Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase

Article abstract of DOI:10.1021/jm0310986

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

Full text of DOI:10.1021/jm0310986

4596
J. Med. Chem. 2005, 48, 4596-4607
Design and Synthesis of the First Generation of Novel Potent, Selective, and in
Vivo Active (Benzothiazol-2-yl)acetonitrile Inhibitors of the c-Jun N-Terminal
Kinase
Pascale Gaillard,† Isabelle Jeanclaude-Etter,† Vittoria Ardissone,# Steve Arkinstall, Yves Cambet,|
Montserrat Camps,‡ Christian Chabert,‡ Dennis Church,† Rocco Cirillo,# Denise Gretener,| Serge Halazy,†
Anthony Nichols,| Cedric Szyndralewiez,§ Pierre-Alain Vitte,§ and Jean-Pierre Gotteland*,†
Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland,
Serono Research Institute, 1-Technology Drive, Rockland, Massachusetts 02370, and LCG-RBM, Serono International S.A.,
10010 Colleretto Giacosa, Turin, Italy
Received November 10, 2003
Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a
critical role in a wide range of diseases including cell death (apoptosis)-related disorders
(neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory
disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening
of our internal compound collection for inhibitors of JNK3 led to the identification of
(benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors.
Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship
(SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the
SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain
a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring
a number of distal combinations in place of the chlorine atom. This led to the observation that
the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and
bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the
potency. Further improvements to the biological and biopharmaceutical profile of the most
promising compounds were performed, resulting in the discovery of compound 59 (AS601245).
The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated
and found to demonstrate efficacy per oral route in an experimental model of rheumatoid
arthritis (RA).
Introduction
Members of the JNK family of kinases are activated
by proinflammatory cytokines such as tumor necrosis
factor-R (TNF-R) and interleukin-1â (IL-1â) as well as
environmental stress, such as anisomycin, UV irradia-
tion, hypoxia, and osmotic shock.3 Three distinct genes
encoding JNKs (jnk1, jnk2, and jnk3) have been identi-
fied, and at least 10 different splice variants exist in
mammalian cells.4 The downstream substrates of JNKs
include the transcription factors c-Jun, ATF-2, Elk1,
NFAT, p53, and a cell death domain protein.5-8 Each
JNK isoform binds to these substrates with different
affinities, suggesting a regulation of signaling pathways
by substrate specificity of different JNKs in vivo.9 JNK1
and JNK2 are widely expressed in a variety of tissues.
In contrast, JNK3 is selectively expressed in the brain
and to a lesser extent in the heart and testis.10,11
The c-Jun N-terminal kinases (JNKs) (also known as
“stress-activated protein kinases”) are members of the
mitogen-activated protein kinase (MAPK) family along
with p38 mitogen-activated protein kinases (p38 ki-
nases) and extracellular signal-regulated kinases (ERKs).
MAP kinases are serine/threonine kinases that are
activated by dual phosphorylation of threonine and
tyrosine residues of the Thr-X-Tyr motif in a loop located
adjacent to the active site.1,2 Phosphorylation of each
MAP kinase is carried out by specific kinases. Activated
MAP kinases then phosphorylate various substrates,
including transcription factors, which in turn regulate
the expression of specific sets of genes and thus mediate
a specific response to the stimulus.
Mice lacking jnk1 or jnk2 exhibit deficits in T-helper
(CD4) cell function.12-14 Double knockout animals are
embryonic lethal, although fibroblasts from these ani-
mals are viable in vitro and exhibit a remarkable
resistance to radiation-induced apoptosis.15 The jnk3
knockout mouse exhibits resistance to kainic acid
induced apoptosis in the hippocampus and to subse-
quent seizures.16 Therefore, JNK activity seems to be
critical for both the immune response and for pro-
grammed cell death,17 and therapeutic inhibition18 of
* To whom correspondence should be addressed. Phone:
+41227069824. Fax: +41227946965. E-mail: jean-pierre.gotteland@
serono.com.
† Department of Chemistry, Serono Pharmaceutical Research In-
stitute.
# Serono International S. A.
Serono Research Institute.
| Department of Screening, Serono Pharmaceutical Research Insti-
tute.
‡ Department of Signaling, Serono Pharmaceutical Research Insti-
tute.
§ Department of Pharmacology, Serono Pharmaceutical Research
Institute.
10.1021/jm0310986 CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/09/2005

Acetonitrile Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4597
Chart 1
Scheme 1a
JNK may provide clinical benefit in a wide range of
apoptosis-related and inflammatory disorders (e.g.,
neurodegenerative diseases,19 ischemia reperfusion in-
juries, multiple sclerosis, rheumatoid arthritis) although
recent evidence also supports the application of JNK
inhibitors in vascular, metabolic,20 and oncological
diseases.21
a Reagents: (a) concentrated HCl/EtOH [1/1], 2 days, room
temp; (b) (1) THF, NaH, 0 °C, 1 h; (2) 2,4-dichloroheteroaryl, THF,
room temp, ON; (c) aminoethylpolystyrene resin, Et3N, dioxane/
DMA [3/1], 70 °C.
Development of JNK inhibitors has gained increas-
ing interest in recent years.18 The anthrapyrazolone
SP600125 is an ATP competitive JNK1, -2, and -3
inhibitor for which a pharmacological profile has been
reported22 with a moderate selectivity over a range of
Ser/Thr and Tyr specific protein kinases.23 Important
contribution to the discovery of JNK inhibitors has been
made very recently by Merck researchers who have
published, for the first time, the JNK3 crystal structure
complex with various JNK small-molecule inhibitors.24
A proprietary drug discovery program at Serono
Pharmaceutical Research Institute was initiated with
the aim of identifying and characterizing small-molecule
JNK inhibitors as novel new chemical entities. We have
reported very recently the identification of (benzoyl-
aminomethyl)thiophene sulfonamide inhibitors such as
AS600292 as the first potent and selective JNK inhibitor
of this class that demonstrates a protective action
against neuronal cell death induced by growth factor
and serum deprivation.25
Scheme 2a
We now report the discovery of inhibitors of JNK iso-
forms, based on the benzothiazol-2ylacetonitrile pyrimi-
dine core, as a novel and unique kinase scaffold. The
structure-activity relationship (SAR) carried out on the
initial hit compound 1, identified by high-throughput
screening and leading to the discovery of compound 59,
and its antiinflammatory pharmacological properties
are the subject of this paper.
a Reagents: (a) MeI, K2CO3, DMSO, room temp, 2 days; (b) H2/
Pd, NaOAc, AcOH, 70 °C, 3 bar, 3 h; (c) 2 M NH3 in EtOH, 150
°C, 3 h.
Chemistry Section
chloropyridine in THF at room temperature under inert
atmosphere with the anion of 3 generated using sodium
hydride in THF (Scheme 1). The substitution of the
benzothiazole nitrogen to obtain compound 11 was
obtained by treating 6 with an excess of methyl iodide
in the presence of potassium carbonate in DMSO
(Scheme 2). The low yield obtained (5%) was due to the
low nucleophilicity of this nitrogen. Removal of the
chlorine atom at position 2 of the pyrimidine, leading
to compound 12, was performed by catalytic hydrogena-
tion of compound 6 using a combination of sodium
acetate and palladium on charcoal as a catalyst in acetic
acid (Scheme 2). An optimal yield of only 13% was
obtained by heating the suspension at 70 °C for 3 h
under 3 bar. The low yield observed was due to
degradation of either the product or the starting mate-
rial. The lack of efficiency of this reaction was probably
due to the presence of the sulfur atom (benzothiazole
moiety) poisoning the catalyst.
Screening of our internal compound collection for the
inhibition of rat JNK3 resulted in the identification of
compounds belonging to the benzothiazol-2ylacetonitrile
series. (3H-Benzothiazol-2-ylidene)(6-bromo-2-chloro-
pyrimidin-4yl)acetonitrile 1 was qualified as a hit and
was the starting point of our medicinal chemistry efforts
(Chart 1).
To investigate the effects on potency induced by small
changes in the structure of 1, cornerstone compounds
(with the exception of the commercially available (1H-
benzimidazol-2yl)acetonitrile analogue 2) were readily
synthesized from commercially available 1,3-benzo-
thiazol-2-ylacetonitrile 3 or (6-trifluoromethylbenzo-
thiazol-2yl)acetonitrile 4 (Scheme 1). Benzothiazol-2-
ylacetic acid ethyl ester 5 was obtained by treatment of
3 at room temperature for 2 days in a 1/1 mixture of
concentrated HCl and EtOH (Scheme 1).26
Derivatives 6-10 were obtained using method A by
reacting the corresponding dichloropyrimidine or di-

4598 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Gaillard et al.
Table 1. Cornerstone Modifications from Screening Hit 1
Scheme 3a
rJNK3
compd
A
X
R1
CN
R2
R3 R4 R5 IC50 (nM)a
a Reagents: (a) R1R2NH, Et3N, EtOH, 70 °C, 1-2 days.
1
2
N
N
N
N
C
N
N
N
N
S
H
Br
Br
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
350
>5000
250
>5000
>5000
993
>5000
>5000
>5000
NH CN
H
Chart 2
6
S
S
S
S
S
S
S
CN
H
7
CN
H
Me
H
8
CN
H
9
CN
CF3
H
H
10
11
12
COOEt
CN
H
H
H
H
Me Cl
CN
H
H
H
a All values in triplicate.
compounds in organic solvents, extending the scope of
chemical transformations that could be investigated.
Going from the benzothiazole group to a benzimid-
azole group led to a drastic drop in activity. The same
effect was seen with the replacement of the cyano group
by an ethyl ester (compound 10) or when a methyl group
was substituted on the nitrogen of the benzothiazole
moiety (compound 11). This latter observation demon-
strates that the benzothiazole acetonitrile moiety binds
to the ATP pocket via its exo tautomeric form because
an intramolecular hydrogen bond is likely to form
between the NH group of the benzothiazole moiety and
one of the nitrogens of the pyrimidine ring (Chart 2).
The outcome of the introduction of a trifluoromethyl
group at position 6 of the benzothiazole moiety was less
drastic 9, although it did induce a loss of potency (990
nM).
The changes investigated in the pyrimidine moiety
were also detrimental to JNK3 potency, as shown by
compound 8. The replacement of one of the pyrimidine
nitrogens at position 4 afforded the inactive pyridine
analogue 8. In addition, the presence of the chorine atom
at position 2 of the pyrimidine moiety seemed necessary
to retain JNK3 activity as shown by compound 12.
Because none of the cornerstone changes attempted
on the scaffold led to any real improvements in JNK3
potency, compound 6, exhibiting an improved potency
and solubility, was considered as the new starting point
to further investigate the SAR.
Compounds 13-62 were synthesized by nucleophilic
substitution using the common intermediate 6. The
replacement of the chlorine by NH2 was performed
under pressure by reacting compound 6 with a 2 M
solution of ammonia in EtOH at 150 °C in a Parr vessel,
affording compound 13 in 51% yield (Scheme 2).
Compounds 14-62 were obtained by displacing the
chlorine atom in position 2 of the pyrimidine moiety
with various amines using the conditions of method B.
All the reactions were performed in ethanol at 70 °C
using Et3N to scavenge the HCl formed (Scheme 3).
