Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity

Article abstract of DOI:10.1021/jm060199b

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38α, and p38δ and showed little inhibitory activity against a panel of 74 kinases.

Full text of DOI:10.1021/jm060199b

J. Med. Chem. 2006, 49, 3563-3580
3563
Aminopyridine-Based c-Jun N-Terminal Kinase Inhibitors with Cellular Activity and Minimal
Cross-Kinase Activity†
Bruce G. Szczepankiewicz,* Christi Kosogof, Lissa T. J. Nelson, Gang Liu, Bo Liu, Hongyu Zhao, Michael D. Serby,
Zhili Xin, Mei Liu, Rebecca J. Gum, Deanna L. Haasch, Sanyi Wang, Jill E. Clampit, Eric F. Johnson, Thomas H. Lubben,
Michael A. Stashko, Edward T. Olejniczak, Chaohong Sun, Sarah A. Dorwin, Kristi Haskins, Cele Abad-Zapatero,
Elizabeth H. Fry, Charles W. Hutchins, Hing L. Sham, Cristina M. Rondinone,‡ and James M. Trevillyan
Metabolic Disease Research, Global Pharmaceutical Research and DiscoVery Organization, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064-6098
ReceiVed February 20, 2006
The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP)
kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses
including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety
of different diseases with an inflammatory component including asthma, stroke, Alzheimer’s disease, and
type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent
kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead
optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range,
activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1000-
fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38R, and p38δ and showed little
inhibitory activity against a panel of 74 kinases.
Introduction
mouse studies lend further evidence to the role of JNK-3 in
neurological disorders.19 Thus, inhibitors of JNK activity have
the potential to provide benefits for patients with many different
chronic diseases. Small molecules with good potency, selectivity
against a broad panel of kinases, and pharmacokinetic profiles
sufficient to achieve good exposure in vivo would be ideal for
probing the role of JNK function in these diseases.
c-Jun N-terminal kinase-1 (JNK-1) is a member of the
mitogen activated protein kinase (MAP kinase) family of
enzymes responsible for the serine/threonine phosphorylation
of intracellular targets. JNK-1 and the other JNK enzymes
JNK-2 and JNK-3 are activated in response to cellular stresses
such as heat shock, irradiation, hypoxia, chemotoxins, and
peroxides. They are also activated in response to various
cytokines and participate in the onset of apoptosis.1,2 Up-
regulation of JNK activity is associated with a number of
different disease states. JNK-1 phosphorylation of IRS-1 at
Ser307 has been shown to down-regulate insulin signaling in
vitro.3,4 Conversely, JNK-1 null mice maintain lower fasting
plasma glucose and insulin levels compared to their wild-type
litter mates when they are high-fat-fed, indicating that these
animals are protected from the development of obesity-induced
insulin resistance.5 These results imply a role for JNK-1 in type
2 diabetes mellitus.6
JNK-2 null mice have been produced by two different
laboratories, and there is a clear role for JNK-2 in immunological
function.7,8 JNK-2, often in concert with JNK-1, has been
implicated in the pathology of autoimmune disorders such as
rheumatoid arthritis9 and asthma.10-15 It also may play a role
in cancer,16 as well as ischemia-reperfusion injury following
myocardial infarction17 or stroke.1 Inhibitors of JNK-1 and
JNK-2 could be useful for treating these diseases, as well as a
broad range of other diseases with an inflammatory component.
JNK-3 has been shown to mediate neuronal apoptosis and
may therefore be involved in the pathology of neurodegenerative
diseases. Inhibitors of JNK-3 could be useful for treating
Parkinson’s disease, Alzheimer’s disease, epilepsy, stroke, and
other diseases of the central nervous system.1,18 JNK-3 knockout
The active sites of JNK-1, -2, and-3 are highly homologous,
but each protein is the product of a different gene. At least 10
different JNK isoforms have been identified, and all of these
are splice variants of JNK-1, -2, or -3. JNK-1 and -2 are
ubiquitously expressed in human tissues, while JNK-3 is
restricted to the brain, heart, and testis. Some cellular proteins
phosphorylated by JNK enzymes include the gene transcription
factors c-Jun, c-Fos, ATF-2, and Elk-1;2 the insulin receptor
substrates IRS-113 and IRS-2; Shc; and Gab-1. JNK enzymes
themselves must be phosphorylated to carry out their functions.
The enzymes MKK-4 and MKK-7 are known to phosphorylate
and activate JNK-1 and -2.1,2 Some of these biochemical events
can serve as the basis for assaying JNK activity in vitro.
There has been considerable effort to identify JNK inhibitors
over the past several years, and several detailed reports have
appeared20-26 (Figure 1). Still more examples have appeared
in the patent literature.27,28 In this work, we report a series
outside the scope of the previous work. The new compounds
are potent JNK inhibitors that bind to the ATP site in an unusual
manner. They also display remarkable selectivity vs several
dozen other kinases, perhaps because of their special binding
mode with JNK-1. These JNK inhibitors may be suitable for
use in animal studies, providing a critical tool to elucidate the
role of JNK in vivo.
Chemistry
† PDB access code: 2GMX.
Most of compounds in this study were prepared by appropri-
* To whom correspondence should be addressed. Phone: (847) 935-
1559. Fax: (847) 938-1674. E-mail: bruce.szczepankiewicz@abbott.com.
‡ Current Address: Hoffman-La Roche Inc., Metabolic Diseases,
340 Kingsland Street, Nutley, NJ 07110.
ate substitution of diaminopyridine 1, available in one step from
29
malononitrile and HBr(g) (Scheme 1). Addition-elimination
with sodium ethoxide at the 2-bromo position gave ether 2.30
10.1021/jm060199b CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/20/2006

3564 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
Scheme 2. Preparation of Acid Chloride 11a
a Reagents and conditions: (a) Br2, AcOH, 25 °C; (b) TBSCl, imidazole,
DMF; (c) (i) n-BuLi/hexanes, THF, -78 °C; (ii) MeSSMe, -78 to 0 °C;
(d) TBAF, THF, 25 °C; (e) H2O2, TFA; (f) 2M H2CrO4, acetone, 25 °C;
(g) SOCl2, reflux.
and triphosgene also reacted with diaminopyridine 2 at N-6,
providing carbamate 7 and, upon treatment of the N-6 isocyanate
with ethylamine, urea 8. Since diaminopyridine 2 is rather
electron-deficient, N-6 did not participate in alkylation or
reductive amination reactions. To prepare an N-6 alkyl deriva-
tive, we started with hydroxypyridine 9.31 A triflate ester was
prepared using PhN(Tf)2, and then addition-elimination with
(2,5-dimethoxyphenyl)ethylamine proceeded smoothly to pro-
vide phenethylaminopyridine 10.
Most of the acid chlorides either were commercially available
or prepared in one step from a readily available acid and either
thionyl chloride or oxalyl chloride. Acid chloride 11 required a
lengthier sequence (Scheme 2). Starting from 2-(2,5-dimethoxy-
phenyl)ethanol32 (12), electrophilic bromination and then protec-
tion of the alcohol with TBSCl gave silyl ether 13. Halogen-
metal exchange with n-butyllithium was followed by addition
of dimethyl disulfide and then removal of the silyl protecting
group with fluoride ion to provide thioether 14. Oxidation of
the thioether to the sulfone was accomplished with hydrogen
peroxide in trifluoroacetic acid, and then oxidation of the alcohol
to the acid with Jones’ reagent provided acid 15. Treatment
with thionyl chloride gave acid chloride 11, which was used
for subsequent acylations.
Figure 1. Previously disclosed c-Jun N-terminal kinase inhibitors.
Scheme 1. Introduction of 2-Pyridyl Substituents and N-6
Elaborationa
We prepared amides 16 and 17 from bromopyridine 1 and
acetyl chloride or (2,5-dimethoxyphenyl)acetyl chloride, re-
spectively (Scheme 3). Attempts to prepare C-2 ethers from
bromide 16 or 17 via alkoxide addition-elimination resulted
in hydrolysis of the amide, so we elected to install the C-2
alkoxy groups first and then acylate the resulting ethers. By
use of conditions similar to those employed for the acylation
of bromopyridine 1, ethers 3 and 4 and thioether 5 were acylated
with acid chlorides to provide acetamides 18 and 19, (2,5-
dimethoxyphenyl)acetamides 20, and amidothioether 21. Other
C-2 substituents could be installed without hydrolysis of the
N-6 amide, so bromides 16 and 17 were suitable starting
materials for the preparation of analogues 22-26. Addition-
elimination of ethylamine or isopropylamine to bromide 16 gave
triaminopyridine 22 or 23, respectively. Suzuki coupling of
bromide 16 with phenylboronic acid gave biarylamide 24, while
Negishi coupling with n-propylzinc iodide gave propylpyridine
25. Reduction of bromide 17 with zinc/HCl provided C-2
unsubstituted pyridine 26.
Acylation of aminopyridine 2 with bromoacetyl bromide
cleanly gave bromoamide 27 (Scheme 4). This intermediate
allowed us to further explore the SAR about the N-6 amide,
using nucleophilic substitution of the bromide to rapidly
introduce further diversity to the series. The best results were
found with amines, so bromide 27 was treated with different
primary or secondary alkylamines to provide analogues 28.
Cyclic amines also smoothly displaced bromide 27, providing
a Reagents and conditions: (a) 1 f 2, 1 M NaOEt in EtOH, 150 °C; (b)
1 f 3, 1 M i-PrONa in i-PrOH, 150 °C; (c) 1 f 4, ca. 1 M Na alkoxide
in parent alcohol or dioxane, 150 °C; (d) 1 f 5, n-BuSNa, DMF, 120 °C;
(e) 2 f 6 acid chloride, pyridine/CH2Cl2, -78 or 25 °C; (f) 2 f 7, methyl
chloroformate, (i-Pr)2EtN, THF, 25 °C; (g) 2 f 8, (i) triphosgene, (i-Pr)2EtN,
THF, 25 °C; (ii) EtNH2 THF, 25 °C; (h) PhN(Tf)2, Et3N, CH2Cl2, (i) (2,5-
dimethoxyphenyl)ethylamine, DMSO, 110 °C.
Similarly, sodium isopropoxide gave ether 3, while other
alkoxides gave alkyl ethers 4, and n-butylthiol sodium salt gave
thioether 5. Acylation of ether 2 with acid chlorides was
regioselective, providing N-6 amides 6. Methylchloroformate

