine was observed. MS m/z 337
°
(M - Boc + H).
General Procedure for the Synthesis of 47a-k, 48a-
e, 49a-d (as exemplified by the synthesis of 4-benzotri-
azolylpiperidinium trifluoroacetates (47a, 48a)). A solution of
44a (2.25 g, 7.45 mmol) in 125 mL of DCM was treated with
25 mL of TFA. The reaction was stirred for 90 min at rt and
evaporated to dryness. The oily residue was treated with
diethyl ether several times, upon which 2.23 g of the corre-
sponding trifluoroacetate of 47a precipitated (99%) as a
colorless solid, which was further neutralized with NH4OH.
1H NMR (DMSO-d6) δ 8.02 (d, J ) 8.4 Hz, 1H), 7.92 (d, J )
8.4 Hz, 1H), 7.52 (t, J ) 8.4 Hz, 1H), 7.38 (t, J ) 8.4 Hz, 1H),
4.91 (m, 1H), 3.09 (d, J ) 12.4 Hz, 2H), 2.69 (t, J ) 12.5 Hz,
2H), 2.19-1.97 (br. m, 5H). MS m/z 203 (M + H), 201.2 (M -
H).
Accordingly 4-benzotriazol-2-ylpiperidinium trifluoroacetate
(48a) could be accessed in quantitative yield. 1H NMR (CDCl3)
δ 8.95 (br s, 1H), 8.73 (br s, 1H), 7.94 (d, J ) 3.4 Hz, 1H), 7.92
(d, J ) 3.0 Hz, 1H), 7.45 (d, J ) 3.4 Hz, 1H), 7.43 (d, J ) 3.0
Hz, 1H), 5.28-5.14 (m, 1H), 3.45 (d, J ) 12.8 Hz, 2H), 3.21 (t,
J ) 10.1 Hz, 2H), 2.48-2.28 (m, 4H), MS m/z 203 (M + H),
201.2 (M - H).
Syntheses of 4-Benzotriazolylpiperidinesulfonamides
50a-k, 51a-e, 52a-d (as exemplified by the synthesis of
N-[5-(4-benzotriazol-1-ylpiperidine-1-sulfonyl)thiophen-2-yl-
methyl]-4-chlorobenzamide (50a) and N-[5-(4-benzotriazol-2-
ylpiperidine-1-sulfonyl)thiophen-2-ylmethyl]-4-chlorobenz-
amide (51a)). To a solution of 44a (2.23 g, 7 mmol) and DIEA
(3.6 mL, 21 mmol) in 45 mL of DCM was added during 1 h a
solution of 2 (2.2 g, 6.3 mmol) in DCM/DMF 9:1 (100 mL). The
reaction mixture was stirred for 3 h at rt. The organic layer
was washed with 0.1 N HCl and extensively washed with
brine. After drying over MgSO4, the solvent was evaporated
to yield 3.3 g of crude 50a. The crude product was recrystal-
lized from DCM/cyclohexane to yield 2.52 g (70%) of pure 50a.
1H NMR (DMSO-d6) δ 9.40 (t, J ) 5.6 Hz, 1H), 8.02 (d, J )
The trifluoroacetate was coupled to the sulfonyl chloride 2
as described in the protocol for the syntheses of 4-benzotri-
azolylpiperidinesulfonamides 50a-k, 51a-e, 52a-d. 50m (13
mg, 60%) was isolated after flash chromatography using
1
cyclohexane/EtOAc as eluent. H NMR (DMSO-d6) δ 9.40 (t,
J ) 6.0 Hz, 1H), 7.90 (d, J ) 8.6 Hz, 2H), 7.82 (d, J ) 7.5 Hz,
1H), 7.68 (d, J ) 8.3 Hz, 1H), 7.61-7.53 (m, 3H), 7.47-7.31
(m, 2H), 7.25 (d, J ) 3.7 Hz, 1H), 4.71 (d, J ) 5.6 Hz, 2H),
4.67-4.49 (m, 1H), 3.85 (br d, J ) 12.2 Hz, 2H), 2.80 (br t, J
) 11.4 Hz, 2H), 2.57-2.37 (m, 2H), 1.99 (d, J ) 10.3 Hz, 2H),
MS m/z 583.1 (M + H); 581.5 (M - H).
Biological Methods. 1. rJNK3 and rJNK2 Enzymatic
Assay. JNK3 and/or -2 assays were performed in 96-well
microtiter (MT) plates, by incubation of 0.5 µg of recombinant,
preactivated GST-JNK3 or GST-JNK2 with 1 µg of recombi-
nant, biotinylated GST-c-Jun and 2 µM 33γ-ATP (2 nCi/µL),
in the presence or absence of sulfonamide inhibitors in a
reaction volume of 50 µL containing 50 mM Tris-HCl, pH 8.0;
10 mM MgCl2; 1 mM dithiothreitol, and 100 µM NaVO4. The
incubation was performed for 120 min at rt and stopped upon
addition of 200 µL of a solution containing 250 µg of strepta-
vidin-coated SPA beads (Amersham, Inc.), 5 mM EDTA, 0.1%
Triton X-100, and 50 µM ATP, in phosphate saline buffer. After
incubation for 60 min at rt, beads were sedimented by
centrifugation at 1500g for 5 min, resuspended in 200 µL of
PBS containing 5 mM EDTA, 0.1% Triton X-100, and 50 µM
ATP, and the radioactivity was measured in a scintillation â
counter, following sedimentation of the beads as described
above. By substituting GST-c Jun for biotinylated GST-1ATF2
or myelin basic protein, this assay could also be used to

6934 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Ru¨ckle et al.
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