It is noteworthy that compounds 1, 2, 6-10, and 12-
62, described in this paper, exist as tautomers. Exten-
sive studies were performed to characterize these
compounds (unpublished results). The 1H NMR in
deuterated DMSO showed that in all cases only the form
with the exocyclic double bond exists in solution, most
likely in configuration E (Chart 2). Indeed, the presence
of a broad exchangeable signal between 10 and 12 ppm
on one hand and the absence of signal in the range of
4-6 ppm on the other hand could correspond only to
the form with the exocyclic double bond. This broad
signal accounting for one proton in the case of the
benzothiazole derivatives (1, 6-10, and 12-62) and two
protons in case of the benzimidazoles 2 could only
correspond to the proton of the enamine.
Results and Discussion
All the results obtained by the displacement of the
chlorine at position 2 of the pyrimidine moiety are
gathered in Table 2. Starting with alkylamines and
cycloalkylamines (12-35), we observed that tertiary
amines were less potent than secondary amines, as
shown by the potency loss observed between compounds
15 and 16 (10-fold), thereby suggesting that the pres-
ence of the pyrimidine “NH” was crucial for activity on
JNK3. This optimized atom arrangement could cor-
respond to the well-described bidentate interaction,
commonplace in several publications on ATP-competi-
tive kinase inhibitors.27,28 The results obtained with the
cyclic amines (17-21) corroborated this hypothesis
because all these compounds exhibited an IC50 of greater
than 2500 nM. In addition, the stronger the donating
capacity of the substituents on the pyrimidine moiety,
the weaker was the potency on rJNK3. Any substituent
Our starting point compound 1 exhibited an IC50 on
rJNK3 of 350 nM as shown in Table 1. To investigate
which parts of the scaffold were necessary for JNK3
activity, cornerstone compounds, like the benzimidazole,
were purchased (analogue 2) or synthesized (compounds
6-12). As shown in Table 1, among the combinations
tested, the only transformation tolerated by the enzyme
involved the removal of the bromine on position 6 of the
pyrimidine moiety, giving rise to compound 6, which
exhibited an IC50 of 250 nM. The replacement of the
bromine by a methyl group at the same position on the
pyrimidine (compound 7) induced a complete loss of
activity. The result obtained with compound 6 was a
real improvement (from a chemistry point of view)
because it did not affect the reactivity of the remaining
chlorine atom but led to an increase in solubility of these

Acetonitrile Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4599
Table 2. Substitution at the Pyrimidinyl-2-chloro Position
The length of the linker, between the pyrimidine and
this polar group identified as being important for
activity, was investigated. The results obtained sug-
gested the presence of a second interaction at the
binding site two to three carbons away from the main
interaction reached by the pyrimidine-NH group. In-
deed, most of the compounds exhibited potency on JNK3
ranging from 400 to 4000 nM. The best potency was
obtained with compounds 32 and 33, bearing morpho-
linyl or 4-methylpiperazinyl moieties linked to the
pyrimidine group by an aminopropyl chain.
Although none of the substitutions described above
led to compounds better than our initial compound 6,
features critical for activity were identified, i.e., the
presence of the pyrimidine-NH (main interaction) and
distal heteroatom (putative second interaction) sepa-
rated by a two to three carbon linker. The influence on
the potency of a number of aromatic or heteroaromatic
groups combining these structural features was then
investigated.
Substitution of the pyrimidine by aromatic or hetero-
aromatic amines with different linker lengths led to
compounds 36-62. The results obtained are in ac-
cordance with our previous observations. Among com-
pounds 36-40 bearing a one-carbon linker, only the
pyridine derivatives 37-39, with a preference for 3- and
4-pyridines, allowed us to retain the potency observed
with the best alkylamines, previously described (com-
pounds 32 and 33). The benzylamine derivative 36 was
20 times less active, confirming the need of a heteroatom
in this part of the molecule, although heteroaromatic
groups having an acidic character like the tetrazole
derivative 40 were not tolerated by the enzyme. These
results most probably suggested that the required
heteroatom acts as a hydrogen bond acceptor (HBA),
explaining the lack of activity of the tetrazolyl analogue.
Increasing the length of the linker by up to two
carbons with the substituted phenethylamine deriva-
tives 41-53 led to potency below 100 nM, particularly
in the case where the phenethylamine was substituted
in the para position with an amine group (51) or a
primary sulfonamide (52). On one hand, naked phen-
ethylamines and phenethylamines substituted with
electron-withdrawing or electron-donating lipophilic
groups (4-F, 3-Cl, 3,4-di-Cl, 4-Br, 4-Me) led to inactive
compounds (44 and 45-48). On the other hand, the
introduction of polar groups (4-OH, 4-OMe, 4-NH2,
4-SO2NH2, 4-NO2) led to compounds exhibiting a po-
tency ranging from 3500 to 40 nM (49-53). Interest-
ingly, the 2- and 3-fluorophenethylamine derivatives 42
and 43 showed a potency of 273 and 1810 nM, respec-
tively, on the enzyme probably because of the HBA
properties of the fluorine atom, thereby corroborating
the hypothesis drawn previously.
rJNK3
compd
R1
R2
IC50 (nM)a
13
14
15
16
17
18
19
20
21
22
23
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
50
51
52
53
54
55
56
57
58
59
60
61
62
H
H
7500
500
H
NH2
Me
Me
H
Me
950
9400
6600
6800
2900
>5000
7200
1300
1490
820
piperazinyl
4-Me-piperazinyl
morpholinyl
pyrolidinyl
4-OH-piperidinyl
(CH2)2N(Me)2
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
(CH2)2NH2
(CH2)2OMe
(CH2)2OH
510
(CH2)2CH3
>5000
3740
760
(CH2)2N-piperidinyl
(CH2)2N-morpholinyl
(CH2)3N(Me)2
1644
707
(CH2)3NH2
(CH2)3OH
660
(CH2)3N-morpholinyl
(CH2)3N-(4-Me-piperazinyl)
(CH2)3N-pyrolidinyl-2-one
(CH2)3NHMe
407
473
1340
1324
6500
650
CH2Ph
CH2-pyridin-2-yl
CH2-pyridin-3-yl
CH2-pyridin-4-yl
CH2-tetrazol-2-yl
(CH2)2Ph
337
340
>5000
>5000
273
(CH2)2-Ph-2-F
(CH2)2-Ph-3-F
1810
>5000
>5000
>5000
>5000
>5000
3500
3080
80
(CH2)2-Ph-4-F
NH(CH2)2-Ph-3-Cl
NH(CH2)2-Ph-3,4-diCl
NH(CH2)2-Ph-4-Br
(CH2)2-Ph-4-Me
(CH2)2-Ph-4-OH
(CH2)2-Ph-4-OMe
(CH2)2-Ph-4-NH2
(CH2)2-Ph-4-SO2NH2
(CH2)2-Ph-4-NO2
(CH2)2-indolyl
41
600
458
(CH2)2-imidazol-4-yl
(CH2)2-imidazol-4N-Me
(CH2)2-imidazol-2N-Me
(CH2)2-pyridin-2-yl
(CH2)2-pyridin-3-yl
(CH2)2-N-1,2,4-triazolyl
(CH2)3-N-imidazolyl
(CH2)3-N-pyrazolyl
80
65
143
250
120
397
147
583
a All values in triplicate.
From these observations, it was clearly shown that
the potency on rJNK3 was improved by the presence of
an aromatic group two carbons away from the amino
pyrimidine moiety and bearing substituents conferring
HBA properties able to reach a second interaction in
the ATP binding pocket.
Phenethyl-like heteroaromatic amine derivatives (54-
60) were further examined for their ability to inhibit
the purified enzyme. As shown in Table 2, N-ethyl-
imidazolyl derivatives, substituted or not (55 to 57), and
that enhanced the electron-deficient character of the
pyrimidine decreased the potency of the inhibitors
within this series (N(Me)2 < NHMe < NHNH2 < Cl).
Further analysis of these moderately active compounds
allowed the extraction of the following information: the
presence of polar groups (OR or NR1R2) within the
lipophilic chains, cyclic or not (17-19, 21, and 35),
contributed to improving the potency if compared with
compounds 20 and 26 bearing, respectively, a pyrroli-
dinyl and n-propyl moiety.

4600 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Gaillard et al.
Table 3
Jurkat cells
rJNK3 % inhibition
compd
R
IC50 (nM)
@10 µM
6
33
38
39
44
42
51
55
56
57
58
59
61
Cl
250
473
337
340
>5000
273
80
90
83
NH(CH2)3N-(4-Me-piperazinyl)
NHCH2-pyridin-3-yl
95
NHCH2-pyridin-4-yl
nda
0
NH(CH2)2-Ph-4-F
NH(CH2)2-Ph-2-F
90
NH(CH2)2-Ph-4-NH2
80
NH(CH2)2-imidazol-4-yl
NH(CH2)2-imidazol-4N-Me
NH(CH2)2-imidazol-2N-Me
NH(CH2)2-pyridin-2-yl
NH(CH2)2-pyridin-3-yl
NH(CH2)3-N-imidazolyl
80
80
65
nda
100
60
Figure 1. Effect of compound 59 on LPS induced TNF-R
143
250
120
147
release.
90
31
monitored by MTS). The observed increase in IC50
values between the biochemical assay and cell-based
assay is most likely a reflection of high ATP levels
present in the cell, although equally we cannot exclude
that this may be due to low cellular permeability of the
compounds. Further IC50 determinations led us to select
the most effective compounds in cells to further char-
acterized their pharmacokinetics properties and in
particular their oral bioavailability. Compound 59 ex-
hibited an acceptable bioavailability in rat (Fz ) 38%)
and was selected as our model compound to further
evaluate, in vivo, the anti-inflammatory efficacy of this
new class of JNK inhibitors.
Compound 59 was tested in a proof of concept in vivo
model of inflammation that measured its ability to
reduce TNFR release induced by lipopolysaccharide
(LPS) in mice in comparison with the broad anti-
inflammatory drug dexamethasone as a positive control.
Compound 59 exhibited a dose-dependent decrease of
TNFR plasma level with an ED50 of 3 mg/kg when
administered by oral route (Figure 1). In light of this
result, 59 could have a high potential in disease models
of inflammation. Indeed, overproduction of proinflam-
matory cytokines in rheumatoid arthritis (RA), such as
TNFR, leads to persistent up-regulation of various
molecules,31 such as metalloproteases, responsible for
the inflammation and destructive processes in the joints.
Inhibition of the signal transduction pathways that
either lead to production of proinflammatory cytokines
or are responsible for their downstream effects could be
an effective route for the treatment of RA. Because the
role of JNK activation and JNK inhibition in the
pathology of RA has already been demonstrated,32-34
compound 59 was tested in an experimental model of
RA.
a Not determined.