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3565
Scheme 4. Preparation of Aminoacetamidesa
Scheme 3. Synthesis of Pyridine C-2 Analoguesa
a Reagents and conditions: (a) bromoacetyl bromide, pyridine, CH2Cl2,
-78 °C; (b) 1° or 2° amine, DMF, 25 °C; (c) 27 f 29, piperidine, DMF,
25 °C; (d) 27 f 30, morpholine, DMF, 25 °C; (e) 27 f 31, 4-hydroxy-
piperidine, DMF, 25 °C; (f) 27 f 31, (i) methyl isonipecotate, DMF,
25 °C; (ii) K2CO3, aqueous MeOH; (g) 32 f 33, n-BuNH2, TBTU, DMF,
25 °C; (h) 32 f 34, i-PrNH2, TBTU, DMF, 25 °C.
Scheme 5. Synthesis of Chloropyridine Analogue 35a
a Reagents and conditions: (a) NaOEt, EtOH, 150 °C; (b) PdCl2CH2-
Cl2dppf, Et3N, 100 atm of CO, CH3OH, 100 °C; (c) NCS; (d) NH3, CH3OH,
25 °C; (e) NaOCl, NaOH, i-PrOH, 25 °C; (f) acid chloride 11, pyr, CH2Cl2,
-78 °C.
a Reagents and conditions: (a) 1 f 16, acetyl chloride, pyridine, 25 °C;
(b) 1 f 17, 2,5-dimethoxyphenylacetyl chloride, pyridine/CH2Cl2, -78 °C;
(c) 3 f 18a, acetyl chloride, pyridine, 25 °C; (b) 3 f 18b, acid chloride
11, pyridine/CH2Cl2, -78 °C; (e) 4 f 19, acetyl chloride, pyridine/CH2Cl2,
-78 or 25 °C; (f) 4 f 20, 2,5-dimethoxyphenylacetyl chloride, pyridine/
CH2Cl2, -78 °C; (g) acetyl chloride, pyridine; (h) 7 f 22, EtNH2, NMP,
150 °C; (i) 7 f 23, i-PrNH2, NMP, 150 °C; (j) 7 f 24, PhB(OH)2, Na2CO3,
Pd(PPh3)4, DMF/THF/H2O; (k) 7 f 25, n-propyl-ZnI, Pd(OAc)2, (o-tol)3P,
DMF, 90 °C; (l) Zn, 1 M HCl(aq)/DMF.
conditions as we did for bromopyridine 1 and then followed
with N-6 acylation. Starting with pentanoylamide 6b, treatment
with sulfuryl chloride gave 5-chloropyridine 41. The N-6
methylation was also straightforward starting from amide 6b,
giving tertiary amide 42. To prepare isomeric pyridine 43, we
started with 2,4,6-trichloropyridine (44)34 and sodium ethoxide.
After isolation of the 4-ethoxypyridine, lithiation and carbox-
ylation were followed by conversion to the corresponding
nicotinamide 45 using standard conditions. Dehydration of the
amide with POCl3 provided the nicotinonitrile, and then the C-2
and C-6 amino groups were simultaneously installed under
autoclave conditions. Acylation completed the synthesis of
amidopyridine 43.
piperidine 29, morpholine 30, hydroxypiperidine 31, and car-
boxypiperidine 32. Carboxypiperidine 32 could be further
elaborated by preparing amides from the carboxylic acid.
Standard peptide coupling conditions were sufficient to produce
amides 33 and 34.
Exploration of the pyridine C-3 position required a different
synthesis of the core (Scheme 5). To prepare 3-chloropyridine
35, we began by heating 4-amino-2,6-dichloropyridine (36) with
sodium ethoxide to displace the C-2 chloride. Then a carboxylate
was installed at C-6 via Pd-mediated carbonylation in methanol,
providing methyl ester 37. Chlorination with N-chlorosuccin-
imide proceeded at C-3, and then the resulting intermediate was
stirred with methanolic ammonia to give an amide. This was
subjected to Hoffmann rearrangement conditions to provide
diaminopyridine 38. Acylation with acid chloride 11 proceeded
as for other aminopyridines, giving amide 35.
Results and Discussion
In our work, the basis for the biochemical assay was JNK-1
mediated phosphorylation of ATF-2.35 High-throughput screen-
ing identified acetamide 6a, with IC50 ) 750 nM (Table 1).
Competition experiments confirmed that this inhibitor binds
competitively and reversibly to the ATP site. This compound
demonstrated a remarkable selectivity profile, showing little
cross-reactivity with any of 74 other non-c-Jun-N-terminal
kinases. Additionally, it showed some inhibition of c-Jun
Changes at C-4 and C-5 were accomplished via other methods
(Scheme 6). To prepare 4-methylpyridine 39, we subjected
chloropyridine 4033 to the same ethoxide addition-elimination

3566 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
Scheme 6. Modification of Pyridine C-4, C-5, and N-1
R-carbon with oxygen or nitrogen completely abolished activity,
as did removal of the amide carbonyl group (7-10). Phenyl-
acetamide 6e was slightly more potent than acetamide 6a, but
further exploration of the amide region was facilitated by the
ready availability of a variety of substituted phenylacetic acids.
Hydrophobic substituents gave some improvement in activity
(6f-k), but the best gains were found with methoxy substituents.
Methoxy regioisomers 6l, 6m, and 6n were all more active than
unsubstituted phenylacetamide 6e. Further gains in potency
could be found upon dimethoxy substitution of the phenyl ring,
with 2,5-dimethoxy analogue 6o being the optimal arrangement.
Disubstitution with other groups such as methyl (6p) or bromide
(6q) did not match these gains in potency. Additional substit-
uents in the 6o 4-phenyl position were tolerated. Acetamide 6r,
sulfone 6s, and bromide 6t were roughly equipotent with 6o,
while nitro compound 6u was slightly less potent than amide
6o. The optimal N-6 amide was the (2,5-dimethoxy-4-methyl-
sulfonylphenyl)acetamide 6s.
Positions and N-6 Alkylationa
A second set of N-6 amides was prepared from bromoamide
27 (Table 2). Since inhibitors could be prepared in one step
from bromoamide 27, we were able to rapidly assemble and
evaluate a set of 2-aminoacetamides. N,N-Disubstitution was
preferred because tertiary amines 28a and 28b were more active
than secondary amine 28c. Linear aliphatic amines were
generally less potent than amide 6a, but we made some gains
by displacing bromoamide 27 with more hydrophilic amines.
Hydroxyethylamine 28d, piperidine 29, morpholine 30, and
hydroxypiperidine 31 all were more potent than acetamide 6a.
Further modification of the piperidine scaffold revealed ad-
ditional gains. Piperidine 4-carboxylic amides were somewhat
more potent than hydroxypiperidine 31, with examples 33 and
34 giving IC50 values below 100 nM. The most potent example
from this set of aminoacetamides was n-butylamidopiperidine
33.
a Reagents and conditions: (a) NaOEt, EtOH, 150 °C; (b) AcCl, pyr, 25
°C; (c) 6b f 41, SO2Cl2, CH2Cl2, 25 °C; (d) 6b f 42, NaH, CH3I, DMF,
25 °C; (e) KOH, EtOH, reflux; (f) (i) n-BuLi, THF -78 °C; (ii) CO2, -78
to 25 °C; (g) SOCl2, reflux; (h) concentrated NH4OH, THF, 25 °C; (i) POCl3,
imidazole, pyr, 0 °C; (j) concentrated NH4OH, dioxane, 130 °C; (k) AcCl,
pyr, 25 °C.
phosphorylation in hepG2 cells. The rapid synthesis of the core
and the modular nature of analogue synthesis allowed us to
quickly explore the SAR about the C-2 and C-6 positions.
Exploration of the amide position revealed that groups larger
than the original acetamide 6a could improve potency (6b).
Branching at the R-position diminished activity (6c), but
â-branching was better tolerated (6d). Replacement of the amide
Three crystal structures of JNK have appeared previously,
facilitating the use of X-ray crystallography to determine the
Table 1. SAR about Amide 6
entry
R
JNK-1 IC50 (nM)
SD
43
63
770
110
140
300
70
170
120
56
4
47
95
59
3
670
1600
16
5
38
JNK-2 IC50 (nM)
SD
pCjun EC50 (nM)
6800
SD
6a
6b
6c
6d
6e
6f
6g
6h
6i
MeCO
n-BuCO
i-PrCO
i-BuCO
PhCH2CO
2-(Cl)PhCH2CO
3-(Cl)PhCH2CO
4-(Cl)PhCH2CO
750
350
4000
1400
570
1100
300
580
400
420
190
120
210
180
45
4400
3000
35
38
77
1100
690
8400
3500
770
4900
900
610
740
790
360
260
110
370
800
590
130
1800
520
270
420
200
89
3000
2-(Me)PhCH2CO
3-(Me)PhCH2CO
4-(Me)PhCH2CO
2-(MeO)PhCH2CO
3-(MeO)PhCH2CO
4-(MeO)PhCH2CO
2,5-(diMeO)PhCH2CO
2,5-(diMe)PhCH2CO
2,5-(diBr)PhCH2CO
4-AcNH-2,5-(diMeO)PhCH2CO
4-(MeSO2)-2,5-(diMeO)PhCH2CO
4-Br-2,5-(diMeO)PhCH2CO
4-NO2-2,5-(diMeO)PhCH2CO
MeOCO
6j
6k
6l
3400
1400
2500
1400
920
960
840
550
340
660
110
56
120
40
6m
6n
6o
6p
6q
6r
6s
6t
310
274
160
>10000
>10000
26
7
21
48
1
280
1300
890
200
170
160
110
150
160
100
6u
7
8
69
14
910
>10000
>10000
>10000
>10000
>10000
>10000
EtNHCO
10
2,5-(diMeO)PhCH2CH2

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3567
Table 2. SAR of Aminoacetamides
entry
X
JNK-1 IC50 (nM)
SD
JNK-2 IC50 (nM)
SD
pCjun EC50 (nM)
5500
SD
28a
28b
28c
28d
29
30
31
33
34
di(Et)N
di(n-butyl)N
n-butylNH
2700
3500
6300
160
510
270
300
69
880
350
2400
46
43
28
150
19
21
6300
>10000
>10000
740
2000
(n-propyl)(2-hydroxyethyl)N
1-piperidinyl
120
670
250
340
7
290
3400
1200
640
180
4-morpholinyl
4-hydroxy-1-piperidinyl
4-(n-butylaminocarbonyl)-1-piperidinyl
4-(i-propylaminocarbonyl)-1-piperidinyl
>10000
87
130
16
3900
1100
Figure 2. X-ray crystal structure of inhibitor 6t with JNK-1 with protein carbon in orange, inhibitor carbon in green, nitrogen in blue, oxygen in
red, sulfur in yellow, and bromine in black. Purple lines denote hydrogen bonds, and black lines denote hydrophobic interactions: (a) ribbon
diagram of 6t bound to JNK-1 with the ATP-binding site highlighted; (b) space filling diagram of 6t bound to JNK-1; (c) stick diagram of 6t-
JNK-1 complex with some ATP-site amino acid residues labeled and interatomic distances in angstroms shown, with some residues removed for
clarity; (d) alternative view of 6t-JNK-1 complex with the pyridine ring shown edge-on. Additional amino acid residues and interatomic distances
in angstroms are shown.
mode of binding for our inhibitors.36-38 An X-ray crystal
structure of bromide 6t bound to JNK-1 reveals some of the
critical binding interactions between the ligand and the enzyme
(Figure 2). The inhibitor occupies the ATP binding site, with
the pyridine ring placed deep within the adenosine binding
region. The C-4 amine forms a weak hydrogen bond with
Glu109, but the hydrogen-bonding interaction is not optimal
because the C-4 amine and Glu109 do not lie in the same plane.
The pyridine ring itself lies in a hydrophobic pocket defined
by Ile86, Leu168, Val158, Val40, and Ile32. The nitrile
protrudes further into this pocket and lies 4.0 Å from Lys55,
forming a weak hydrogen bond with the side chain amine. The
ethyl ether is oriented out of the adenosine binding region and
toward the ribose binding region on the surface of the protein.
There are no clear hydrogen-bonding interactions between the
ether oxygen and the protein. The inhibitor amide carbonyl
oxygen lies 2.6 Å from the backbone amide NH group of
Met111 but not in the same plane as the backbone amide. Thus,
the inhibitor makes two out-of-plane hydrogen bonds with the
hinge region (residues 109-111). The phenyl ring makes a
hydrophobic interaction with Ile32, while the 4-bromo and
5-methoxy substituents are pointed toward the solvent. Ad-
ditional residues on the protein surface offer the potential for
hydrogen-bonding interactions with substituents in the 4- or
5-phenyl position. It is possible that hydrogen bond acceptors
in the 4-phenyl position (e.g., 6r and 6s) take advantage of these
interactions. Unfortunately we could not confirm this hypothesis
because high-quality X-ray data could not be obtained with
inhibitors bearing such 4-phenyl substituents.
The X-ray data revealed that the ethyl ether was pointed
toward the ribose binding region of JNK-1. The ribose binding
site is rich in potential hydrogen-bonding interactions, and so

3568 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Table 3. Pyridine C-2 SAR
Szczepankiewicz et al.
JNK-1
JNK-2
pCjun
entry
R1
R2
IC50 (nM)
SD
43
53
1
IC50 (nM)
SD
EC50 (nM)
SD
6a
17
Me
EtO
Br
i-PrO
i-PrO
750
1700
310
1100
>10000
470
110
6800
3000
2,5-(diMeO)PhCH2
18a
18b
19a
19b
19c
19d
19e
20a
20b
20c
21
Me
140
2
(2,5-(diMeO)-4-(MeSO2)-Ph)CH2
14
2
64
650
24
Me
Me
Me
Me
(2-Me2N)CH2CH2O
(2-CO2H)CH2CH2O
(3-tetrahydrofuryl)O
>10000
4800
890
>10000
190
88
1000
120
11
13
45
690
300
200
37
4900
2400
400
1000
920
15
2900
4663
4500
240
BuO
MeO
2100
2200
74
110
400
8900
2400
910
Me
2,5-(diMeO)PhCH2
2,5-(diMeO)PhCH2
2,5-(diMeO)PhCH2
HOCH2CH2O
(MeO)CH2CH2O
(2-MeSO2)CH2CH2O
1600
1400
520
920
270
740
>10000
>10000
3000
38
70
Me
Me
Me
Me
n-BuS
NHEt
NH(i-Pr)
Ph
n-Pr
H
22
23
24
25
410
1400
1900
>10000
2600
7700
Me
>10000
26
2,5-(diMeO)PhCH2
58
5100
1100
accessing this region was the focus of SAR efforts about the
ether. Believing that more polar groups would provide stronger
interactions with the hydrogen-bonding groups available in the
ribose binding pocket, we added a variety of functional groups
to the end of the ethyl chain (Table 3). 2-Hydroxyethyl and
2-methoxyethyl ethers 20a and 20b were both approximately
as potent as ethyl ether 6o, but there was no gain in potency
with these modifications. Other changes we explored at the ethyl
2-position included methyl sulfone 20c, amine 19a, and car-
boxylic acid 19b. In each case, installation of these groups
diminished activity. Variation in the spacing between the ether
oxygen atom and the distal functional group also failed to
improve potency. In the hope of mimicking the ribose furan
skeleton, we prepared tetrahydrofuryl analogue 19c, but this too
was less potent than ethyl ether 6a. Despite these attempts,
efforts to access the nearby ribose binding pocket did not result
in significant gains in potency. An ether linkage was optimal
at C-2 because bromide 17, thioether 21, amine (22 and 23),
aryl 24, alkyl 25, or hydrogen 26 substitution gave compounds
with diminished activity. Variation of the alkyl ether chain length
showed that neither a longer alkyl ether (19d) nor a methyl
ether (19e) provided additional potency. However, a branched
ether did provide a boost in potency. Isopropyl ether 18a showed
about a 2-fold improvement over the corresponding ethyl ether
6a. The optimal group at the C-2 position was the isopropyl
ether. When this was combined with the optimal C-6 amide,
the resulting compound 18b showed IC50 ) 14 nM.
Changes elsewhere about the diaminopyridine core generally
were not tolerated (Table 4). As expected from the X-ray
structure, replacement of the 4-amino group with a methyl
substituent resulted in a loss of activity (39). Simultaneous
deletion of the C-3 nitrile and the C-4 amino groups (46)39
likewise abolished JNK inhibition. The pyridine nitrogen atom
of 6u did not appear to be interacting with the protein surface,
so we prepared 2,6-diaminopyridine 43 in the hope of gaining
another H-bond with the hinge region. Unfortunately, 43 was
devoid of activity. Chlorination of the unsubstituted C-5 position
also was not tolerated (41). N-Methylation of the N-6 amide
abolished activity as well (42). One change that was successful
involved replacement of the C-3 nitrile with a chloride.
Chloropyridine 35 showed an IC50 value of 36 ( 14 nM,
comparable to cyanopyridine 6s. Lead amide 6a therefore
contains the optimal ring pattern substitution with a C-2 ether,
a C-3 nitrile, a C-4 amine, and a C-6 amide.
Compounds with IC50 < 200 nM vs JNK-1 were screened
for their ability to inhibit c-Jun phosphorylation in HepG2 cells
(Tables 1-4). Because c-Jun is a known substrate for JNK-1,
this assay provides a direct measurement of JNK-1 inhibition
in whole cells. In general, compounds showed EC50 values about
15-fold less potent than their IC50 values. There was no
correlation between inhibitor membrane permeability (data not
shown) and the EC50 to IC50 ratio. This indicates that cell
penetration alone does not explain the drop in cellular potency
relative to the ATF-2 phosphorylation assay. The intracellular
concentration of ATP (10 mM) is greater than that used in the
in vitro assays (5 µM), and this may account for most of the
discrepancy between the two assay formats. Most of the
phenylacetamides tested showed cellular activity, with the
relative potencies in the enzymatic assay translating to a similar
rank order in whole cells.
Perhaps the most remarkable feature of this series is its
exquisite kinase selectivity profile. Previously, investigators at
Serono published JNK inhibitors with some kinase selectivity,
and these represent the most selective JNK inhibitors in the
literature.22,25 We screened several compounds against a panel
of 74 different kinases and found that cross-reactivity was
minimal, with the exception of JNK-2 and JNK-3 (Table 5). Ki
values for JNK-1 were in the single-digit nanomolar range for
the most potent compounds, but these showed measurable
activity against only a few other members of the panel. Where
cross-reactivity could be found, the compounds were well over
100-fold selective. A possible explanation for this selectivity
profile can be discerned from the crystal structure of the
enzyme-inhibitor complex. Most kinase inhibitors form strong
and clearly defined hydrogen bonds to the hinge region.40-42
Our compounds form two suboptimal hydrogen bonds with the
JNK-1 hinge region, deriving much of their potency from other
interactions within the ATP binding site (Figure 2). The weak
hydrogen-bonding interaction with the hinge region could limit
the ability of our JNK inhibitors to bind to other kinases.43
Although the kinases we screened represent only about 15% of
the human kinome, this level of selectivity is uncommon among

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3569
Table 4. Pyridine N-1, C-3, C-4, and C-5 Modifications and N-6 Amide Alkylation
kinase inhibitors. Such selectivity offers an important advantage
for probing the role of JNK inhibition in vivo.
indicate that the hydrogen-bonding network of these kinase
ATP-site ligands is different from many other kinase inhibitors,
and the unusual binding mode may be responsible for their
kinase selectivity. We have prepared compounds 6s and 35 with
cellular activity and oral bioavailability. These selective JNK
inhibitors can serve as important probes to discern what role
JNK plays in a host of diseases. Evaluation of the new
compounds in a variety of disease models will give us a better
understanding of what the physiological implications of JNK
inhibition are, and we are eager to make these inhibitors
available for such studies. Our own in vivo results with JNK
inhibitors in inflammation and diabetes models will be disclosed
at a later date.
Pharmacokinetic profiles were determined for JNK inhibitors
6o, 6s, 18b, and 35 in Sprague-Dawley rats (Table 6). Inhibitor
6o showed a short half-life of roughly 1 h, with rapid clearance
and barely measurable bioavailability. Microsomal incubation
studies revealed that oxidative metabolism was very rapid with
this compound, indicating that the pharmacokinetic profile could
be improved if oxidative metabolism could be suppressed.
Attempts to improve the microsomal stability of these phenyl-
acetamides revealed that substitution of the phenylacetamide
with a methylsulfonyl group greatly lowered the rate of oxidative
metabolism. Inhibitor 6s, which bears a 4-methylsulfonyl group
on the 2,5-dimethoxyphenyl ring, showed a somewhat better
half-life than 6o and markedly better bioavailability. Although
it is not clear whether this is due to improved first-pass
metabolism or better absorption, the presence of the methyl-
sulfone group clearly is beneficial to the pharmacokinetic profile
of the JNK inhibitors. The most potent JNK-1 inhibitor,
compound 18b, was less orally bioavailable than 6s, and it was
also cleared more rapidly than ethyl ethers 6o and 6s. This is
evidence that the isopropyl ether is more metabolically labile
than the corresponding ethyl ether. 3-Chloropyridine analogue
35 showed the best oral bioavailability (F ) 49%), half-life
(2.1 h), and drug exposure level (AUC ) 1.47 µg‚h/mL) of the
four compounds we evaluated. The oral bioavailability and
reasonable drug exposures demonstrated with 6s and 35 make
them candidates for further in vivo testing in JNK-related disease
models.
Experimental Section
General. Thin-layer chromatography systems were the same as
those used for column chromatography, with Rf ≈ 0.3. Column
chromatography was performed under 5 psi of N2 using 230-400
mesh silica gel or with an Analogix Intelli Flash 280 MPLC system
using prepacked, disposable silica gel columns. Analytical HPLC
traces and compounds purified by HPLC were obtained by elution
through a C18 reverse-phase column with a gradient of 5 mM
aqueous ammonium acetate/acetonitrile or 0.1% aqueous TFA/
acetonitrile as eluants. Concentration gradients started at 0%
acetonitrile and 100% aqueous TFA or at 5% acetonitrile and 95%
aqueous medium. Microwave heating was performed using a
Personal Chemistry Emrys Optimizer microwave apparatus, and
1
reaction pressures were not allowed to exceed 20 atm. H NMR
data are tabulated in the following order: multiplicity (s, singlet;
d, doublet; t, triplet; m, multiplet; b, broad), number of protons,
coupling constants in Hz.
4,6-Diamino-2-ethoxynicotinonitrile (2).30 To a 5 mL sealable
heavy-walled glass tube suitable for microwave heating containing
5 mL of absolute ethanol was added 150 mg (6.5 mmol) of sodium
Conclusion
We have identified several potent JNK inhibitors with
excellent kinase selectivity profiles. X-ray crystallographic data