N-ethyl-2- and -3-pyridines (58 and 59) led to com-
pounds with a potency ranging from 250 to 65 nM. The
results obtained with the N-ethylimidazolyl derivatives
confirmed that the presence of a nitrogen with HBA
properties was necessary to get a potency below 100 nM,
as shown by the difference in activity exhibited by
compounds 55 and 56 on one hand and compound 57
on the other hand, in which the HBA was forced into
the position 4 of the imidazole moiety by introduction
of a methyl group at position 2. This observation was
emphasized by the weaker activity shown by the amino-
ethylindolyl derivatives 54 where the nitrogen behaves
as a hydrogen bond donor (HBD). The marked decrease
in potency noticed for the N-ethyltriazole derivative 60
(400 nM) might suggest that decreasing the electron
density of the nitrogen HBA is detrimental for activity,
thus weakening this second interaction. Finally, extend-
ing the linker to three carbons (61 and 62) did not
further influence the activity.
The introduction of these N-ethyl heteroaromatic
substituents allowed access to potent JNK3 inhibitors
with improved physicochemical properties, hence im-
proving the druglike properties of the compounds of this
series compared to our hit compound 6. Indeed, the
introduction of another protonable center allowed a
dramatic increase in the solubility in aqueous media of
these compounds, as shown by compound 59, which is
soluble at over 100 mg/mL in saline as a dimesylate salt.
As anticipated for the high identity of the ATP binding
region of the different JNK isoforms, none of the
compounds mentioned above demonstrated specificity
when tested against the rat or the human versions of
JNK1, -2, or -3 (results not shown).
Compound 59 induced significant anti-inflammatory
effects when orally administered in a therapeutic dosing
regimen (i.e., after disease onset) in a mouse model of
collagen induced adjuvant (CIA) rheumatoid arthritis.
The compound elicited significant depression of paw
swelling and a clinical score at 60 mg/kg (Figure 2),
while the reference compound indomethacin completely
inhibited paw swelling.
The activity of the most potent compounds was then
assessed in a Jurkat T-cell assay (Table 3) where JNK
has been reported to regulate the transcription of the
IL-2 gene.29,30 Jurkat cells from a human T lymphoblast
cell line were treated with phorbol-12 myristate-13
acetate (PMA) plus ionomycin to induce IL-2 production.
Compounds 6, 33, 42, 57, and 59 demonstrated almost
complete inhibition at 10 µM IL-2 secretion in this assay
(no cell toxicity was observed at this concentration as
As well as reducing joint swelling, histopathological
analysis revealed that 59 preserved joint areas (de-

Acetonitrile Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4601
Figure 2. Effect of compound 59 on the clinical course (A) and joint histology (B) in an experimental CIA model.
Table 4. Compound 59 Kinase Selectivity Profilea
selective, we can assume that its in vivo effects are
mainly due to the selective inhibition of the JNK
isoforms, as we have demonstrated in other in vivo
models of ischemia reperfusion injuries such as myo-
cardial infarction35 or cerebral ischemia.36
kinases
JNK3
JNK2
JNK1
IC50 (µM)
kinases
MKK7b
IC50 (µM)
0.07
0.22
0.15
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
MKK4
MAPKAP-K2
ERK1
MEK1
PI3Kg
PDK1
AKT
c-SRC
c-Raf
CDK2/CycA
P38a
1.1
1.2
1.4
4.8
Conclusion
Altogether, we have identified a novel class of (benzo-
thiazol-2-yl)acetonitrile derivatives as potent and selec-
tive JNK1, -2, -3 inhibitors. SAR studies initially led to
the discovery of compound 6, which was further opti-
mized. It was clearly shown that the potency on rJNK3
was improved by the presence of an aromatic group, two
carbons away from the aminopyrimidine moiety and
bearing substituents conferring HBA properties able to
reach a second interaction in the ATP binding pocket.
The introduction of aminoethyl heteroaromatic substit-
uents allowed access to very potent JNK3 inhibitors
with improved physicochemical properties, thereby
improving the druglike properties of compounds of this
chemical series to provide 59. This compound demon-
strated in vivo reduction of TNF-R production and
arthritis severity, confirming the ever increasing po-
tential of JNK inhibitors to effectively act as anti-
inflammatory agents.
p70S6K
P56Lck
CHK1
EGF
MKK6
RSK-2
ROCK-II
Blk
MSK1
SGK
5-10
5-10
5-10
5-10
5-10
5-10
IKKb
PKC
PRAK
a [ATP] ) 10 µM.
creasing cartilage erosion) and reduced synovium in-
flammation in a significant manner at 60 mg/kg. This
clearly demonstrates the potential of JNK inhibitors for
the treatment of anti-inflammatory diseases. Selectivity
of compound 59 was tested against a large panel of
kinases (Table 4). It exhibited 10- to 20-fold selectivity
over c-src, CDK2, and c-Raf and above 50- to 100-fold
selectivity over a range of Ser/Thr and Tyr protein
kinases. Although 59 was established as moderately

4602 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Gaillard et al.
HPLC (condition b, 254 nm) 92%, tR ) 13.91 min; 1H NMR
(DMSO-d6) δ 13.22 (br s, 1H, exchangeable, H9), 7.96 (d, J )
7.79 Hz, 1H, H3), 7.63 (d, J ) 8.17 Hz, 1H, H4), 7.48-7.42
(m, 1H, H2), 7.30-7.25 (m, 1H, H1), 7.07 (s, 1H, H12), 2.39
(s, 3H, CH3 pyrimidine).
1,3-Benzothiazol-2-yl(6-chloro-2-pyridyl)acetonitrile (8).
Yield ) 36.2%; HPLC (314 nm) 99.6%, tR ) 7.02 min (10 min
TFA); MS m/z (APCI+) 286.0; 1H NMR (DMSO-d6) δ 12.44 (s,
1H, H9), 7.64 (d, J ) 7.54 Hz, 1H, H3), 7.57 (t, J ) 7.91 Hz,
1H, H pyridine), 7.31 (d, J ) 7.91 Hz, 1H, H pyridine), 7.20-
7.14 (m, 1H, H2), 7.07 (d, J ) 8.29 Hz, 1H, H pyridine), 7.00-
6.96 (m, 1H, H5), 6.86 (d, J ) 7.54 Hz, 1H, H4). Anal.
(C14H8ClN3S) C, H, N.
(2-Chloropyrimidin-4-yl)[5-(trifluoromethyl)-1,3-benzo-
thiazol-2-yl]acetonitrile (9). Yield ) 58.6%; MS m/z 352.6
(M - 1); HPLC (condition c, max plot) 99.8%, tR ) 5.96 min;
1H NMR (DMSO-d6) δ 13.40 (s, 1H, H9), 8.37 (d, J ) 5.66 Hz,
1H, H13), 8.21 (d, J ) 8.29 Hz, 1H, H3), 7.87 (s, 1H, H4), 7.60
(d, J ) 7.91 Hz, 1H, H2), 7.23-7.19 (m, 1H, H12). Anal. (C14H6-
ClFN4S) C, H, N.
1,3-Benzothiazol-2-yl(2-chloro-pyrimidin-4-yl)acetic
Acid Ethyl Ester (10). To a suspension of NaH (60% in oil,
0.039 g, 0.94 mmol) in dry THF (1 mL) was added dropwise a
solution of benzothiazol-2-ylacetic acid ethyl ester (5) (0.104
mg, 0.47 mmol) in dry THF (1 mL), and the suspension was
stirred for 1 h at room temperature under an inert atmosphere.
A solution of 2,4-dichloropyrimidine (0.070 g, 047 mmol) in
THF (1 mL) was added dropwise. After the mixture was stirred
for 30 min at room temperature under argon, the reaction was
stopped by addition of water. The product was extracted with
AcOEt (4×). Then the organics were washed with water and
brine (4×) until neutral pH was attained and dried over MgSO4
before removal of the solvent. The solid obtained was dried
overnight in a vacuum. The solid was taken up in ether and
warmed. The precipitate was filtered off and washed with
ether and dried at 40 °C in a vacuum to give 44 mg of the title
compound as a beige powder. Yield ) 28%; HPLC (247 nm)
97.8% tR ) 6.76 min (10 min TFA); MS m/z (APCI-) 332.0; 1H
NMR (DMSO-d6) δ 13.62 (br s, 1H, H9), 8.42 (br d, 1H, H13),
7.96-7.93 (m, 2H, H3, H4), 7.87-7.84 (m, 1H, H12), 7.49-
7.44 (m, 1H, H2), 7.34-7.29 (m, 1H, H1), 4.36 (q, J ) 7.16 Hz,
2H, COOCH2CH3), 1.37 (t, J ) 7.16 Hz, 3H, COOCH2CH3).
(2-Chloro-4-pyrimidinyl)(3-methyl-1,3-benzothiazol-
2(3H)-ylidene)ethanenitrile (11). To a solution of 6 (0.1 g,
0.35 mmol) in dry DMSO (1 mL) was added dry K2CO3 (48
mg, 0.35 mmol) and then methyl iodide (0.02 mL, 0.35 mmol).
The suspension was shaken at room temperature for 2 days.
The precipitate formed after addition of water, was filtered
off, and then washed with water until neutral pH was attained.
The crude residue, dried under vacuum at 40°C, was triturated
in warm acetonitrile, filtered off, and then dried under vacuum
at 40°C, affording 5.6 mg (5%) of the title compound as a yellow
powder. MS m/z 623 (2M + Na); HPLC (condition a, 388 nm)
99%, tR ) 5.31 min; 1H NMR (DMSO-d6) δ 8.01 (d, J ) 7.7 Hz,
1H, H3), 7.83 (d, J ) 8.0 Hz, 1H, H4), 7.79 (d, J ) 7.4 Hz, 1H,
H13), 7.45-7.40 (m, 1H, H2), 7.31-7.24 (m, 1H, H1), 6.81 (d,
J ) 7.4 Hz, 1H, H12), 3.67 (s, 3H, CH3).
We have further concentrated our efforts on the
identification of compounds with an improved biophar-
maceutical profile and kinase selectivity profile to fully
dissect the pharmacological potential of this new class
of JNK inhibitors for the treatment of inflammatory
diseases,37 and this will be the subject of a future paper.
Experimental Section
General Experimental Methods. Procedures. Melting
points were measured with a Bu¨chi B-545 melting point
apparatus and were uncorrected. 1H NMR spectra were
recorded on a Brucker DPX 300 MHz spectrometer. The
following data were reported: chemical shift δ in ppm using
residual DMSO-d6 as internal standard (2.49 ppm), multiplic-
ity (s ) singlet, d ) doublet, t ) triplet, q ) quadruplet, m )
multiplet), coupling constant (s) in hertz, and integration. MS
data provided were obtained using a Perkin-Elmer API 150
EX (APCI) mass spectrometer. Analytical HPLC was per-
formed using an HPLC Waters Symmetry C8 50 mm × 4.6
mm column. Conditions were the following: (a) MeCN/H2O,
0.09% TFA, from 0% to 100% (10 min); (b) MeCN/H2O, 0.09%
TFA, from 0% to 100% (20 min); (c) MeCN/H2O, 0.09% TFA,
from 5% to 100% (10 min), max plot 230-400 nm; (d) MeCN/
H2O, from 5% to 100% (10 min), max plot 230-400 nm.
In the experimental procedures, “purification by preparative
HPLC” refers to dissolving the crude product in DMF and in
the following purifying solvents: solvent A, 0.1% TFA in
acetonitrile; solvent B, 0.1% TFA in water; 0-100% solvent A
over 50 min (flow rate 50 mL/min; UV monitored at 240 nM).