3570 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
Table 5. Kinase Selectivity Data for Diaminopyridine-Based JNK Inhibitors (Ki in nM)a
Ki (nM)
Ki (nM)
kinase
6a
6o
6s
24
18b
35
kinase
6a
6o
6s
24
18b
35
AKT1
AKT2
AKT3
AMPK
Abl
Aurora1
Aurora2
Blk
CDC2
CDC42
CDK2
CDK5
CHK1
CHK2
CK2
CSF1R
CTAK1
CaMK4
CK1δ
Dyrk1A
EGFR
EMK
ERK2
ErbB2
FGFR
FGFR3
Flt1
Flt3
Flt4
Fyn
GSK3R
GSK3â
ITK
IKKR
IKKâ
>8200 >8200 >8200 >8200 >8200 >8200 JAK3
>8400 >8400 >8400 >8400 >8400 >8400 JNK-1
>9500 >9500 >9500 >9500 >9500 >9500 JNK-2
>9800 >9800 >9800 >9800 >9800 >9800 JNK-3
>1700 >1700 >1700 >1700 >1700 >1700
190
440
520
2
4
3
9
550
1900
3300
1
4
3
13
61
52
72
18
>710
>710
>710
>710
>710
>710 Kdr
>8200 >8200 >8900 >8200 >8900 >8900
>2700 >2700 >2700 >2700 >2700 >2700
>9600 >9600 >9600 >9600 >9600 >9600
>1700 >1700 >1700 >1700 >1700 >1700
>6900 >6900 >6900 >6900 >6900 >6900 Lck
>6700 >6700 >6700 >6700 >6700 >6700 Limk1
>1500 >1500 >1500 >1500 >1500 >1500 Lyn
>8900 >8900 >8900 >8900 >8900 >8900 MAPK(APK2) >8400 >8400 >8400 >8400 >8400 >8400
>9500 >9500 >9500 >9500 >9500 >9500 MK3
>8600 820 >8600 1300 >8600 >8600 Mst2
>9200 >9200 >9200 >9200 >9200 >9200
>8800 >8800 >8800 >8800 >8800 >8800
>9300 >9300 >9300 >9300 >9300 >9300 Nek2
>8800 >8800 >8800 >8800 >8800 >8800 P70S6K
>9500 >9500 >9500 >9500 >9500 >9500 PAK1
>5800 >5800 >5800 >5800 >5800 >5800 PAK4
>2500 >2500 >2500 >2500 >2500 >2500 PDK1
>6700 >6700 >6700 >6700 >6700 >6700 PKA
>8600 >8600 >8600 >8600 >8600 >8600 PKCδ
>7800 >7800 >7800 >7800 >7800 >7800 PKCγ
>6900 >6900 >6900 >6900 >6900 >6900 PKCú
>1800 >1800 >1800 >1800 >1800 >1800 Pim1
>7500 >7500 >7500 >7500 >7500 >7500 Pim2
>920
>6700
>920 >9200 >9200 >9200 >9200
3400 >6700 >6700 >6700 >6700
>9700 >9700 >9700 >9700 >9700 >9700
>5500 >5500 >5500 >5500 >5500 >5500
>3800 >3800 >3800 >3800 >3800 >3800
>7500 >7500 >7500 >7500 >7500 >7500
>8900 >8900 >8900 >8900 >8900 >8900
>8800
>8800 >8800 >8800 >8800
>8300 >8300 >8300 >8300 >8300 >8300
>9200 >9200 >9200 >9200 >9200 >9200
>1400 >1400 >1400 >1400 >1400 >1400
>8800
4700
5800 >8800
3900 >8800 Pkd2
2500
5300 >9000 >9000
950 >9000
>1400 >1400 >1400 >1400 >1400 >1400 Plk1
>4700 >4700 >4700 >4700 >4700 >4700 Plk3
>1900 >1900 >1900 >1900 >1900 >1900 Rock1
>8900 >8900 >8900 >8900 >8900 >8900
>8000 >8000 >8000 >8000 >8000 >8000
>9100 >9100 >9100 >9100 >9100 >9100
>7500 >7500 >7500 >7500 >7500 >7500
>440
>440
>440
>440
>440
>440 Rock2
>2500 >2500 >2500 >2500 >2500 >2500 RSK2
>7100 >7100 >7100 >7100 >7100 >7100 SGK
>7400 >7400 >7400 >7400
3300 >7400
>9400 >9400 >9400 >9400 >9400 >9400
>1900 >1900 >1900 >1900 >1900 >1900
>2500 >2500 >2500 >2500 >2500 >2500
>4500 >4500 >4500 >4500 >4500 >4500
>1900 >1900 >1900 >1900 >1900 >1900
>1400 >1400 >1400 >1400 >1400 >1400
>1200 >1200 >1200 >1200 >1200 >1200
>2900 >2900 >2900 >2900 >2900 >2900
>6900 >6900 >6900 >6900 >6900 >6900
>7500 >7500 >7500 >7500 >7500 >7500
>810
>810
>810
>810
>810
>810 Src
>8900 >8900 >8900 >8900 >8900 >8900 TrkA
>7100 >7100 >7100 >7100 >7100 >7100 TrkB
>1500 >1500 >1500
970 >1500 >1500 Tyk2
>3800 >3800 >3800 >3800 >3800 >3800 ZipK
>3200 >3200 >3200 >3200 >3200 >3200 cKit
insulin R >5200 >5200 >5200 >5200 >5200 >5200 cMet
IRAK4
JAK2
>9900 >9900 >9900 >9900 >9900 >9900 p38δ
>1500 >1500 >1500 >1500 >1500 >1500 p38γ
a Kinases showing measurable inhibition are in italics.
Table 6. Pharmacokinetic Parameters for JNK Inhibitors in
Sprague-Dawley Rats (5 mg/kg Dose)
all of the sodium had reacted. Then 1.00 g (4.71 mmol) of 1 was
added. The mixture was heated at 160 °C for 20 min and then
concentrated in vacuo. The residue was taken up in 10 mL of water
and then extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were extracted with saturated NaHCO3(aq) (1 × 10
mL) and then brine (1 × 10 mL), dried over MgSO4, filtered, and
concentrated to a solid. This was purified via MPLC, eluting with
a 30-50% ethyl acetate/hexanes gradient to give 380 mg (42%)
iv
po
a
b
c
e
f
g
h
compd t1/2 Vss Vb AUCd CLp t1/2 Cmax Tmax AUCi F j
6o
1.4 1.2 2.6 4.1
1.2 1.1 0.31 0.033 0.11
2.7
31
5.7
49
6s
18b
35
2
2
1.5 4.7 3.5
1.6 8.6 2.2
1.5 1.5 1.36 0.5
3.0 2.9 0.08 0.75
1.7 2.1 1.42 0.67
1.1
0.12
1.5
0.8 1.6 2.0 3.0
1
of a white solid. H NMR (300 MHz, DMSO-d6) δ 6.16 (s, 2H),
a Intravenous half-life (hours). b Intravenous volume of distribution at
steady state (L/kg). c Intravenous volume of distribution (L/kg). d Area under
the curve or total drug exposure after intravenous dosing from t ) 0 to t )
∞ (µg‚h/mL). e Plasma clearance after intravenous dosing ((L/h)/kg). f Oral
half-life (hours). g Maximum plasma concentration after oral dosing (µM).
h Time from oral dosing to maximum plasma concentration (hours). i Area
under the curve or total drug exposure after oral dosing from t ) 0 to t )
∞ (µg‚h/mL). j Oral bioavailability (%).
6.11 (s, 2H), 5.30 (s, 1H), 5.11-5.28 (septet, J ) 6.1 Hz, 1H),
1.23 (d, J ) 6.1 Hz, 6H). MS (ESI) m/e ) 151 (M - Pr + H)+,
193 (M + H)+.
4,6-Diamino-2-butylsulfanylnicotinonitrile (5). To a suspension
of 60% NaH (16 mg, 0.4 mmol) in DMF (1 mL) was added
n-butylthiol (22 µL, 0.2 mmol) followed by 1 (43 mg, 0.2 mmol).
The mixture was heated with an oil bath at 120 °C for 30 min.
Then it was cooled and partitioned between ethyl acetate and water.
The organic phase was washed with brine (×3), dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified via
silica gel chromatography, eluting with ethyl acetate/hexane (1:1)
metal. The mixture was stirred until all of the sodium had reacted,
and then 1.00 g (4.71 mmol) of 2-bromo-4,6-diaminonicotinonitrile
(1)29 was added. The tube was sealed, and the mixture was heated
with a microwave apparatus at 150 °C for 10 min, then cooled and
diluted with 15 mL of water. The orange precipitate that formed
was washed with water until pH 7 was attained and then dried in
vacuo at 105 °C to give 0.545 g (65%) of a light-orange solid. 1H
NMR (300 MHz, DMSO-d6) δ 6.19 (s, 2H), 6.14 (s, 2H), 5.32 (s,
1H), 4.23 (q, 2H, J ) 7.1 Hz), 1.25 (t, 3H, J ) 7.1 Hz); MS (ESI)
m/e ) 151 (M - Et + H)+, 179 (M + H)+.
1
to provide the thioether as a pale-yellow solid (23 mg, 52%). H
NMR (300 MHz, DMSO-d6) δ 6.28 (s, 2H), 6.19 (s, 2H), 5.42 (s,
1H), 3.09 (t, J ) 7.1 Hz, 2H), 1.63-1.52 (m, 2H), 1.44-1.32 (m,
2H), 0.89 (t, J ) 7.1 Hz, 3H); MS (ESI) m/e ) 223 (M + H)+;
221 (M - H)-.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)acetamide (6a). To
50 mg (0.28 mmol) of 2 dissolved in 1 mL of pyridine was added
60 µL (0.84 mmol) of acetyl chloride. The mixture was stirred at
ambient temperature for 2 h and then diluted with 7 mL of water.
The precipitate was filtered and washed with 5 mL of water. The
4,6-Diamino-2-isopropoxynicotinonitrile (3). To a 5 mL seal-
able heavy-walled glass tube suitable for microwave heating
containing 5 mL of 2-propanol was added 150 mg (6.5 mmol) of
sodium metal. The mixture was stirred and heated under N2 until

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3571
supernatant was extracted with ethyl acetate (3 × 5 mL). Then the
combined ethyl acetate layers were back-extracted with brine (1 ×
5 mL), dried over MgSO4, filtered, and concentrated in vacuo to a
solid. This was combined with the precipitated product, dissolved
in methanol/ethyl acetate, and concentrated. Most of the remaining
pyridine was removed by coevaporation with toluene in vacuo. The
product was purified via silica gel chromatography, eluting with
50% ethyl acetate/hexanes to give 31 mg (50%) of a white solid.
1H NMR (300 MHz, DMSO-d6) δ 10.73 (s, 1H), 7.17 (s, 1H), 6.88
(s, 2H), 4.32 (q, 2H, J ) 7.1 Hz), 2.07 (s, 3H), 1.29 (t, 3H, J ) 7.1
Hz); MS (ESI) m/e ) 221 (M + H)+, m/e ) 219 (M - H)-. Anal.
(C10H12N4O2) C, H, N.
Pentanoic Acid (4-Amino-5-cyano-6-ethoxypyridin-2-yl)amide
(6b). To 50 mg (0.28 mmol) of 2 in 1 mL of pyridine was added
7 drops of valeryl chloride via pipet. After being stirred at ambient
temperature for 18 h, the mixture was diluted with 7 mL of water
and stirred for 1 h. The suspension was subjected to centrifugation
(800g for 1 min), and then the solvent was decanted. After
resuspension and centrifugation once more, the product was purified
via reversed-phase HPLC (aqueous TFA gradient). The purified
product was dissolved in 5 mL of ethyl acetate, extracted with
NaHCO3(aq) (1 × 1 mL), then brine (1 × 1 mL), dried over MgSO4,
filtered, and concentrated to 21 mg (29%) of a white solid. 1H NMR
(300 MHz, DMSO-d6) δ 10.03 (s, 1H), 7.20 (s, 1H), 6.87 (s, 2H),
4.33 (q, J ) 7.1 Hz, 2H), 2.36 (t, J ) 7.5 Hz, 2H), 1.39-1.73 (m,
2H), 1.16-1.42 (m, 5H), 0.88 (t, J ) 7.3 Hz, 3H); MS (ESI)
m/e ) 263 (M + H)+. Anal. (C13H18N4O2) C, H, N.
d6) δ 10.38 (s, 1H), 7.41 (m, 2H), 7.31 (m, 2H), 7.13 (s, 1H), 6.89
(s, 2H), 4.35 (q, 2H, J ) 7.1 Hz), 3.90 (s, 2H), 1.31 (t, 3H, J ) 7.1
Hz); MS (ESI) m/e ) 303 (M - Et + H)+, 331 (M + H)+, 329
(M - H)-. Anal. (C16H15ClN4O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(3-chlorophen-
yl)acetamide (6g). This was prepared according to the same
procedure as 6f, substituting (3-chlorophenyl)acetic acid for (2-
chlorophenyl)acetic acid. The product was purified via reverse-
phase HPLC, eluting with a 0-70% CH3CN gradient in 0.1%
1
aqueous TFA. Yield, 8 mg (14%); H NMR (300 MHz, DMSO-
d6) δ 10.35 (s, 1H), 7.38 (m, 1H, J ) 1.7 Hz), 7.31 (m, 3H), 7.15
(s, 1H), 6.90 (s, 2H), 4.34 (q, 2H, J ) 7.0 Hz), 3.73 (s, 2H), 1.31
(t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 303 (M - Et + H)+, 331
(M + H)+, 329 (M - H)-. Anal. (C16H15ClN4O2‚0.07TFA) C, H,
N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(4-chlorophen-
yl)acetamide (6h). This was prepared according to the same
procedure as 6f, substituting (4-chlorophenyl)acetic acid for (2-
chlorophenyl)acetic acid. Yield, 8 mg (14%); 1H NMR (300 MHz,
DMSO-d6) δ 10.33 (s, 1H), 7.35 (m, 4H), 7.15 (s, 1H), 6.90 (s,
2H), 4.34 (q, 2H, J ) 7.1 Hz), 3.71 (s, 2H), 1.30 (t, 3H, J ) 7.1
Hz); MS (ESI) m/e ) 303 (M - Et + H)+, 331 (M + H)+, 329
(M - H)-. Anal. (C16H15ClN4O2‚0.15CH3OH) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-o-tolylaceta-
mide (6i). This was prepared according to the same procedure as
6f, substituting (2-methylphenyl)acetic acid for (2-chlorophenyl)-
1
acetic acid. Yield, 10 mg (10%); H NMR (300 MHz, DMSO-d6)
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)isobutyramide (6c).
This was prepared according to the same procedure described for
6a, substituting isobutyryl chloride for acetyl chloride. The crude
product was recrystallized from methanol to give 40 mg (69%) of
δ 10.28 (s, 1H), 7.19 (t, 1H, J ) 7.5 Hz), 7.16 (s, 1H), 7.07 (m,
3H), 6.89 (s, 2H), 4.34 (q, 2H, J ) 7.1 Hz), 3.65 (s, 2H), 2.28 (s,
3H), 1.30 (t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 283 (M - Et +
H)+, 311 (M + H)+, 309 (M - H)-. Anal. (C17H18N4O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-m-tolylaceta-
mide (6j). This was prepared according to the same procedure as
6f, substituting (3-methylphenyl)acetic acid for (2-chlorophenyl)-
acetic acid. The product was purified via MPLC, eluting with 30%
ethyl acetate in hexanes followed by trituration with ethyl acetate.
1
a white solid. TLC (30% ethyl acetate/hexanes); H NMR (300
MHz, DMSO-d6) δ 10.01 (s, 1H), 7.20 (s, 1H), 6.87 (s, 2H), 4.33
(q, 2H, J ) 7.1 Hz), 2.75 (m, 1H, J ) 6.8 Hz), 1.29 (t, 3H, J ) 7.1
Hz), 1.05 (d, 6H, J ) 7.1 Hz); MS (ESI) m/e ) 221 (M - Et +
H)+, 249 (M + H)+, 247 (M - H)-. Anal. (C12H16N4O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-3-methylbutyra-
mide (6d). This was prepared according to the same procedure
described for 6a, substituting 3-methylbutyryl chloride for acetyl
chloride. The crude product was purified by reverse-phase HPLC,
eluting with a gradient of 5-100% CH3CN in 0.1% aqueous TFA
to give 12 mg (16%) of a white solid. TLC (30% ethyl acetate/
1
Yield, 10 mg (10%); H NMR (500 MHz, DMSO-d6) δ 10.31-
10.28 (s, 1H), 7.20 (t, 1H, J ) 7.6 Hz), 7.16 (s, 1 H), 7.13-7.08
(m, 2H), 7.05 (d, 1H, J ) 7.3 Hz), 6.89 (s, 2 H), 4.34 (q, 2H, J )
7.0 Hz), 3.65 (s, 2H), 2.28 (s, 3H), 1.30 (t, 3H, J ) 7.0 Hz); MS
(ESI) m/e ) 283 (M - Et + H)+, 311 (M + H)+, 309 (M - H)-.
Anal. (C17H18N4O2‚0.11EtOAc) C, H, N.
1
hexanes); H NMR (300 MHz, DMSO-d6) δ 10.01 (s, 1H), 7.22
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-p-tolylaceta-
mide (6k). This was prepared according to the same procedure as
6f, substituting (4-methylphenyl)acetic acid for (2-chlorophenyl)-
acetic acid. The product was purified by trituration with hot
2-propanol. Yield, 20 mg (19%); 1H NMR (300 MHz, DMSO-d6)
δ 10.26 (s, 1H), 7.15 (m, 5H), 6.88 (s, 2H), 4.34 (q, 2H, J ) 7.1
Hz), 3.64 (s, 2H), 2.27 (s, 3H), 1.30 (t, 3H, J ) 7.1 Hz); MS (ESI)
m/e ) 283 (M - Et + H)+, 311 (M + H)+, 309 (M - H)-. Anal.
(C17H18N4O2‚0.09i-PrOH) C, H, N.
(s, 1H0, 6.87 (s, 2H), 4.33 (q, 2H, J ) 7.1 Hz), 2.25 (d, 2H, J )
7.1 Hz), 2.03 (m, 1H), 1.29 (t, 3H, J ) 7.1 Hz), 0.90 (d, 6H, J )
6.4 Hz); MS (ESI) m/e ) 263 (M + H)+, 261(M - H)-. Anal.
(C13H18N4O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-phenylaceta-
mide (6e). This was prepared according to the same procedure
described for 6a, substituting phenylacetyl chloride for acetyl
chloride. The crude product was recrystallized from methanol to
give 8 mg (11%) of a white solid. TLC (30% ethyl acetate/hexanes);
1H NMR (300 MHz, DMSO-d6) δ 10.31 (s, 1H), 7.26 (m, 5H),
7.16 (s, 1H), 6.89 (s, 2H), 4.34 (q, 2H, J ) 7.1 Hz), 3.70 (s, 2H),
1.30 (t, 1H, J ) 7.1 Hz,); MS (ESI) m/e ) 269 (M - Et + H)+,
297 (M + H)+, 295 (M - H)-. Anal. (C16H16N4O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2-chlorophen-
yl)acetamide (6f). To a solution of 29 mg (0.17 mmol) of
(2-chlorophenyl)acetic acid in 0.6 mL of CH2Cl2 was added 20 µL
(0.23 mmol) of oxalyl chloride. The mixture was allowed to stand
at ambient temperature for 20 min, and then the acid chloride
solution was added to 30 mg (0.17 mmol) of 1 in 0.6 mL of pyridine
at ambient temperature. The mixture was swirled and then allowed
to stand at ambient temperature for 1 h. Next, 1 mL of water was
added. Then the mixture was concentrated to dryness in vacuo.
The residue was partitioned between EtOAc (10 mL) and water (5
mL), and then the water layer was removed. The organic phase
was extracted with water (1 × 5 mL), saturated NaHCO3(aq) (1 ×
5 mL), and brine (1 × 5 mL), dried over MgSO4, filtered, and
concentrated to a solid. This was recrystallized from methanol to
give 10 mg (18%) of white crystals. 1H NMR (300 MHz, DMSO-
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2-methoxyphen-
yl)acetamide (6l). This was prepared according to the same
procedure as 6f, substituting (2-methoxyphenyl)acetic acid for (2-
chlorophenyl)acetic acid. The product was purified by trituration
1
with hot 2-propanol. Yield, 10 mg (9%); H NMR (300 MHz,
DMSO-d6) δ 10.10 (s, 1H), 7.24 (m, 1H), 7.17 (dd, 1H, J ) 7.5,
1.7 Hz), 7.13 (s, 1H), 6.97 (d, 1H, J ) 7.1 Hz), 6.89 (m, 3H), 4.34
(q, 2H, J ) 7.1 Hz), 3.76 (s, 3H), 3.68 (s, 2H), 1.31 (t, 3H, J ) 7.1
Hz); MS (ESI) m/e ) 299 (M - Et + H)+, 327 (M + H)+, 325
(M - H)-. Anal. (C17H18N4O3‚0.08EtOAc) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(3-methoxyphen-
yl)acetamide (6m). This was prepared according to the same
procedure as 6f, substituting (3-methoxyphenyl)acetic acid for (2-
chlorophenyl)acetic acid. The product was recrystallized from 1
mL of methanol to give 9 mg (16%) of a colorless solid. 1H NMR
(300 MHz, DMSO-d6) δ 10.28 (s, 1H), 7.22 (m, 1H), 7.16 (m,
1H), 6.89 (m, 4H), 6.81 (m, 1H), 4.34 (q, 2H, J ) 7.1 Hz), 3.74 (s,
3H), 3.67 (s, 2H), 1.31 (t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 299
(M - Et + H)+, 327 (M + H)+, 349 (M + Na)+. Anal.
(C17H18N4O3) C, H, N.

3572 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(4-methoxyphen-
yl)acetamide (6n). This was prepared according to the same
procedure as 6f, substituting (4-methoxyphenyl)acetic acid for (2-
chlorophenyl)acetic acid. Yield, 9 mg (16%); 1H NMR (300 MHz,
DMSO-d6) δ 10.24 (s, 1H), 7.23 (d, 2H, J ) 8.8 Hz), 7.15 (s, 1H),
6.87 (d, 4H, J ) 8.8 Hz), 4.34 (q, 2H, J ) 7.1 Hz), 3.72 (s, 3H),
3.62 (s, 2H), 1.30 (t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 299 (M -
Et + H)+, 327 (M + H)+, 349 (M + Na)+. Anal. (C17H18N4O3‚
0.17CH3OH) C, H, N.
were back-extracted with ether (1 × 5 mL). The ether layers were
set aside, and the NaOH layer was made acidic by addition of 12
M HCl(aq) (ca. 1.5 mL). The acidic suspension was then extracted
with diethyl ether (3 × 5 mL). Then a final set of ether layers was
back-extracted with brine (1 × 5 mL), dried, and concentrated to
173 mg of a white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.57
(s, 1H), 7.66 (d, J ) 2.7 Hz, 1H), 7.56 (d, J ) 8.8 Hz, 1H), 7.42
(dd, J ) 8.8, 2.7 Hz, 1H), 3.74 (s, 1 H); MS (ESI) m/e ) 249
(M - CO2H)-.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxy-
phenyl)acetamide (6o). This was prepared according to the same
procedure as 6f, substituting (2,5-dimethoxyphenyl)acetic acid for
(2-chlorophenyl)acetic acid. The product was purified by recrys-
tallization from ethanol. Yield, 11 mg (18%); 1H NMR (300 MHz,
DMSO-d6) δ 10.10 (s, 1H), 7.12 (s, 1H), 6.90 (m, 3H), 6.79 (m,
2H), 4.34 (q, 2H, J ) 7.1 Hz), 3.70 (s, 3H), 3.69 (s, 3H), 3.66 (s,
2H), 1.31 (t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 329 (M - Et +
H)+, 357 (M + H)+, 355 (M - H)-. Anal. (C18H20N4O4‚0.10EtOH)
C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethyl-
phenyl)acetamide (6p). This was prepared according to the same
procedure as 6f, substituting 2,5-dimethylphenylacetic acid for (2-
chlorophenyl)acetic acid. The product was purified by trituration
with hot methanol. Yield, 9 mg (16%); 1H NMR (300 MHz,
DMSO-d6) δ 10.26 (s, 1H), 7.15 (s, 1H), 7.04 (m, 2H), 6.93 (m,
3H), 4.35 (q, 2H, J ) 7.1 Hz), 3.70 (s, 2H), 2.23 (s, 3H), 2.21 (s,
3H), 1.31 (t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 297 (M - Et +
H)+, 325 (M + H)+, 323 (M - H)-. Anal. (C18H20N4O2‚
0.09EtOAc) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dibromo-
phenyl)acetamide (6q). This was prepared according to the same
procedure as 6f from 100 mg of 1 and 170 mg of 2,5-dibromo-
phenylacetic acid. The product was purified via reversed-phase
HPLC followed by trituration with hot methanol. Yield, 20 mg
1
(8%); H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 7.64 (d, J
) 2.7 Hz, 1H), 7.56 (d, J ) 8.5 Hz, 1H), 7.42 (dd, J ) 8.5, 2.4
Hz, 1H), 7.11 (s, 1H), 6.90 (s, 2H), 4.35 (q, J ) 7.1 Hz, 2H), 3.93
(s, 2H), 1.32 (t, J ) 7.1 Hz, 3H); MS (ESI) m/e ) 450 (M + H)+.
Anal. (C16H14Br2N4O2‚0.39H2O) C, H, N.
2-(4-Acetylamino-2,5-dimethoxyphenyl)-N-(4-amino-5-cyano-
6-ethoxypyridin-2-yl)acetamide (6r). To a suspension of 250 mg
(0.623 mmol) of 6u in 10 mL of glacial acetic acid and 2 mL of
water at 90 °C was added 500 mg (7.65 mmol) of Zn dust. The
mixture was heated at 90 °C for 30 min. Then the amber solution
was decanted from the excess Zn, and the solvents were removed
in vacuo. The residue was suspended in water amd filtered, and
the precipitate was washed with additional water. The product was
purified by dissolution in 10 mL of MeOH and 0.5 mL of 1 M
HCl(aq), and then addition of 0.3 mL of 2 M NaOH gave a yellow
precipitate. This was filtered, washed with methanol, and dried in
2,5-Dibromobenzyl Alcohol. To an ice-cooled solution of 2.80
g (10.0 mmol) of 2,5-dibromobenzoic acid in 5 mL of THF was
added 11 mL of 1.0 M BH3‚THF. The mixture was warmed to
ambient temperature and stirred for 24 h. The mixture was cooled
with an ice bath. Then 4 mL of water was added, and the solution
was stirred until bubbling ceased. The solvents were removed in
vacuo, and then the residue was taken up in 30 mL of diethyl ether.
This was extracted with 20 mL of 1 M NaOH and then with 2 M
NaOH (2 × 10 mL) and brine (1 × 10 mL), dried, and concentrated
1
vacuo to give 100 mg (43%) of the aminophenylacetamide. H
NMR (300 MHz, DMSO-d6) δ 9.78 (s, 1H), 7.12 (s, 1H), 6.88 (s,
2H), 6.67 (s, 1H), 6.35 (s, 1H), 4.69 (s, 2H), 4.32 (q, J ) 6.9 Hz,
2H), 3.68 (s, 3H), 3.64 (s, 3H), 3.51 (s, 2H), 1.30 (t, J ) 7.1 Hz,
3H); MS (ESI) m/e ) 372 (M + H)+.
To a solution of 20 mg (0.054 mmol) of N-(4-amino-5-cyano-
6-ethoxypyridin-2-yl)-2-(4-amino-2,5-dimethoxyphenyl)aceta-
mide in 1 mL of pyridine and 1 mL of CH2Cl2 at -78 °C was
added a solution of 100 µL (1.40 mmol) of acetyl chloride in 1
mL of CH2Cl2. The mixture was stirred at -78 °C for 5 min. Then
2 mL of water was added, and the mixture was warmed until the
ice melted. The solvents were removed in vacuo, and the residue
was suspended in 4 mL of water. This was placed in a centrifuge
for 1 min at 2000 rpm. Then the water was decanted. This was
repeated with 4 mL portions of water until the aqueous suspension
was at neutral pH. The resulting wet solid was taken up in methanol
and concentrated to dryness. The solid was recrystallized from
methanol to give 5 mg (22%) of the acetamide as a white solid. 1H
NMR (300 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.11 (s, 1H), 7.72 (s,
1H), 7.12 (s, 1H), 6.92 (s, 1H), 6.87 (s, 2H), 4.34 (q, J ) 7.1 Hz,
2H), 3.76 (s, 3H), 3.66 (s, 3H), 3.65 (s, 2H), 2.08 (s, 3H), 1.31 (t,
J ) 7.1 Hz, 3H); MS (ESI) m/e ) 414 (M + H)+. Anal.
(C20H23N5O5‚0.53H2O) C, H, N.
1
to 2.49 g (94%) of a white solid. H NMR (300 MHz, DMSO-d6)
δ 7.65 (d, J ) 2.4 Hz, 1H), 7.53 (d, J ) 8.5 Hz, 1H), 7.40 (dd,
J ) 8.5, 2.4 Hz, 1H), 5.58 (t, J ) 5.4 Hz, 1H), 4.49 (d, J ) 5.4
Hz, 2H).
2,5-Dibromobenzyl Chloride. To a solution of 2.49 g (9.36
mmol) of 2,5-dibromobenzyl alcohol in 15 mL of DMF was added
794 mg (18.7 mmol) of lithium chloride. After the lithium chloride
had dissolved, 1.2 mL (16.4 mmol) of thionyl chloride was added.
After spontaneously warming, the solution was stirred at ambient
temperature for 1.5 h and then poured into 75 mL of water. The
precipitate was collected, washed with water (2 × 20 mL), and
then taken up in 50 mL of hexanes. The residual water was drained,
and the organic layer was extracted with brine (1 × 10 mL), dried,
1
and concentrated to 2.42 g (91%) of a white solid. H NMR (300
MHz, DMSO-d6) δ 7.88 (d, J ) 2.4 Hz, 1H), 7.64 (d, J ) 8.5 Hz,
1H), 7.51 (dd, J ) 8.5, 2.4 Hz, 1H), 4.79 (s, 2H); MS (ESI) m/e )
305 (M + Na)+.
N-(4-Amino-5-cyano-6-isopropoxypyridin-2-yl)-2-(4-methane-
sulfonyl-2,5-dimethoxyphenyl)acetamide (6s). This was prepared
from 2 and acid chloride 11 according to the procedure for 6f. The
(2,5-Dibromophenyl)acetonitrile.44 To 2.42 g (8.51 mmol) of
2,5-dibromobenzyl chloride in 10 mL of DMSO was added 600
mg (9.2 mmol) of powdered potassium cyanide. The mixture was
stirred at ambient temperature for 24 h and then poured into 75
mL of water. The aqueous mixture was stirred for 30 min, and
then the precipitate was collected. The crude product was purified
via silica gel chromatography (90:10 hexane/EtOAc) to give 1.16
g (50%) of a solid. 1H NMR (300 MHz, DMSO-d6) δ 7.77 (d, J )
2.4 Hz, 1H), 7.66 (d, J ) 8.5 Hz, 1H), 7.53 (dd, J ) 8.5, 2.4 Hz,
1H), 4.08 (s, 2H); MS (ESI) m/e ) 273 (M - H)-.
1
mixture was run at -78 °C. Yield, 34 mg (25%); H NMR (300
MHz, DMSO-d6) δ 10.27 (s, 1H), 7.30 (s, 1H), 7.26 (s, 1H), 7.10
(s, 1H), 6.88 (s, 2H), 4.35 (q, J ) 7.1 Hz, 2H), 3.89 (s, 3H), 3.82
(s, 2H), 3.76 (s, 3H), 3.24 (s, 3H), 1.32 (t, J ) 7.1 Hz, 3H); MS
(ESI) m/e ) 435 (M + H)+. Anal. (C19H22N4O6S‚0.25CH3OH) C,
H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(4-bromo-2,5-
dimethoxyphenyl)acetamide (6t). This was prepared according to
the same procedure as 6f from 100 mg (0.562 mmol) of 1 and 202
mg (0.734 mmol) of 4-bromo-2,5-dimethoxyphenylacetic acid.45
Trituration with methanol gave 46 mg (19%) of a colorless solid.
1H NMR (300 MHz, DMSO-d6) δ 10.16 (s, 1H), 7.18 (s, 1H), 7.11
(s, 1H), 7.04 (s, 1H), 6.87 (s, 2H), 4.34 (q, J ) 7.0 Hz, 2H), 3.77
2,5-Dibromophenylacetic Acid. To 274 mg (1.0 mmol) of (2,5-
dibromophenyl)acetonitrile was added 4 mL of 60% (w/w) H2-
SO4(aq). The mixture was stirred at reflux for 3 h and then was
cooled, diluted with 10 mL of water, and extracted with diethyl
ether (2 × 15 mL). The combined ether layers were back-extracted
with 1 M NaOH (2 × 5 mL), and then the combined NaOH layers