Preparative HPLC was performed on a Water Prep LC 4000
system using two different column sizes depending on the
quantity to purify: small column (up to 200 mg), Xterra Prep
MS C8, 10 µm, 50 mm × 30 mm; large column (from 200 to
1000 mg), Xterra Prep MS C8, 10 µm, 50 mm × 30 mm. Signals
were detected using a Waters 2487 absorbance detector with
dual wavelength.
Elemental analyses were performed with a VarioEL CHN
analyzer from Elementar.
Benzothiazol-2-ylacetic Acid Ethyl Ester (5). A solution
of benzothiazol-2ylacetonitrile (3) in a mixture of EtOH and
concentrated HCl [1/1] (30 mL) was stirred at room temper-
ature. After 2 days, EtOH was evaporated. The acidic aqueous
phase was extracted (3×) with AcOEt. Then the organics were
washed with brine, and the solvent was evaporated. The oily
residue was dried in a vacuum at 40 °C overnight.
Method A: 1,3-Benzothiazol-2-yl-(2-chloro-4-pyrimidin-
yl)acetonitrile (6). To a stirred suspension of NaH (60% in
oil, 9.2 g, 0.23 mol) in dry THF (200 mL) was added dropwise
under inert atmosphere a solution of 1,3-benzothiazol-2-
ylacetonitrile (20 g, 0.15 mol) in dry THF (200 mL). After the
mixture was stirred for 1 h and 30 min at room temperature,
a solution of 2,4-dichloropyrimidine (17.1 g, 0.15 mol) in dry
THF (200 mL) was added dropwise. The reaction mixture was
allowed to stir under inert atmosphere at room temperature
until complete disappearance of the starting material. The
reaction was quenched by addition of water, and the THF was
evaporated. Water was added, and the suspension was slightly
acidified with aqueous 1 M HCl to pH∼4.0. The precipitate
obtained was filtered off, washed thoroughly with water until
neutral pH was attained, and then washed with hexane to
remove the oil. The crude solid was dried under vacuum at 40
°C, affording 28 g (84%) of the title compound (6) as a brown
powder: mp 246 °C dec; MS m/z 286.8 (M + 1); HPLC
1,3-Benzothiazol-2-yl(pyrimidin-4-yl)acetonitrile (12).
To a solution of 6 (0.1 g, 0.35 mmol) in acetic acid was added
sodium acetate (29 mg, 0.35 mmol) and palladium on charcoal
(20 mg). The suspension was heated to 70 °C under hydrogen
at 3.5 bar for 3 h. After cooling to room temperature, the
suspension was filtered through Celite and the acetic acid was
evaporated. The bright-yellow powder was taken up in AcOEt
and 10% aqueous NaOH. After three extractions, the organic
phases were thoroughly washed with brine and then dried over
MgSO4 and concentrated to dryness. After purification by
preparative HPLC and drying under vacuum at 50 °C, 12 mg
(13%) of the title compound were obtained as a yellow powder.
MS m/z 253.2 (M + 1); HPLC (condition c, max plot) 98%, tR
1
(condition a, 268 nm) 97%, tR ) 5.66 min; H NMR (DMSO-
d6) δ 13.25 (br s, 1H, exchangeable, H9), 8.09 (d, J ) 4.14 Hz,
1H, H13), 7.90 (d, J ) 7.53 Hz, 1H, H3), 7.61 (d, J ) 7.92 Hz,
1H, H4), 7.39-7.34 (m, 1H, H2), 7.20-7.15 (m, 1H, H1), 6.96
(br d, 1H, H12). Anal. (C13H7ClN4S) C, H, N.
By use of method A described above and the appropriate
starting material and reagents, compounds 7-9 could be
obtained.
1,3-Benzothiazol-2-yl-(2-chloro-6-methyl-4-pyrimidin-
yl)acetonitrile (7). Yield ) 42.8%; MS m/z 300.8 (M + 1);
1
) 3.35 min; H NMR (DMSO-d6) δ 11.24 (br s, H9), 8.61 (s,
1H, H pyrimidine), 8.00-7.73 (m, 3H, H13, H3, H4), 7.44-
7.39 (m, 1H, H2), 7.29-7.23 (m, 1H, H1), 6.90 (br d, 1H, H12).

Acetonitrile Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4603
(2-Aminopyrimidin-4-yl)(1,3-benzothiazol-2-yl)aceto-
nitrile (13). A suspension of 6 (0.1 g, 0.35 mmol) in a 2 M
solution of ammonia in EtOH (10 mL) was heated to 150 °C
in a Parr vessel for 3 h. The solution was cooled to room
temperature, and the yellow precipitate formed was filtered
off and then washed thoroughly with 1:1 EtOH/water and
water. The precipitate was dried in a vacuum at 40 °C,
affording 48 mg (51%) of the title compound as a yellow
powder. MS m/z 268.0 (M + 1); HPLC (condition c, max plot)
95%, tR ) 3.20 min; 1H NMR (DMSO-d6) δ 10.92 (br s, 1H,
exchangeable, H9), 7.79 (d, J ) 7.16 Hz, 1H, H3), 7.70 (d, J )
7.01 Hz, 1H, H4), 7.42-7.15 (m, 5H, H13, H1, H2, pyrimidine
NH2 exchangeable), 6.34 (d, J ) 7.54 Hz, 1H, H12).
7.29-7.24 (m, 1H, H1), 6.60 (d, J ) 5.8 Hz, 1H, H12), 4.95-
4.70 (m, 2H, CH2-piperazine), 4.42-3.68 (m, 1H, CF3CO2H),
3.67-3.50 (m, 2H, CH2-piperazine), 3.48-3.31 (m, 2H, CH2-
piperazine), 3.26-3.05 (m, 2H, CH2-piperazine), 2.86 (s, 3H,
NCH3).
1,3-Benzothiazol-2-yl[2-(4-morpholinyl)-4-pyrimidinyl]-
acetonitrile (Trifluoroacetate) (19). Yield ) 55%; MS m/z
338.0 (M + 1); HPLC (condition a, 270 nm) 99%, tR ) 3.51
min; 1H NMR (DMSO-d6) δ 7.94 (d, J ) 7.8 Hz, 1 H, H3), 7.79
(br d, 1H, H13), 7.70 (d, J ) 8.0 Hz, 1H, H4), 7.45-7.40 (m,
1H, H2), 7.30-7.25 (m, 1H, H12), 6.54 (d, J ) 6.3 Hz, 1H, H12),
4.40-3.65 (m, 9H, CH2 morpholine and CF3CO2H)
1,3-Benzothiazol-2-yl[2-(1-pyrrolidinyl)-4-pyrimidinyl]-
MethodB: 1,3-Benzothiazol-2-yl(2-{[2-(1H-imidazol-4-yl)-
ethyl]amino}-4-pyrimidinyl)acetonitrile (Bis(trifluoro-
acetate)) (55). To a suspension of 6 (0.1 g, 0.35 mmol) in dry
EtOH (3 mL) was added Et3N (0.05 mL, 0.35 mmol) and
histamine (0.078 g, 0.70 mmol). After sonication, the yellow
solution was shaken at 70 °C for 3 days. The yellow precipitate
formed was filtered off and washed with H2O (2×) and then
with EtOH (3×) and dried under vacuum at 40 °C, affording
47 mg (37%) of the title compound as a bright-yellow powder:
mp 257-258°C. This powder was taken up in a mixture of
DCM and TFA. The yellow fluffy solid formed by addition of
Et2O was filtered off, washed with Et2O (3×), then dried under
vacuum at 40 °C, affording 36 mg (29%) of the title compound
55 as a yellow powder: mp 247-249°C; MS m/z 362.0 (M +
acetonitrile (20). Yield ) 27%, mp 270-272 °C; MS m/z 322.0
1
(M + 1); HPLC (condition a, 372 nm) 98%, tR ) 3.90 min; H
NMR (DMSO-d6) δ 11.30 (v br s, 1 H, exchangeable, H9), 7.86
(d, J ) 7.2 Hz, 1H), 7.66 (d, J ) 7.9 Hz, 1H, H3), 7.46 (d, J )
6.8 Hz, 1H, H13), 7.35-7.30 (m, 1H, H2), 7.17-7.12 (m, 1H,
H1), 6.33 (d, J ) 6.8 Hz, 1H, H12), 3.90-3.45 (m, 4H, CH2-
pyrrolidine), 2.08-1.94 (m, 4H, CH2-pyrrolidine).
1,3-Benzothiazol-2-yl[2-(4-hydroxy-1-piperidinyl)-4-pyr-
imidinyl]acetonitrile (Trifluoroacetate) (21). Yield ) 14%;
MS m/z 352 (M + 1); HPLC (condition a, 271 nm) 97%, tR
)
3.21 min; 1H NMR (DMSO-d6) δ 7.95 (d, J ) 7.79 Hz, 1H, H3),
7.72-7.65 (m, 2H, H4 and H13), 7.45-7.40 (m, 1H, H2), 7.28-
7.23 (m, 1H, H1), 6.48 (, d, J ) 6.07 Hz, 1H, H12), 4.60-3.75
(m, 5H, CH2piperidine, HOCH-piperidine, CF3CO2H), 3.58-
3.51 (m, 2H, CH2-piperidine), 1.95-1.82 (m, 2H, CH2-piperi-
dine), 1.55-1.42 (m, 2H, CH2-piperidine).
1
1); HPLC (condition a, 265 nm) 98%, tR ) 2.87 min; H NMR
(DMSO-d6) d 14.25 (br s, 2H, exchangeable), 11.05 (br s, 1H,
exchangeable), 9.03 (s, 1H), 7.94-7.87 (m, 1 H), 7.74-7.71 (m,
2H), 7.57-7.52 (m, 2H), 7.42-7.37 (m, 1H), 7.24-7.19 (m, 1H),
6.40 (d, J ) 7.1 Hz, 1H), 3.97-3.55 (m, 3H), 3.11-3.05 (m,
2H). Anal. (C18H15N7S‚2C2HF3O2) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(dimethylamino)ethyl]ami-
no}-4-pyrimidinyl)acetonitrile (Bis(trifluoroacetate)) (22).
Yield ) 30%; MS m/z 339.0 (M + 1); HPLC (condition a, 270
1
nm) 99%, tR ) 2.69 min; H NMR (DMSO-d6) δ 11.85 (v br s,
By use of method B described above for compound 55 and
the appropriate starting material and reagents, the following
compounds could be obtained:
1,3-Benzothiazol-2-yl(2-hydrazino-4-pyrimidinyl)aceto-
nitrile (Trifluoroacetate) (14). Yield ) 60%; MS m/z 283.0
(M + 1); HPLC (271 nm) 98%, tR ) 3.17 min; 1H NMR (DMSO-
d6) δ 9.78 (br s, 1H, exchangeable, H9), 7.89-7.75 (m, 4H, H13,
H4, H3, pyrimidineNHNH2), 7.48-7.43 (m, 1H, H2), 7.32-
7.27 (m, 1H, H1), 6.53 (br d, 1H, H12), 4.25-3.40 (m, 1H,
CF3CO2H).
1H, exchangeable, H9), 9.59 (br s, 1H), 7.90 (br d, 2H, H3 and
NH-pyrimidine), 7.73 (d, J ) 7.9 Hz, 1H, H4), 7.60 (br d, 1H,
H13), 7.43-7.38 (m, 1H, H2), 7.27-7.22 (m, 1H, H1), 6.43 (d,
J ) 6.8 Hz, 1H, H12), 4.25-3.70 (m, 3H, NCH2CH2 and
CF3CO2H), 3.51-3.41 (m, 2H, NCH2CH2), 2.87 (s, 6H,
N(CH3)2).
1,3-Benzothiazol-2-yl{2-[(2-aminoethyl)amino]pyrimi-
din-4-yl}(acetonitrile (Bis(trifluoroacetate)) (23). Yield )
86%; MS m/z 311.0 (M + 1); HPLC (condition a, 382 nm) 95%,
tR ) 2.64 min; 1H NMR (DMSO-d6) δ 11.60 (v br s, 1H,
exchangeable, H9), 7.95-7.86 (m, 3H, H3, NH-pyrimidine and
CF3CO2H), 7.73 (d, J ) 7.91 Hz, 1H, H4), 7.59 (br d, 1H, H13),
7.43-7.37 (m, 1H, H2), 7.28-7.22 (m, 1H, H1), 6.43 (d, J )
7.16 Hz, 1H, H12), 5.50-4.00 (m, 3H, exchangeable, CH2NH2
and CF3CO2H), 3.90-3.70 (m, 2H, NCH2CH2), 3.25-3.10 (m,
2H, NCH2CH2).
1,3-Benzothiazol-2-yl[2-(methylamino)-4-pyrimidinyl]-
acetonitrile (Trifluoroacetate) (15). Yield ) 11%; MS m/z
282.0 (M + 1); HPLC (condition a, 270 nm) 97%, tR ) 3.39
1
min; H NMR (DMSO-d6) d 11.70 (v br s, 1 H, exchangeable,
H9), 8.15-7.90 (m, 2H [1 + 1 exchangeable], H3, pyrimidine
NH), 7.85-7.55 (m, 2H, H4, H13), 7.46-7.41 (m, 1H, H2),
7.30-7.25 (m, 1H, H1), 6.43 (d, J ) 6.0 Hz, 1H, H12), 4.81-
3.78 (m, 1 H), 3.10 (s, 3H, NHCH3).
1,3-Benzothiazol-2-yl{2-[(2-methoxyethyl)amino]-4-
pyrimidinyl}acetonitrile (24). Yield ) 54%; MS m/z 326.0
1,3-Benzothiazol-2-yl[2-(dimethylamino)-4-pyrimidinyl]-
1
(M + 1); HPLC (condition a, 273 nm) 99%, tR ) 3.66 min; H
acetonitrile (16). Yield ) 12%; MS m/z 295.8 (M + 1); HPLC
1
NMR (DMSO-d6) δ 10.83 (s, 1H, H9), 7.85 (d, J ) 7.54 Hz,
1H, H3), 7.72 (d, J ) 7.91 Hz, 1H, H4), 7.60 (br s, 1H, NH-
pyrimidine), 7.44 (d, J ) 6.78 Hz, 1H, H13), 7.38-7.33 (m,
1H, H2), 7.22-7.16 (m, 1H, H1), 6.33 (d, J ) 7.16 Hz, 1H, H12),
3.82-3.74 (m, 2H, NCH2CH2), 3.62 (t, J ) 5.27 Hz, 2H,
NCH2CH2), 3.31 (s, 3H, OCH3).
(condition a, 270 nm) 99%, tR ) 3.50 min; H NMR (DMSO-
d6) δ 11.20,(br s, 1 H, exchangeable, H9), 7.88 (d, J ) 7.76 Hz
1H, H3), 7.69 (d, J ) 8 Hz 1H, H4), 7.50-7.40 (m, 1H, H13),
7.38-7.33 (m, 1H, H2), 7.20-7.16 (m, 1H, H1), 6.38 (d, J )
6,9 Hz, 1H, H12), 3.26 (s, 6H, N(CH3)2). Anal. (C15H13N5S‚
0.2H2O) C, H, N.
1,3-Benzothiazol-2-yl{2-[(2-hydroxyethyl)amino]-4-
pyrimidinyl}acetonitrile (25). Yield ) 80%; MS m/z 312.2
1,3-Benzothiazol-2-yl[2-(1-piperazinyl)-4-pyrimidinyl]-
acetonitrile (Bis(trifluoroacetate)) (17). Yield ) 37%; MS
m/z 337.2 (M + 1); HPLC (condition a, 271 nm) 96%, tR ) 2.58
min; 1H NMR (DMSO-d6) δ 9.13 (br s, NH-piperazine,
CF3COOH), 7.96 (br d, 1H, H13), 7.90 (d, J ) 7.74 Hz, 1 H,
H3), 7.65 (d, J ) 8.0 Hz, 1H, H4), 7.45-7.40 (m, 1H, H2), 7.29-
7.24 (m, 1H, H1), 6.58 (d, J ) 6.0 Hz, 1H, H12), 4.70-3.60
(m, 5H, CH2-piperazine, CF3COOH), 3.38-3.20 (m, 4H, CH2-
piperazine).
1,3-Benzothiazol-2-yl[2-(4-methyl-1-piperazinyl)-4-pyr-
imidinyl]acetonitrile (Bis(trifluoroacetate)) (18). Yield )
30%; MS m/z 351.0 (M + 1); HPLC (condition a, 271 nm) 99%,
tR ) 2.54 min; 1H NMR (DMSO-d6) δ 10.10 (br s, 1H,
exchangeable, H9), 8.05 (br d, 1H, H13), 7.88 (d, J ) 7.8 Hz,
1 H, H3), 7.63 (d, J ) 8.0 Hz, 1H, H4), 7.45-7.40 (m, 1H, H2),
1
(M + 1); HPLC (condition a, 273 nm) 99%, tR ) 3.16 min; H
NMR (DMSO-d6) δ 10.85 (s, 1H, H9), 7.86 (d, J ) 7.91 Hz,
1H, H3), 7.71 (d, J ) 7.91 Hz, 1H, H4), 7.57 (br s, 1H, NH-
pyrimidine), 7.44 (d, J ) 7.16 Hz, 1H, H13 7.38-7.32 (m, 1H,
H2, 7.21-7.16 (m, 1H, H1, 6.32 (d, J ) 7.16 Hz, 1H, H12),
4.92 (br s, 1H, OH), 3.68-3.45(m, 4H, NCH2CH2OH).
1,3-Benzothiazol-2-yl[2-(propylamino)-4-pyrimidinyl]-
acetonitrile (26). Yield ) 81%; MS m/z 310.0 (M + 1); HPLC
1
(condition a, 273 nm) 95%, tR ) 4.04 min; H NMR (DMSO-
d6) δ 10.91 (br s, 1H, H9), 7.84 (d, J ) 7.54 Hz, 1H, H3, 7.71
(d, J ) 8.29 Hz, 1H, H4), 7.62 (br s, 1H, NH-pyrimidine), 7.42
(d, J ) 6.78 Hz, 1H, H13), 7.38-7.32 (m, 1H, H2), 7.21-7.16
(m, 1H, H1), 6.31 (d, J ) 7.53 Hz, 1H, H12), 3.42-3.33 (m,

4604 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Gaillard et al.
2H, NCH2CH2CH3), 1.71-1.64 (m, 2H, NCH2CH2CH3),
1.02-0.97 (m, 3H, NCH2CH2CH3).
1,3-Benzothiazol-2-yl(2-{[3-(2-oxopyrrolidin-1-yl)propyl]-
amino}pyrimidin-4-yl)acetonitrile (trifluoroacetate) (34).
Yield ) 65%; MS m/z 3.93.0 (M + 1); HPLC (condition a, 254
1,3-Benzothiazol-2-yl{2-[(2-piperidin-1-ylethyl)amino]-
pyrimidin-4-yl}acetonitrile (Bis(trifluoroacetate)) (27).
Yield ) 12%; mp 228-230 °C; MS m/z 379.0 (M + 1); HPLC
(condition a, 270 nm) 99.9%, tR ) 2.84 min; 1H NMR (DMSO-
d6) δ 9.18 (br s, 1H, H9), 7.89-7.87 (m, 2H, H3 and H13), 7.74
(d, J ) 7.91 Hz, 1H, H4), 7.62 (br s, 1H, NH-pyrimidine), 7.44-
7.39 (m, 1H, H2), 7.29-7.24 (m, 1H, H1), 6.45 (d, J ) 7.17
Hz, 1H, H12), 5.25-4.30 (m, 1H, CF3CO2H), 4.05-3.93 (m,
2H, NCH2CH2N-piperidine), 3.63-3.50 (m, 2H, NCH2CH2N-
piperidine), 3.48-3.37 (m, 2H, piperidine), 3.07-2.92 (m, 2H,
piperidine), 1.90-1.30 (m, 6H, piperidine).
1,3-Benzothiazol-2-yl{2-[(2-morpholin-4-ylethyl)amino]-
pyrimidin-4-yl}acetonitrile (Bis(trifluoroacetate)) (28).
Yield ) 53%; MS m/z 381.0 (M + 1); HPLC (condition a, 254
nm) 99.5%, tR ) 2.80 min; 1H NMR (DMSO-d6) δ 10.9 (v br s,
1 H, exchangeable, H9), 8.00-7.98 (m, 2H, H3 and H13), 7.73
(d, J ) 7.9 Hz, 1H, H4), 7.58 (br s, 1H, NH-pyrimidine), 7.42-
7.37 (m, 1H, H2), 7.27-7.22 (m, 1H, H1), 6.55 (d, J ) 7.15,
1H, H12), 4.24-3.18 (m, 12H, NCH2CH2N-morpholine); 1H
NMR (D2O) δ 7.59 (d, J ) 7.9 Hz, 1H), 7.39-7.29 (m, 2H),
7.20-7.15 (m, 2H), 6.20 (d, J ) 6.8, 1H), 3.89-3.82 (m, 6H),
3.45-3.32 (m, 6H).
1
nm) 98%, tR ) 3.52 min; H NMR (DMSO-d6) δ 11.50 (v br s,
1H, exchangeable), 8.15-8.02 (br s, 1H), 7.95-7.60 (m, 4H),
7.46-7.41 (m, 1H), 7.31-7.25 (m, 1H), 6.45 (br s, 1H), 3.70-
3.50 (m, 2H), 3.48-2.28 (m, 4H), 2.23-2.17 (m, 2H), 1.92-
1.84 (m, 4H). Anal. (C20H20N6O1S) C, H, N.
1,3-Benzothiazol-2-yl(2-{methyl[3-(methylamino)propyl]-
amino}pyrimidin-4-yl)acetonitrile (Bis(trifluoroacetate))
(35). Yield ) 11%; MS m/z 353.2 (M + 1); HPLC (condition a,
1
272 nm) 96%, tR ) 2.88 min; H NMR (DMSO-d6) δ 11.30 (v
br s, 1H, exchangeable, H9), 8.50-8.25 (br s, 2H, H3 and
CF3CO2H), 7.92 (d, J ) 7.91 Hz, 1H, H4), 7.72-7.68 (m, 2H,
H12 and NH-pyrimidine), 7.43-7.37 (m, 1H, H2), 7.27-7.21
(m, 1H, H1), 6.46 (d, J ) 6,8 Hz, 1H, H12), 4.00-3.65 (m, 2H,
NH and CF3CO2H), 3.22 (s, 3H, CH2CH2CH2NHCH3), 3.10-
2.85 (m, 2H, CH2CH2CH2NHCH3), 2.63-2.51 (m, 2H,
CH2CH2CH2NHCH3), 2.10-1.80 (m, 2H, CH2CH2CH2-
NHCH3).