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3573
(s, 3H), 3.71 (s, 3H), 3.69 (s, 2H), 1.31 (t, J ) 7.0 Hz, 3H); MS
(ESI) m/e ) 436 (M + H)+. Anal. (C18H19BrN4O4‚0.12EtOAc) C,
H, N.
To 31 mg (0.10 mmol) of trifluoromethanesulfonic acid 4-amino-
5-cyano-6-ethoxypyridin-2-yl ester and 91 mg (0.50 mmol) of
2-(2,5-dimethoxyphenyl)ethylamine was added DMSO (250 µL).
The mixture was heated in a sealed vessel in a microwave reactor
at 110 °C for 30 min. The mixture was taken up in EtOAc (20
mL) and washed with H2O (2 × 15 mL). The organic layer was
concentrated in vacuo and purified by MPLC (20-60% ethyl acetate
gradient in hexanes) to provide 4-amino-6-[2-(2,5-dimethoxyphen-
yl)ethylamino]-2-ethoxynicotinonitrile (27 mg, 79%). 1H NMR (300
MHz, DMSO-d6) δ 6.84-6.90 (m, 2H), 6.75 (t, J ) 3.39 Hz, 1H),
6.73 (s, 1H), 6.13 (s, 2H), 5.32 (s, 1H), 4.30 (q, J ) 6.89 Hz, 2H),
3.73 (s, 3H), 3.67 (s, 3H), 3.28-3.30 (m, 2H), 2.75 (dd, J ) 8.48,
6.10 Hz, 2H), 1.28 (t, J ) 7.12 Hz, 3H); MS (ESI) m/e ) 365
(M + Na)+, 343 (M + H)+. Anal. (C18H22N4O3) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxy-
4-nitrophenyl)acetamide (6u). To 241 mg (1.00 mmol) of (2,5-
dimethoxy-4-nitrophenyl)acetic acid46 was added 2 mL of thionyl
chloride. The mixture was stirred at reflux for 1 h, during which
time the starting acid dissolved completely. Then the excess thionyl
chloride was concentrated in vacuo to give a yellow solid. This
was taken up in 3 mL of CH2Cl2 and concentrated in vacuo to
give the acid chloride. To a solution of 356 mg (2.00 mmol) of 1
in 6 mL of pyridine and 6 mL of CH2Cl2 at -78 °C was added a
solution of 1.0 g (3.9 mmol) of (2,5-dimethoxy-4-nitrophenyl)acetyl
chloride in 6 mL of CH2Cl2. The mixture was stirred at -78 °C
for 20 min, and then 5 mL of water was added. The mixture was
warmed until the ice had melted, and then the solvents were
removed in vacuo. The residue was taken up in 75 mL of ethyl
acetate and extracted with 1 M HCl (3 × 10 mL). Next, 30 mL of
hexanes was added, and the organic phase was extracted with
saturated NaHCO3(aq) (3 × 10 mL) and then brine (1 × 10 mL),
dried over MgSO4, filtered, and concentrated in vacuo to a yellow
solid. This was triturated with 10 mL of hot methanol to give 479
mg (60%) of the phenylacetamide as fine yellow crystals. 1H NMR
(300 MHz, DMSO-d6) δ 10.30 (s, 1H), 7.49 (s, 1H), 7.32 (s, 1H),
7.10 (s, 1H), 6.89 (s, 2H), 4.35 (q, J ) 7.1 Hz, 2H), 3.86 (s, 3H),
3.82 (s, 2H), 3.77 (s, 3H), 1.32 (t, J ) 7.1 Hz, 3H); MS (ESI)
m/e ) 401 (M + H)+. Anal. (C18H19N5O5‚0.58H2O) C, H, N.
(4-Methanesulfonyl-2,5-dimethoxyphenyl)acetyl Chloride (11).
To 300 mg (1.09 mmol) of acid 15 was added 4 mL of thionyl
chloride. The mixture was heated at reflux for 20 min and then
concentrated in vacuo. The residue was taken up in 5 mL of CH2-
Cl2 and concentrated 2 times to give the acid chloride.
[2-(4-Bromo-2,5-dimethoxyphenyl)ethoxy]-tert-butyldimeth-
ylsilane (13). To a solution of 3.16 g (12.1 mmol) of 2-(4-bromo-
2,5-dimethoxy)phenylethanol (12)47 in 15 mL of N,N-dimethyl-
formamide was added 1.65 g (24.2 mmol) of imidazole and then
2.19 g (14.5 mmol) of tert-butyldimethylsilyl chloride. The mixture
was stirred at ambient temperature for 1.5 h, and then it was poured
into 100 mL of water. The aqueous suspension was extracted with
hexanes (3 × 20 mL). Then the combined hexanes layers were
back-extracted with water (1 × 20 mL), 5% (w/w) NH4OH (2 ×
20 mL), 1 M HCl(aq) (2 × 20 mL), and brine (1 × 20 mL), dried
over MgSO4, filtered, and concentrated in vacuo to an oil. This
was heated at 110 °C at ca. 1 mmHg for 1 h to remove any volatile
impurities, giving 4.38 g (96%) of the silyl ether as a colorless oil.
1H NMR (300 MHz, DMSO-d6) δ 7.14 (s, 1H), 6.96 (s, 1H), 3.76
(s, 3H), 3.74 (s, 3H), 3.72 (t, J ) 6.8 Hz, 2H), 2.72 (t, J ) 6.8 Hz,
2H), 0.82 (s, 9H), -0.04 (s, 6H).
(4-Amino-5-cyano-6-ethoxypyridin-2-yl)carbamic Acid Meth-
yl Ester (7). To a solution of 50 mg (0.28 mmol) of 1 in 1 mL of
tetrahydrofuran was added 100 µL (0.57 mmol) of N,N-diisopro-
pylethylamine and then three drops of methylchloroformate. The
mixture was stirred at ambient temperature for 5 min and then
diluted with 7 mL of water. The precipitate was collected, washed
with water, and recrystallized from methanol to give 17 mg (24%)
1
of a white solid. H NMR (300 MHz, DMSO-d6) δ 9.87 (s, 1H),
2-(2,5-Dimethoxy-4-methylsulfanylphenyl)ethanol (14). To a
solution of 4.37 g (11.6 mmol) of 13 in 25 mL of tetrahydrofuran
at -78 °C and under N2 was added 6 mL (15 mmol) of 2.5 M
n-butyllithium in hexanes. The mixture was stirred at -78 °C for
10 min, and then 2.3 mL (25.5 mmol) of dimethyl disulfide was
added. The mixture was warmed to ambient temperature over 30
min. Then 3 mL of water was added, and the solution was
concentrated in vacuo. The residue was taken up in 50 mL of
hexanes, extracted with water (3 × 20 mL) and then brine (1 × 20
mL), dried over MgSO4, filtered, and concentrated in vacuo to a
white solid. This was taken up in 30 mL of tetrahydrofuran, and to
the solution was added 4.5 g (ca. 15.7 mmol) of tetrabutylammo-
nium fluoride hydrate. After 2.5 h, the solution was concentrated
in vacuo to a solid. This was washed with water (3 × 20 mL) and
then hexanes (2 × 20 mL). The solid was dissolved in ethyl acetate,
dried over MgSO4, and filtered. The ethyl acetate was removed in
vacuo, and the residue was purified via silica gel chromatography,
eluting with a 20-40% ethyl acetate/hexanes gradient to give 1.70
6.89 (s, 1H), 6.86 (s, 2H), 4.30 (q, J ) 7.1 Hz, 2H), 3.65 (s, 3H),
1.28 (t, J ) 7.1 Hz, 3H); MS (ESI) m/e ) 237 (M + H)+. Anal.
(C10H12N4O3‚0.4CH3OH) C, H, N.
1-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-3-ethylurea (8). To
a solution of 50 mg (0.28 mmol) of 1 in 1 mL of tetrahydrofuran
was added 100 µL (0.57 mmol) of N,N-diisopropylethylamine and
then 28 mg (0.094 mmol) of triphosgene. The white suspension
was stirred at ambient temperature for 15 min, and then 0.5 mL
(0.5 mmol) of 1.0 M ethylamine in THF was added. The suspension
cleared, and the solution was stirred at ambient temperature for 5
min. The mixture was concentrated in vacuo, and the white solid
residue was suspended in 5 mL of water. This was extracted with
warm ethyl acetate (15 mL). Then the ethyl acetate layer was back-
extracted with water (1 × 5 mL) and brine (1 × 5 mL), dried over
MgSO4, filtered, and concentrated in vacuo to a solid. This was
recrystallized from ethyl acetate to give an impure solid containing
some symmetrical urea. Pure product was obtained by reversed-
1
1
phase HPLC to give 7 mg (10%) of the urea as a white solid. H
g (64%) of a white solid. H NMR (300 MHz, DMSO-d6) δ 6.79
NMR (300 MHz, DMSO-d6) δ 8.88 (s, 1H), 7.32 (t, J ) 5.1 Hz,
1H), 6.75 (s, 2H), 6.55 (s, 1H), 4.27 (q, J ) 7.1 Hz, 2H), 3.02-
3.22 (m, 2H), 1.30 (t, J ) 7.1 Hz, 3H), 1.06 (t, J ) 7.1 Hz, 3H);
MS (ESI) m/e ) 250 (M + H)+. Anal. (C11H15N5O2S‚0.18H2O)
C, H, N.
4-Amino-6-[2-(2,5-dimethoxyphenyl)ethylamino]-2-ethoxyni-
cotinonitrile (10). To a solution of 1.1 g (6.1 mmol) of 4-amino-
2-ethoxy-6-hydroxynicotinonitrile (9)31 in 10 mL of CH2Cl2 was
added 1.4 g (14 mmol) of Et3N and then 2.8 g (7.0 mmol) of
N-phenyl-bis(trifluoromethanesulfonimide). The mixture was stirred
at ambient temperature for 4 h, and then it was extracted with water,
dried over MgSO4, and concentrated in vacuo. The residue was
purified via MPLC (0-60% ethyl acetate gradient in hexanes) to
give 900 mg (60%) of trifluoromethanesulfonic acid 4-amino-5-
cyano-6-ethoxypyridin-2-yl ester. 1H NMR (300 MHz, DMSO-d6)
δ 7.66 (broad s, 2H), 6.30 (s, 1H), 4.31 (q, J ) 7.2 Hz, 2H), 1.30
(t, J ) 7.2 Hz, 3H).
(s, 1H), 6.72 (s, 1H), 4.58 (t, J ) 5.3 Hz, 1H), 3.76 (s, 3H), 3.74
(s, 3H), 3.46-3.57 (m, 2H), 2.68 (t, J ) 7.3 Hz, 2H), 2.39 (s, 3H);
MS (ESI) m/e ) 211 (M - OH)+.
(4-Methanesulfonyl-2,5-dimethoxyphenyl)acetic Acid (15). To
a solution of 1.70 g (7.44 mmol) of 2-(2,5-dimethoxy-4-methyl-
sulfanylphenyl)ethanol in 12 mL of trifluoroacetic acid was added
1.82 mL (17.8 mmol) of 9.8 M H2O2(aq). The mixture was stirred
at ambient temperature for 4 h, and then NaHSO3(aq) was added to
destroy any excess H2O2, until starch-KI paper showed no excess
oxidant. The mixture was concentrated in vacuo, and the residue
was taken up in 50 mL of methanol. To the solution was added 6
g (43.4 mmol) of K2CO3 and 10 mL of water. Then the mixture
was stirred at ambient temperature for 10 min. The solvents were
removed in vacuo, and the residue was taken up in 50 mL of ethyl
acetate. This was filtered to remove the inorganic salts. Then the
salts were washed with ethyl acetate (2 × 5 mL). The combined
filtrate and washings were extracted with brine (2 × 20 mL), water