1,3-Benzothiazol-2-yl[2-(benzylamino)pyrimidin-4-yl]-
acetonitrile (Trifluoroacetate) (36). Yield ) 78%; MS m/z
358.0 (M + 1); HPLC (condition a, 254 nm) 99.2%, tR ) 4.40
1
min; H NMR (DMSO-d6) δ 8.30 (br t, 1H), 7.84 (d, J ) 7.53
Hz, 1H), 7.71 (d, J ) 7.91 Hz, 1H), 7.65 (br d, 1H), 7.46-7.34
(m, 5H), 7.28-7.22 (m, 2H), 6.47 (d, J ) 7.14 Hz, 1H), 5.2-
4.5 (m, 1H), 4.86 (br d, 2H). Anal. (C20H15N5S‚C2HF3O2‚0.2H2O)
C, H, N.
1,3-Benzothiazol-2-yl(2-{[3-(dimethylamino)propyl]-
amino}pyrimidin-4-yl)acetonitrile (Bis(trifluoroacetate))
(29). Yield ) 54%; mp 204-205°C; MS m/z 353.0 (M + 1);
HPLC (condition a, 272 nm) 98%, tR ) 2.75 min; 1H NMR
(DMSO-d6) δ 11.4 (v br s, 1H), 9.43 (s, 1H), 7.94 (d, J ) 7.91
Hz, 1H), 7.90 (br s, 1H), 7.73 (d, J ) 7.91 Hz, 1H), 7.55 (br d,
1H), 7.43-7.37 (m, 1H), 7.27-7.22 (m, 1H), 6.39 (d, J ) 7.14
Hz, 1H), 4.80-4.00 (m, 1H), 3.71-3.60 (m, 2H), 3.24-3.13 (m,
2H), 2.78 (d, J ) 4.14 Hz, 6H), 2.11-1.99 (m, 2H). Anal.
(C18H20N6S‚2C2HF3O2‚0.3H2O) C, H, N.
{2-[(3-Aminopropyl)amino]pyrimidin-4-yl}(1,3-benzo-
thiazol-2-yl)acetonitrile (Bis(trifluoroacetate)) (30). Yield
) 62%; MS m/z 325.0 (M + 1); HPLC (condition a, 382 nm)
90.0%, tR ) 2.67 min; 1H NMR (DMSO-d6) δ 11.60 (v br s, 1H,
exchangeable, H9), 7.99 (br s, 1H, exchangeable, CF3CO2H),
7.90 (d, J ) 7.92 Hz, 1H, H3), 7.75-7.49 (m, 3H, H13 and H4
and NH-pyrimidine), 7.44-7.37 (m, 1H, H2), 7.27-7.22 (m,
1H, H1), 6.39 (d, J ) 7.16 Hz, 1H, H12), 5.50-4.00 (m, 3H,
exchangeable, NH2 and CF3CO2H), 3.80-3.5 (m, 2H, CH2CH2-
CH2NH2), 3.00-2.80 (m, 2H, CH2CH2CH2NH2), 2.10-1.80
(m, 2H, CH2CH2CH2NH2).
1,3-Benzothiazol-2-yl{2-[(2-pyridinylmethyl)amino]-4-
pyrimidinyl}acetonitrile (Bis(trifluoroacetate)) (37). Yield
) 52%; mp ) 250°C dec; MS m/z 359.0 (M + 1); HPLC
1
(condition a, 266 nm) 99%, tR ) 2.84 min; H NMR (DMSO-
d6) δ 8.66 (d, J ) 4.9 Hz, 1H), 8.38 (br s, 1H), 7.99-7.94 (m,
1H), 7.82 (d, J ) 7.91 Hz, 1H), 7.71-7.69 (m, 2H), 7.64 (d, J
) 7.91 Hz, 1H), 7.45-7.38 (m, 2H), 7.28-7.23 (m, 1H), 6.48
(d, J ) 6.78 Hz, 1H), 5.00 (br s, 2H), 5.15-4.05 (m, 2H). Anal.
(C19H14N6S‚2C2HF3O2) C, H, N.
1,3-Benzothiazol-2-yl{2-[(pyridin-3-ylmethyl)amino]-
pyrimidin-4-yl}acetonitrile (Bis(trifluoroacetate)) (38).
Yield ) 46%; MS m/z 359.0 (M + 1); HPLC (condition c, max
plot) 99.7%, tR ) 2.56 min; 1H NMR (DMSO-d6) δ 8.87 (s, 1H),
8.64 (d, J ) 4.9 Hz, 1H), 8.29-8.26 (m, 2H), 7.84-7.63 (m,
4H), 7.41-7.36 (m, 1H), 7.25-7.20 (m, 1H), 6.46 (d, J ) 7.16
Hz, 1H), 4.97 (br d, 2H). Anal. (C19H14N6S‚2C2HF3O2‚0.5H2O)
C, H, N.
1,3-Benzothiazol-2-yl{2-[(pyridin-4-ylmethyl)amino]-
pyrimidin-4-yl}acetonitrile (Bis(trifluoroacetate)) (39).
Yield ) 46.5%; MS m/z 359.0 (M + 1); HPLC (condition c, max
1,3-Benzothiazol-2-yl{2-[(3-hydroxypropyl)amino]-
pyrimidin-4-yl}acetonitrile (31). Yield ) 44%; MS m/z 326.0
(M + 1); HPLC (condition c, max plot) 99%, tR ) 3.26 min; 1H
NMR (DMSO-d6) δ 10.81 (br s, 1H, exchangeable, H9), 7.84
(d, J ) 7.54 Hz, 1H, H13), 7.71 (d, J ) 8.29 Hz, 1H, H3), 7.49
(br s, 1H, exchangeable, NH-pyrimidine), 7.43-7.32 (m, 2H,
H2 and H4), 7.21-7.15 (m, 1H, H1), 6.32 (d, J ) 7.20 Hz, 1H,
H12), 4.65-4.50 (br s, 1H, exchangeable, OH), 3.80-3.50 (m,
4H CH2CH2CH2OH), 1.90-170 (m, 2H CH2CH2CH2OH),
Anal. (C16H15N5OS‚0.2H2O) C, H, N.
1
plot) 99%, tR ) 2.55 min; H NMR (DMSO-d6) δ 8.77 (d, J )
6.4 Hz, 1H), 8.33 (br t, 1H), 7.95-7.93 (br d, 2H), 7.79 (d, J )
7.54 Hz, 1H), 7.69-7.62 (m, 2H), 7.39-7.34 (m, 1H), 7.24-
7.19 (m, 1H), 6.43 (d, J ) 7.17 Hz, 1H), 5.05 (br d, 2H), 5.6-
4.4 (br s, 1H). Anal. (C19H14N6S‚2C2HF3O2‚0.4H2O) C, H, N.
1,3-Benzothiazol-2-yl{2-[(1H-tetraazol-5-ylmethyl)ami-
no]pyrimidin-4-yl}acetonitrile (Trifluoroacetate) (40).
Yield ) 31%; MS m/z 447.8 (M - 1); HPLC (condition c, max
plot) 99%, tR ) 2.60 min; 1H NMR (DMSO-d6) δ 8.40-8.29 (br
d, 1H, exchangeable), 7.78-7.60 (m, 4H), 7.41-7.36 (m, 1H),
7.27-7.21 (m, 1H), 6.47 (d, J ) 6.78 Hz, 1H), 5.25-5.05 (m,
2H). Anal. (C15H11N9S‚C2HF3O2‚0.5H2O) C, H, N.
1,3-Benzothiazol-2-yl{2-[(3-morpholin-4-ylpropyl)ami-
no]pyrimidin-4-yl}acetonitrile (Bis(trifluoroacetate)) (32).
Yield ) 73.9%; MS m/z 408.2 (M + 1); HPLC (condition a, 272
1
nm) 99.6%, tR ) 2.77 min; H NMR (DMSO-d6) δ 9.66 (br s,
1H), 7.94 (d, J ) 7.91 Hz, 1H), 7.86 (br s, 1H), 7.73 (d, J )
7.91 Hz, 1H), 7.55 (br d, 1H), 7.43-7.38 (m, 1H),7.27-7.22
(m, 1H), 6.40 (d, J ) 7.17 Hz, 1H), 4.15-3.52 (m, 7H [6 + 1]),
3.49-3.38 (m, 2H), 3.29-3.19 (m, 2H), 3.16-3.00 (m, 2H),
2.15-2.01 (m, 2H). Anal. (C20H22N6OS‚2C2HF3O2‚0.5H2O) C,
H, N.
1,3-Benzothiazol-2-yl{2-[(2-phenylethyl)amino]-4-
pyrimidinyl}acetonitrile (Trifluoroacetate) (41). Yield )
46.1%; mp ) 256 °C dec; MS m/z 371.8 (M + 1); HPLC
1
(condition a, 270 nm) 99%, tR
) 4.64 min; H NMR (DMSO-
d
6) δ 11.04 (br s, 1H), 7.71 (d, J ) 8.29 Hz, 1H), 7.64-7.61 (m,
2H), 7.45 (d, J ) 7.16 Hz, 1H), 7.37-7.32 (m, 5H), 7.29-7.26
(m, 1H), 7.20-7.16 (m, 1H), 6.33 (d, J ) 7.17 Hz, 1H), 3.94-
3.81 (m, 2H), 2.99 (t, J ) 7.54 Hz, 2H). Anal. (C21H17N5S‚
C2HF3O2‚0.3H2O) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(2-fluorophenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (42). Yield ) 76%; MS
1,3-Benzothiazol-2-yl(2-{[3-(4-methylpiperazin-1-yl)pro-
pyl]amino}pyrimidin-4-yl)acetonitrile (Tris(trifluoro-
acetate)) (33). Yield ) 65.2%; MS m/z 408.0 (M + 1); HPLC
(condition a, 272 nm) 99.2%, tR ) 2.67 min; 1H NMR (DMSO-
d6) δ 7.94-7.91 (m, 2H), 7.74 (d, J ) 7.92 Hz, 1H), 7.56 (br d,
1H), 7.43-7.38 (m, 1H), 7.27-7.22 (m, 1H), 6.40 (d, J ) 7.17
Hz, 1H), 4.95-4.05 (m, 1H), 3.71-3.60 (m, 2H), 3.54-3.15 (m,
4H), 3.02-2.86 (m, 4H), 2.75 (s, 3H), 2.03-1.91 (m, 2H). Anal.
(C21H25N7S‚3C2HF3O2‚0.5H2O) C, H, N.
m/z 390.2 (M + 1); HPLC (condition c, max plot) 99.7%, tR
)
4.41 min; 1H NMR (DMSO-d6) δ 7.88 (br t, 1H), 7.75-7.68 (m,
2H), 7.60 (br d, 1H), 7.45-7.40 (m, 2H), 7.33-7.16 (m, 4H),

Acetonitrile Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4605
6.44 (d, J ) 7.17 Hz, 1H), 4.20-3.60 (m, 3H), 3.04 (t, J ) 7.14
Hz, 2H). Anal. (C21H16FN5S) C, H, N.