3574 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
(2 × 20 mL), saturated NaHCO3(aq) (1 × 20 mL), and again with
brine (1 × 20 mL), dried over MgSO4, filtered, and concentrated
to 1.33 g (69%) of a solid. 1H NMR (300 MHz, DMSO-d6) δ 7.27
(s, 1H), 7.16 (s, 1H), 4.67 (t, J ) 5.4 Hz, 1H), 3.89 (s, 3H), 3.79
(s, 3H), 3.51-3.66 (m, 2H), 3.21 (s, 3H), 2.79 (t, J ) 7.0 Hz, 2H);
MS (ESI) m/e ) 261 (M + H)+.
50 mg (23%) of a yellow foam. The foam was taken up in 0.6 mL
of pyridine and 0.6 mL of CH2Cl2 and then cooled to -78 °C.
Next, 16 mL (0.23 mmol) of acetyl chloride was added, and the
mixture was stirred at -78 °C for 10 min. Water (1 mL) was added,
and the mixture was allowed to warm until the ice had melted.
Then the solvents were removed in vacuo. The residue was taken
up in 5 mL of 2 M NaOH(aq) and then extracted with ethyl acetate
(6 × 5 mL). The combined organic layers were back-extracted with
brine (1 × 5 mL), dried over MgSO4, filtered, and concentrated to
a solid. Recrystallization from methanol gave 12 mg (20%) of a
white solid. 1H NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H), 7.23
(s, 1H), 6.94 (s, 2H), 4.40-4.43 (t, J ) 5.83 Hz, 2H), 2.64-2.67
(t, J ) 5.83 Hz, 2H), 2.27 (s, 6H), 2.13 (s, 3H); MS (ESI) m/e )
264 (M + H)+, 262 (M - H)-. Anal. (C12H17N5O2) C, H, N.
3-(6-Acetylamino-4-amino-3-cyanopyridin-2-yloxy)propion-
ic Acid (19b). 4,6-Diamino-2-(3-hydroxypropoxy)nicotinonitrile
was prepared according to the procedure described for 2, substituting
propylene glycol for ethanol. The yield was 617 mg (63%). From
200 mg (0.96 mmol) of this glycol ether and 400 µL (5.63 mmol)
of acetyl chloride, both N-6 acylation and O acylation were
accomplished according to the procedure described for 6a. To the
intermediate acetoxypropyl ester in 10 mL of warm 95:5 MeOH/
H2O was added 400 mg (2.89 mmol) of K2CO3. The hydrolysis
was complete upon dissolution of the starting ester with gentle
heating. The solvents were removed in vacuo, and then the residue
was taken up in 30 mL of ethyl acetate and 5 mL of water. Gentle
heating was necessary to dissolve the product. The layers were
separated, and the organic layer was extracted with 1 M HCl(aq) (1
× 5 mL), saturated NaHCO3(aq) (2 × 5 mL), and brine (1 × 5 mL),
dried, and concentrated to 135 mg (56%) of a white solid. To a
suspension of 50 mg (0.20 mmol) of this alcohol in 5 mL of
2-butanone was added 1 mL (2.0 mmol) of Jones’ reagent. The
mixture was stirred at ambient temperature for 2.5 h, and then the
solvent was removed in vacuo. The residue was suspended in 5
mL of water, and then NaHSO3(s) was added until the solution was
blue. The precipitate was collected, washed with water, suspended
in ethyl acetate, and concentrated in vacuo to 25 mg (47%) of a
white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.34 (s, 1H), 10.12
(s, 1H), 7.19 (s, 1H), 6.90 (s, 2H), 4.45 (t, J ) 6.4 Hz, 2H), 2.70
(t, J ) 6.4 Hz, 2H), 2.07 (s, 3H); MS (ESI) m/e ) 265 (M + H)+,
263 (M - H)-. Anal. (C11H12N4O4‚0.08EtOAc) C, H, N.
To a solution of 1.33 g (5.11 mmol) of 2-(4-methanesulfonyl-
2,5-dimethoxyphenyl)ethanol in 25 mL of acetone was added 6.5
mL (13.0 mmol) of 2.0 M H2CrO4(aq). After 1 h, an additional 2
mL (4.0 mmol) of 2.0 M H2CrO4(aq) was added. Then the mixture
was stirred for another 2 h. The excess chromic acid was quenched
by addition of 10 mL of water, followed by NaHSO3(s) to discharge
the yellow-orange color. The solvents were removed in vacuo, and
the residue was taken up in 25 mL of water and then extracted
with ethyl acetate (3 × 35 mL). The combined ethyl acetate layers
were back-extracted with water (2 × 10 mL) and brine (1 × 10
mL), dried over MgSO4, filtered, and concentrated to a solid. This
was recrystallized from 30 mL of ethyl acetate to give 453 mg
1
(32%) of a white solid. H NMR (300 MHz, DMSO-d6) δ 12.36
(s, 1H), 7.30 (s, 1H), 7.26 (s, 1H), 3.89 (s, 3H), 3.78 (s, 3H), 3.62
(s, 2H), 3.24 (s, 3H); MS (ESI) m/e ) 275 (M + H)+.
N-(4-Amino-6-bromo-5-cyanopyridin-2-yl)acetamide (16). Bro-
mopyridine 1 (100 mg, 0.47 mmol) was dissolved in 1.0 mL of
anhydrous pyridine. AcCl (225 µL, 2.35 mmol) was added dropwise
slowly to control heat evolution. The reaction mixture was shaken
overnight at ambient temperature. Water (5 mL) was added to the
reaction mixture. The resulting brown solid was filtered, washed
with cold water, and dried in a vacuum oven to give 100 mg (83%)
of the acetamide. 1H NMR (300 MHz, DMSO-d6) δ 10.65 (s, 1H),
7.51 (s, 1H), 7.30 (s, 2H), 2.06 (s, 3H); MS (ESI) m/e ) 255
(M + H)+, 257 (M + H)+.
N-(4-Amino-6-bromo-5-cyanopyridin-2-yl)-2-(2,5-dimethoxy-
phenyl)acetamide (17). This was prepared according to the
procedure described for 6f, substituting 456 mg of (2,5-dimethoxy-
phenyl)acetic acid for (2-chlorophenyl)acetic acid and 212 mg (1.00
mmol) of bromopyridine 1 for ethoxypyridine 2. Recrystallization
from EtOAc gave 73 mg (19%) of a colorless solid. 1H NMR (300
MHz, DMSO-d6) δ 10.69 (s, 1H), 7.47 (s, 1H), 7.28 (s, 2H), 6.88
(m, 1H), 6.78 (m, 2H), 3.69 (s, 3H), 3.68 (s, 3H), 3.65 (s, 2H); MS
(ESI) m/e ) 391, 393 (M + H)+. Anal. (C16H15BrN4O3‚0.12EtOAc)
C, H, N.
N-(4-Amino-5-cyano-6-isopropoxypyridin-2-yl)acetamide (18a).
18a was prepared according to the procedure for 6a, substituting
isopropyl ether 3 for ethyl ether 2. The product was recrystallized
N-[4-Amino-5-cyano-6-(tetrahydrofuran-3-yloxy)pyridin-2-yl]-
acetamide (19c). A sealable tube suitable for microwave heating
was charged with 176 mg (2.00 mmol) of 3-hydroxytetrahydrofuran,
3 mL of dioxane, and 60 mg (1.5 mmol) of 60% NaH in mineral
oil. The mixture was stirred for 1 h, and then 213 mg (1.00 mmol)
of 1 was added. The mixture was heated in a microwave reactor at
190 °C for 10 min and then concentrated in vacuo. The residue
was taken up in 10 mL of ethyl acetate and extracted with water
(2 × 5 mL) and brine (1 × 5 mL), dried, and concentrated. The
product was purified via MPLC, eluting with a 50-100% ethyl
acetate in hexanes gradient to give 44 mg (20%) of the ether as a
solid. From 44 mg (0.20 mmol) of this ether and 5 drops of acetyl
chloride, the product was prepared according to the procedure for
6a. Recrystallization from MeOH gave 10 mg (19%) of a white
1
from methanol to give 12 mg (11%) of a white solid. H NMR
(300 MHz, DMSO-d6) δ 10.05 (s, 1H), 7.15 (s, 1H), 6.84 (s, 2H),
5.27 (m, 1H), 2.06 (s, 3H), 1.28 (d, 6H, J ) 6.4 Hz); MS (ESI)
m/e ) 193 (M - Pr + H)+, 235 (M + H)+, 233 (M - H)-. Anal.
(C11H14N4O2) C, H, N.
N-(4-Amino-5-cyano-6-isopropoxypyridin-2-yl)-2-(4-methane-
sulfonyl-2,5-dimethoxyphenyl)acetamide (18b). 18b was prepared
according to the procedure for 6f from 160 mg (0.547 mmol) of
acid chloride 11 and 55 mg (0.29 mmol) of 3. The acylation was
performed at -78 °C. Recrystallization from methanol gave 49 mg
1
(38%) of a white solid. H NMR (300 MHz, DMSO-d6) δ 10.25
(s, 1H), 7.30 (s, 1H), 7.26 (s, 1H), 7.08 (s, 1H), 6.86 (s, 2H), 5.30
(m, 1H), 3.89 (s, 3H), 3.82 (s, 2H), 3.76 (s, 3H), 3.24 (s, 3H), 1.30
(d, J ) 6.4 Hz, 6H); MS (ESI) m/e ) 449 (M + H)+. Anal.
(C20H24N4O6S‚0.33CH3OH) C, H, N.
1
solid. H NMR (300 MHz, DMSO-d6) δ 10.06 (s, 1H), 7.18 (s,
1H), 6.92 (s, 2H), 5.46 (m, 1H), 3.82 (m, 4H), 2.22 (m, 1H), 2.07
(s, 3H), 1.99 (m, 1H); MS (ESI) m/e ) 263 (M + H)+. Anal.
(C12H14N4O3) C, H, N.
N-[4-Amino-5-cyano-6-(2-(dimethylamino)ethoxy)pyridin-2-
yl]acetamide (19a). To 2 mL of dimethylaminoethanol in a sealable
tube suitable for microwave heating was added 40 mg (1.7 mmol)
of sodium. The mixture was heated gently until all of the sodium
had reacted. Then it was cooled to ambient temperature. Next, 212
mg (1.0 mmol) of 1 was added, and the mixture was heated at 150
°C for 15 min in a microwave reactor. The solvent was removed
in vacuo, and the residue was taken up in 2 M NaOH and then
extracted with ethyl acetate (3×). The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated
to an oily residue. This was purified via silica gel chromatography,
eluting with CH2Cl2/MeOH/concentrated NH4OH (10:1:0.1) to give
N-(4-Amino-6-butoxy-5-cyanopyridin-2-yl)acetamide (19d).
4,6-Diamino-2-butoxynicotinonitrile was prepared according to the
procedure for 3, substituting n-butanol for 2-propanol. The butyl
ether was purified via silica gel chromatography, eluting with 50%
ethyl acetate/hexanes to give a white solid. This was acylated
according to the procedure for 6a. The product was recrystallized
1
from 1 mL of methanol to give 16 mg (27%) of a white solid. H
NMR (300 MHz, DMSO-d6) δ 10.07 (s, 1H), 7.17 (s, 1H), 6.89 (s,
2H), 4.27 (t, 2H, J ) 6.6 Hz), 2.06 (s, 3H), 1.67 (m, 2H), 1.40 (m,
2H), 0.93 (t, 3H, J ) 7.5 Hz); MS (ESI) m/e ) 193 (M - Bu +
H)+, 249 (M + H)+, 247 (M - H)-. Anal. (C12H16N4O2) C, H, N.