4.9-4.2 (v br s, 1H), 3.94-3.82 (m, 2H), 2.99 (t, J ) 7.16 Hz,
2H). Anal. (C21H18N6S‚2C2HF3O2‚0.7H2O) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(3-fluorophenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Bis(trifluoroacetate))
(43). Yield ) 34%; MS m/z 390.0 (M + 1); HPLC (condition c,
4-[2-({4-[1,3-Benzothiazol-2-yl(cyano)methyl]pyrimidin-
2-yl}amino)ethyl]benzenesulfonamide (52). Yield ) 80%;
MS m/z 449.0 (M - 1); HPLC (condition c, max plot) 99%, tR
) 3.28 min; 1H NMR (DMSO-d6) δ 11.20 (v br s, 1H,
exchangeable, H9), 7.81-7.65 (m, 5H, H13, H3, H4 and
4-H2N2SO2-Ph), 7.57-7.50 (m, 3H, H2NSO2 and NH-
pyrimidine), 7.44-7.22 (m, 4H, H1, H2 and 4-H2N2SO2-Ph),
6.43 (d, J ) 6.78 Hz, 1H, H12), 4.10-3.80 (m, 2H, CH2CH2N),
3.20-3.00 (m, 2H, CH2CH2N).
1
max plot) 98.4%, tR ) 4.72 min; H NMR (DMSO-d6) δ 7.90-
7.67 (m, 3H, H3 and H4 and H13), 7.57 (br d, 1H, NH-
pyrimidine), 7.44-7.36 (m, 2H, 3-F-Ph), 7.27-7.16 (m, 3H, H2
and 3-F-Ph), 7.12-7.06 (m, 1H, H1), 6.42 (d, J ) 7.14 Hz, 1H,
H12), 4.50-3.70 (m, 3H, CH2CH2N and CF3CO2H), 3.05-
3.00 (m, 2H, CH2CH2N).
1,3-Benzothiazol-2-yl(2-{[2-(4-fluorophenyl)ethyl]-
1,3-Benzothiazol-2-yl{2-[(2-{4-[hydroxy(oxido)amino]-
phenyl}ethyl)amino]pyrimidin-4-yl}acetonitrile (53). Yield
) 33.8%; MS m/z 417.2 (M + 1); HPLC (condition c, max plot)
98.7%, tR ) 4.21 min; 1H NMR (DMSO-d6) δ 8.21 (d, J ) 8.66
Hz, 2H, 4-NO2-Ph), 7.88 (br s, 1H, H13), 7.75-7.68 (m, 2H,
H3 and H4), 7.61 (d, J ) 8.66 Hz, 2H, 4-NO2-Ph), 7.54 (br d,
1H, NH-pyrimidine), 7.42-7.37 (m, 1H, H2), 7.26-7.21 (m,
1H, H1), 6.41 (d, J ) 7.17 Hz, 1H, H12), 3.85-3.70 (m, 2H,
CH2CH2N), 3.55-3.10 (m, 1H, CF3CO2H), 3.18-3.14 (m, 2H,
CH2CH2N).
amino}pyrimidin-4-yl)acetonitrile (44). Yield ) 73%; MS
m/z 389.8 (M + 1); HPLC (condition c, max plot) 99.6%, tR
)
4.72 min; 1H NMR (DMSO-d6) δ 7.75-7.70 (m, 3H, H3 and
H4 and H13), 7.59 (br d, 1H, NH-pyrimidine), 7.45-7.34 (m,
3H, H2 and 4-F-Ph), 7.29-7.24 (m, 1H, H1), 7.20-7.14 (m,
2H, 4-F-Ph), 6.43 (d, J ) 7.17 Hz, 1H, H12), 4.32-3.65 (m,
3H [2 + 1], CH2CH2N and CF3CO2H), 2.99 (t, J ) 7.16 Hz,
2H, CH2CH2N).
1,3-Becnzothiazol-2-yl(2-{[2-(3-chlorophenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Bis(trifluoroacetate))
(45). Yield ) 70%; MS m/z 406.0 (M + 1); HPLC (condition c,
1,3-Benzothiazol-2-yl(2-{[2-(1H-indol-3-yl)ethyl]amino}-
pyrimidin-4-yl)acetonitrile (TFA) (54). Yield ) 60.6%; MS
1
max plot) 99.7%, tR ) 4.91 min; H NMR (DMSO-d6) δ 7.85-
m/z 411.0 (M + 1); HPLC (condition a, 272 nm) 99.9%, tR
)
1
7.73 (m, 3H), 7.60 (br d, 1H), 7.41-7.24 (m, 7H), 6.44 (d, J )
7.17 Hz, 1H), 3.98-3.50 (m, 3H), 3.04-2.99 (m, 2H). Anal.
(C21H16ClN5S‚C2HF3O2) C, H, N.
4.94 min; H NMR (DMSO-d6) δ 11.25 (very br s, 1H), 10.98
(s, 1H), 7.92-7.79 (m, 1H), 7.71 (d, J ) 7.92 Hz, 1H), 7.63-
7.57 (m, 2H), 7.42-7.37 (m, 3H), 7.28 (br d, 1H), 7.21-7.16
(m, 1H), 7.10-7.05 (m, 1H), 6.97-6.92 (m, 1H), 6.44 (d, J )
7.17 Hz, 1H), 4.60-3.70 (m, 3H [2 + 1]), 3.12 (t, J ) 7.15 Hz,
2H). Anal. (C23H18N6S‚C2HF3O2‚0.4H2O) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(3,4-dichlorophenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Trifluoroacetate) (46).
Yield ) 56%; MS m/z 440.0 (M + 1); HPLC (condition c, max
1
plot) 99.6%, tR ) 5.15 min; H NMR (DMSO-d6) δ 7.75-7.73
1,3-Benzothiazol-2-yl(2-{[2-(1-methyl-1H-imidazol-4-
yl)ethyl]amino}pyrimidin-4-yl)acetonitrile (56). Yield )
58.8%; MS m/z 376.0 (M + 1); HPLC (condition a, 272 nm)
(m, 3H), 7.62-7.58 (m, 3H), 7.45-7.40 (m, 1H), 7.33-7.25 (m,
2H), 6.44 (d, J ) 7.17 Hz, 1H), 4.20-3.60 (m, 3H), 3.01 (t, J )
6.78 Hz, 2H). Anal. (C12H15Cl2N5S‚C2HF3O2) C, H, N.
1
99.3%, tR ) 3.09 min; H NMR (DMSO-d6) δ 11.20 (very br s,
1H, H9), 9.00 (s, 1H, imidazolyl-4-CH3), 7.90 (br s, 1H, H13),
7.78-7.72 (m, 2H, H3 and H4), 7.58-7.54 (m, 2H, imidazolyl-
4-CH3 and NH-pyrimidine), 7.43-7.38 (m, 1H, H2), 7.27-7.22
(m, 1H, H1), 6.41 (d, J ) 7.17 Hz, 1H, H12), 4.60-4.10 (m,
1H, CF3CO2H), 3.93-3.85 (m, 2H, CH2CH2N), 3.83 (s, 3H,
imidazolyl-4-CH3), 3.11-3.02 (m, 2H, CH2CH2N).
1,3-Benzothiazol-2-yl(2-{[2-(1-methyl-1H-imidazol-5-
yl)ethyl]amino}pyrimidin-4-yl)acetonitrile (Bis(trifluoro-
acetate)) (57). Yield ) 46%; mp 219-220 °C; MS m/z 376.0
(M + 1); HPLC (condition a, 270 nm) 99.8%, tR ) 2.73 min; 1H
NMR (DMSO-d6) δ 11.15 (br s, 1H, H9), 9.03 (s, 1H, imidazole),
7.83 (br s, 1H, H13), 7.74-7.64 (m, 3H, H3 and H4 and
imidazole), 7.54 (br d, 1H, NH-pyrimidine), 7.41-7.36 (m, 1H,
H2), 7.24-7.18 (m, 1H, H1), 6.41 (d, J ) 7.14 Hz, 1H, H12),
4.50-3.88 (m, 3H, CH2CH2N and CF3CO2H), 3.78 (s, 3H,
imidazolyl-2-CH3), 3.10-3.05 (m, 2H, CH2CH2N).
1,3-Benzothiazol-2-yl(2-{[2-(4-bromophenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Trifluoroacetate) (47).
Yield ) 96%; MS m/z 450.0 (M + 1); HPLC (condition c, max
1
plot) 99.6%, tR ) 5.01 min; H NMR (DMSO-d6) δ 7.81-7.68
(m, 3H), 7.60 (br d, 1H), 7.53 (d, J ) 8.28 Hz, 2H), 7.45-7.40
(m, 1H), 7.30-7.25 (m, 3H), 6.43 (d, J ) 7.16 Hz, 1H), 4.12-
3.55 (m, 3H), 3.01-2.96 (m, 2H). Anal. (C21H16BrN5S‚C2HF3O2)
C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(4-methylphenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Trifluoroacetate) (48).
Yield ) 72%; MS m/z 386.0 (M + 1); HPLC (condition c, max
plot) 100% tR ) 4.66 min; 1H NMR (DMSO-d6) δ 7.87 (br s,
1H, H13), 7.76-7.67 (m, 3H, H3, H4 and NH-pyrimidine),
7.47-7.42 (m, 1H, H2), 7.32-7.27 (m, 1H, H1), 7.23-7.14 (m,
4H, 4-CH3-Ph), 6.47 (d, J ) 7.14 Hz, 1H, H12), 4.20-3.55 (m,
3H, CH2CH2N and CF3CO2H), 2.98-2.93 (m, 2H,CH2CH2N),
2.30 (s, 3H, 4-CH3-Ph).
1,3-Benzothiazol-2-yl(2-{[2-(4-hydroxyphenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Trifluoroacetate) (49).
Yield ) 76%; mp 258-261°C; MS m/z 388.0 (M + 1); HPLC
(condition a, 272 nm) 98.8%, tR ) 4.00 min; 1H NMR (DMSO-
d6) δ 9.25 (br s, 1H), 7.75-7.69 (m, 2H), 7.59 (br d, 1H), 7.45-
7.40 (m, 1H), 7.29-7.24 (m, 1H), 7.12 (d, J ) 8.29 Hz, 1H),
6.74 (d, J ) 8.29 Hz, 1H) 6.43 (d, J ) 6.78 Hz, 1H), 3.89-3.71
(m, 2H), 2.90-2.85 (m, 2H). Anal. (C21H17N5OS‚C2HF3O2‚
0.7H2O) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(2-pyridinyl)ethyl]amino}-
4-pyrimidinyl)acetonitrile (Bis(trifluoroacetate)) (58).
Yield ) 80%; mp ) 247°C dec; MS m/z 373.2 (M + 1); HPLC
1
(condition a, 266 nm) 99%, tR ) 2.85 min; H NMR (DMSO-
d6) δ 8.70 (d, J ) 5.28 Hz, 1H), 8.15-8.10 (m, 1H), 7.88 (br s,
1H), 7.75-7.72 (m, 2H), 7.67 (d, J ) 7.91 Hz, 1H), 7.61-7.50
(m, 2H), 7.44-7.39 (m, 1H), 7.28-7.23 (m, 1H), 6.44 (d, J )
7.14 Hz, 1H), 4.65-3.60 (m, 3H), 3.32-3.28 (m, 2H). Anal.
(C20H16N6S‚2C2HF3O2) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(3-pyridinyl)ethyl]amino}-
4-pyrimidinyl)acetonitrile (Bis(trifluoroacetate)) (59).
Yield ) 74%; MS m/z 373.0 (M + 1); HPLC (condition a, 263
1,3-Benzothiazol-2-yl(2-{[2-(4-methoxyphenyl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (Bis(trifluoroacetate))
(50). Yield ) 69%; MS m/z 402.0 (M + 1); HPLC (condition c,
1
nm) 99%, tR ) 2.92 min; H NMR (DMSO-d6) δ 8.81 (d, J )
1
max plot) 99.6%, tR ) 4.33 min; H NMR (DMSO-d6) δ 7.75-
1.13 Hz, 1H, H-pyridine), 8.71 (dd, J ) 5.27 Hz, J ) 1.13 Hz,
1H, H-pyridine), 8.31 (d, J ) 7.91 Hz, 1H, H-pyridine), 7.94
(br s, 1H, NHCH2), 7.85-7.73 (m, 3H, H3/H4/H13), 7.60 (br
d, 1H, H-pyridine), 7.45-7.40 (m, 1H, H1), 7.29-7.24 (m, 1H,
H2), 6.43 (d, J ) 7.14 Hz, 1H, H12), 6.00-4,40 (m, 2H,
CF3CO2H), 4.05-3.87 (m, 2H, NCH2), 3.19-3.15 (m, 2H,
NCH2CH2). Anal. (C23H18N6S‚2 C2HF3O2‚1H2O) C, H, N.
1,3-Benzothiazol-2-yl(2-{[2-(1H-1,2,4-triazol-1-yl)ethyl]-
amino}pyrimidin-4-yl)acetonitrile (60). Yield ) 77.6%; MS
7.69 (m, 3H), 7.61 (br d, 1H), 7.40-7.35 (m, 1H), 7.24-7.17
(m, 3H), 6.86 (d, J ) 8.28 Hz, 2H), 6.38 (d, J ) 7.16 Hz, 1H),
4.15-3.65 (m, 3H), 3.69 (s, 3H), 2.9-2.85 (m, 2H). Anal.
(C22H19N5OS‚2C2HF3O2‚0.4H2O) C, H, N.
(2-{[2-(4-Aminophenyl)ethyl]amino}pyrimidin-4-yl)-
(1,3-benzothiazol-2-yl)acetonitrile (Bis(trifluoroacetate))
(51). Yield ) 73%. (salt); MS m/z 387.2 (M + 1); HPLC
(condition c, max plot) 98.3%, tR ) 3.02 min; 1H NMR (DMSO-
d6) δ 7.93 (br d, 1H), 7.75-7.66 (m, 2H), 7.55 (br d, 1H), 7.43-
7.36 (m, 3H), 7.26-7.17 (m, 3H), 6.40 (d, J ) 7.16 Hz, 1H),
m/z 361.2 (M - 1); HPLC (condition c, max plot) 99.4%, tR
)
2.79 min; 1H NMR (DMSO-d6) δ 8.59 (s, 1H, triazole), 8.09 (s,

4606 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Gaillard et al.
1H, triazole), 7.98-7.82 (m, 2H, H13, H3), 7.75 (d, J ) 7.91
Hz, 1H, H4), 7.67 (br d, 1H, NH-pyrimidine), 7.48-7.43 (m,
1H, H2), 7.33-7.28 (m, 1H, H1), 6.51 (d, J ) 7.16 Hz, 1H, H12),
5.05-4.25 (m, 3H [2 + 1], CH2CH2N and CF3CO2H), 4.10-
3.98 (m, 2H, CH2CH2N).
treatment with compound 59 (30, 10, 3, and 0.3 mg/kg in 0.5%
CMC/0.25% Tween-20/water). Groups of six mice were used.
Fifteen minutes later, endotoxins (O111:B4 Sigma, 0.3 mg/
kg) were intraperitoneally injected. Heparinized whole blood
was collected by decapitation. TNF-R was determined in
plasma by ELISA (R & D Systems, Abdingdon, U.K.). Control
animals received 0.5% CMC/0.25% Tween-20 (10 mL/kg) as
the vehicle. Data obtained from experiments were expressed
as the mean ( SEM and analyzed using one-way analysis of
variance (ANOVA) followed by Dunnett’s t-test.
4. In Vivo Mouse Model of Collagen-Induced Rheu-
matoid Arthritis. DBA/1 male mice, 8-12 weeks old, were
primed (day 0) by injecting intradermally at the base of the
tail 0.2 mL of an emulsion composed of 0.2 mg of bovine type
II collagen in complete Freund’s adjuvant containing 0.2 mg
of mycobacterium tuberculosis. Starting from day 18 of the
study, each animal was examined daily and each paw was
scored from 0 to 2 for the presence of inflammation in digits,
and from 0 to 3 for the presence of oedema in both forepaws
and hind paws (paw thickness was measured by means of a
precision calliper). The maximum achievable score was 14.
Upon appearance of disease signs (score g 1.5), the animals
were assigned to different treatment groups.
1,3-Benzothiazol-2-yl(2-{[3-(1H-imidazol-1-yl)propyl]-
amino}-4-pyrimidinyl)acetonitrile (Trifluoroacetate) (61).
Yield ) 57%; MS m/z 376.0 (M + 1); HPLC (condition a, 270
1
nm) 98%, tR ) 2.80 min; H NMR (DMSO-d6) δ 11.60 (v br s,
1 H, exchangeable, H9), 9.18 (s, 1H, imidazole), 8.13 (br s, 1H,
NH-pyrimidine), 7.92-7.85 (m, 2H, H4 and imidazole), 7.74-
7.59 (m, 3H, H3, H13 and imidazole), 7.43-7.39 (m, 1H, H2),
7.28-7.23 (m, 1H, H1), 6.40 (d, J ) 7.2 Hz, 1H, H12), 5.05-
4.20 (m, 1H, CF3CO2H), 4.35 (t, J ) 6.8 Hz, 2H, NCH2-
CH2CH2), 3.70-3.45 (m, 2H, NCH2CH2CH2), 2.35-2.10 (m,
2H, NCH2CH2CH2).
1,3-Benzothiazol-2-yl(2-{[3-(1H-pyrazol-1-yl)propyl]-
amino}pyrimidin-4-yl)acetonitrile (62). Yield ) 70%; MS
m/z 374.0 (M - 1); HPLC (condition c, max plot) 94.8%, tR
)
1
3.40 min; H NMR (DMSO-d6) δ 8.01-7.85 (m, 2H, H3, NH-
pyrimidine), 7.78-7.73 (m, 2H, H4 and pyrazole), 7.62 (br d,
1H, H13), 7.46-7.43 (m, 2H, H2 and pyrazole), 7.31-7.26 (m,
1H, H1), 6.45 (d, J ) 7.17 Hz, 1H, H12), 6.22 (s, 1H, pyrazole),
4.30-3.85 (m, 3H, NCH2CH2CH2 and CF3CO2H), 3.62-3.48
(m, 2H, NCH2CH2CH2), 2.21-2.06 (m, 2H, NCH2CH2CH2).
Biological Methods. 1. rJNK3 Enzymatic Assay. GST-
JNK3 (1 mg/mL) was incubated overnight at room temperature
with 0.05 mg/mL of GST-JNKK2 in a solution containing 200
µM ATP-γ-S, 1 mM DTT, 10 mM MgCl2, and 100 µM Na3VO4,
followed by dialysis against 50 mM Tris-HCl, pH 8, 150 mM
NaCl, and 5 mM DTT overnight at 4 °C to remove the ATP-
γ-S.
Treatment. Compound 59 (bis(trifluoroacetate) salt) was
tested at doses of 20 and 60 mg/kg per oral route using saline
as vehicle. Indomethacin was included as a reference com-
pound at a dose of 2 mg/kg, orally. All treatments continued
for 7 days.
Histology. Twenty-four hours after the last treatment,
animals were sacrificed by an overdose of anesthetic, and the
first limb to become arthritic was removed, identified, fixed
in neutral buffered 10% formalin, and decalcified in formic acid
solution (50%). This paw was then embedded in paraffin,
sectioned, and stained with hematoxylin and eosin. Three
slides per animal were prepared and scored according to the
following scale: for erosion score, 0 ) no destruction, 1-2 )
localized cartilage erosions, 3 ) more extended erosions, 4 )
general destruction; for inflammation scores, 0 ) no inflam-
mation, 1-2 ) slight thickening of lining layer and/or some
infiltrating cells in sublining layer, 3 ) thickening of lining
layer and more pronounced influx of cells in sublining layer
with presence of cells in the synovial space, 4) synovium
highly infiltrated with many inflammatory cells.
rJNK3 assays are performed in 96-well low-binding Corning
microtiter (MT) plates: 0.5 µg of recombinant, preactivated
GST-JNK3 were incubated with 1 µg of recombinant, bio-
tinylated GST-c-Jun and 2 µM 33γ-ATP (2 nCi/µL) in the
presence or absence of compounds and 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 for 120 min and at room
temperature. The reaction was stopped by the addition of 200
µL of a solution containing 250 µg of streptavidine-coated SPA
beads (Amersham, Inc.), 5 mM EDTA, 0.1% Triton X-100, and
50 µM ATP in phosphate saline buffer and further incubated
at room temperature for 60 min. After incubation 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 after letting the beads settle for an
additional 60 min at room temperature.
2. Jurkat Cell Assay. Jurkat cells, from a human T cell
leukemia cell line (American Type Culture Collection, no. TIB
152), were cultured in RPMI 1640 medium (Gibco, BRL)
supplemented with 10% heat-inactivated fetal calf serum,
glutamine, and penicillin/streptomycin. The cells were diluted
in the medium to give 2 × 106 cells/mL, and afterward, they
were plated (2 × 105 cells/well) in a 96-well plate containing
different concentrations of test compound (final concentration
of compounds of 10, 3, 1, 0.3, 0.1 µM in 0.1% DMSO). This
mixture was incubated for 30 min at 37 °C in a humidified
CO2 atmosphere. Cells were then treated with 10 µL of PMA
+ ionomycine (0.1 and 1 µM final concentration) in all wells
except the negative control. In wells without compounds, 10
µL of RPMI 2% DMSO ()0.1% final) is added. Cells were
incubated for 24 h at 37 °C, and then the supernatant was
harvested (freeze at -20 °C if not used the same day) prior to
performing an IL-2 ELISA test on the supernatant.
Acknowledgment. We thank the Analytical Chem-
istry group for their support in physicochemical param-
eter determination, and we acknowledge the role of the
Serono Early Safety Evaluation group in providing in
vitro ADME (absorption, distribution, metabolism, and
excretion) and DMPK (drug metabolism and pharmaco-
kinetics) data. Grace Shomade is acknowledged for
proofreading the manuscript.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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