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3575
N-(4-Amino-5-cyano-6-methoxypyridin-2-yl)acetamide (19e).
4,6-Diamino-2-methoxynicotinonitrile was prepared according to
the procedure for 2, substituting methanol for ethanol. The methyl
ether was purified via silica gel chromatography, eluting with 60:
40 hexanes/ethyl acetate to give 114 mg (70%) of a white solid.
To a solution of 50 mg (0.30 mmol) of the methyl ether in 1 mL
of pyridine was added 3 drops of acetyl chloride. The mixture was
stirred for 2 h at ambient temperature, and then 10 mL of water
was added. After the mixture was stirred for 10 min, the precipitate
was collected and washed with water. Recrystallization from 1 mL
N-(4-Amino-5-cyano-6-(ethylamino)pyridin-2-yl)acetamide (22).
To a solution of 1 g (22 mmol) of ethylamine in 5 mL of
N-methylpyrrolidinone in a sealable tube suitable for microwave
heating was added 250 mg (0.98 mmol) of 16. The mixture was
heated in a microwave apparatus at 150 °C for 10 min and then
poured into 50 mL of water. The suspension was extracted with
ethyl acetate (3 × 10 mL). Then 25 mL of hexanes was added,
and the organic layer was back-extracted with water (2 × 10 mL)
and brine (1 × 10 mL), dried, and concentrated to a waxy solid.
This was triturated with diethyl ether and then recrystallized from
methanol to give 15 mg (7%) of white crystals. 1H NMR (300 MHz,
DMSO-d6) δ 9.73 (s, 1H), 6.82 (s, 1H), 6.46 (s, 2H), 6.32 (t, J )
5.8 Hz, 1H), 3.34 (m, 2H), 2.04 (s, 3H), 1.09 (t, J ) 7.0 Hz, 3H);
MS (ESI) m/e ) 220 (M + H)+. Anal. (C10H13N5O‚0.08CH3OH)
C, H, N.
1
of methanol gave 17 mg (27%) of a white solid. H NMR (300
MHz, DMSO-d6) δ 10.13 (s, 1H), 7.19 (s, 1H), 6.92 (s, 2H), 3.85
(s, 3H), 2.07 (s, 3H); MS (ESI) m/e ) 207 (M + H)+, 205 (M -
H)-. Anal. (C9H10N4O2‚0.13H2O) C, H, N.
N-[4-Amino-5-cyano-6-(2-hydroxyethoxy)pyridin-2-yl]-2-(2,5-
dimethoxyphenyl)acetamide (20a). 4,6-Diamino-2-(2-hydroxy-
ethoxy)nicotinonitrile was prepared according to the procedure for
3, substituting ethylene glycol for 2-propanol. Purification of the
pyridyl ether was not necessary. Acylation of 100 mg (0.51 mmol)
of the pyridyl ether was accomplished according to the same
procedure as for 6f, substituting (2,5-dimethoxyphenyl)acetic acid
for (2-chlorophenyl)acetic acid and running the reaction at -78
°C. The product was purified by reversed-phase HPLC, eluting with
a 5-100% CH3CN gradient in 0.1% TFA(aq) to give 13 mg (7%)
of a white solid. 1H NMR (300 MHz, DMSO-d6) δ 10.10 (s, 1H),
7.13 (s, 1H), 6.90 (m, 3H), 6.79 (m, 2H), 4.81 (t, 1H, J ) 5.4 Hz),
4.31 (m, 2H), 3.69 (m, 8H), 3.66 (s, 2H); MS (ESI) m/e ) 329
(M - EtOH + H)+, 373 (M + H)+, 371 (M - H)-. Anal.
(C18H20N4O5‚0.49TFA) C, H, N.
N-[4-Amino-5-cyano-6-(2-methoxyethoxy)pyridin-2-yl]-2-(2,5-
dimethoxyphenyl)acetamide (20b). 4,6-Diamino-2-(2-methoxy-
ethoxy)nicotinonitrile was prepared according to the procedure for
3, substituting 2-methoxyethanol for 2-propanol. Purification of the
pyridyl ether was not necessary. Acylation was accomplished
according to the same procedure as for 6f, substituting (2,5-
dimethoxyphenyl)acetic acid for (2-chlorophenyl)acetic acid. The
product was recrystallized from 3 mL of ethyl acetate to give 30
mg (38%) of a white solid. 1H NMR (300 MHz, DMSO-d6) δ 10.11
(s, 1H), 7.14 (s, 1H), 6.90 (m, 3H), 6.79 (m, 2H), 4.42 (m, 2H),
3.70 (s, 3H), 3.69 (s, 3H), 3.64 (m, 4H), 3.31 (s, 3H); MS (ESI)
m/e ) 329 (M - CH2CH2OCH3 + H)+, 387 (M + H)+, 385 (M -
H)-. Anal. (C19H22N4O5) C, H, N.
N-(4-Amino-5-cyano-6-isopropylaminopyridin-2-yl)aceta-
mide (23). This was prepared according to the procedure for 22
from 167 mg of 2-propylamine and 50 mg of 16. The mixture was
heated at 200 °C for 30 min. The product was purified via silica
gel chromatography, eluting with 35% EtOAc/hexanes to give 15
mg (33%) of a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.74
(s, 1H); 6.83 (s, 1H), 6.47 (s, 2H), 5.83 (d, J ) 8.24 Hz, 1H),
4.25-4.30 (m, 1H), 2.04 (s, 3H), 1.14 (d, J ) 6.40 Hz, 6H); MS
(ESI) m/e ) 234 (M + H)+, 232 (M - H)-. Anal. (C11H15N5O‚
0.1H2O) C, H, N.
N-(4-Amino-5-cyano-6-phenylpyridin-2-yl)acetamide (24). A
mixture of bromide 16 (45 mg, 0.18 mmol), Na2CO3 (38 mg, 0.35
mmol), and Pd(PPh3)4 (10 mg, 0.009 mmol) was stirred in DMF/
THF/H2O (1:1:0.5) under nitrogen in a microwave reactor vial.
Phenylboric acid (26 mg, 0.21 mmol) was added. The resulting
mixture was capped and heated at 130 °C for 20 min in a microwave
reactor. The crude mixture was partitioned between EtOAc and
water. The organic layer was washed with water and brine, dried
over Na2SO4, and evaporated in vacuo. The crude residue was
purified by reversed-phase HPLC. The purified product was
transferred to a vial in methanol and then concentrated to give 33
mg (72%) of the phenylpyridine. 1H NMR (300 MHz, DMSO-d6)
δ 10.44 (s, 1H), 7.74-7.68 (m, 2H), 7.55 (s, 1H), 7.52-7.46 (m,
3H), 6.98 (s, 2H), 2.08 (s, 3H); MS (ESI) m/e 253 (M + H)+, 251
(M - H)-. Anal. (C14H12N4O‚0.11CH3OH) C, H, N.
N-(4-Amino-5-cyano-6-propylpyridin-2-yl)acetamide (25). To
2.1 g (32 mmol) of freshly activated zinc dust was added 5.4 mL
of DMF and then 930 mg of n-propyl iodide. The mixture was
stirred under a nitrogen atmosphere at 85 °C for 20 min and then
cooled, and the excess zinc was allowed to settle.
N-[4-Amino-5-cyano-6-(2-methanesulfonylethoxy)-2-pyridinyl]-
2-(2,5-dimethoxyphenyl)acetamide (20c). 4,6-Diamino-2-(2-meth-
ylsulfanylethoxy)nicotinonitrile was prepared according to the
procedure for 3, substituting 2-methylthioethanol for 2-propanol.
Purification via silica gel chromatography (1:1 EtOAc/hexanes)
gave 186 mg (83%). Acylation was accomplished according to the
procedure for 6f, substituting (2,5-dimethoxyphenyl)acetic acid for
(2-chlorophenyl)acetic acid. The reaction was run at -78 °C.
Purification via silica gel chromatography (40% EtOAc/hexanes)
gave 67 mg (76%). To the amide (19 mg, 0.049 mmol) dissolved
in 2 mL of glacial acetic acid was added 6 equiv of 30% hydrogen
peroxide. After being heated at 50 °C for 2 h, the mixture was
stirred at ambient temperature for 8 h. Water was added. Then the
precipitate was collected, washed with additional water, and dried
To 128 mg of 16, 6 mg (0.027 mmol) of palladium(II) acetate,
and 30 mg (0.098 mmol) of tri(o-tolyl)phosphine was added 0.5
mL of DMF. The mixture was put under N2 and stirred, and then
2.0 mL of the n-propylzinc iodide solution was added via syringe.
The mixture was heated at 90 °C for 10 min and then poured into
a stirred mixture of 10 mL of water and 10 mL of ethyl acetate.
The metal salts were filtered away through diatomaceous earth,
and then the layers were separated. The organic layer was extracted
with water (2 × 10 mL) and brine (1 × 10 mL), dried over MgSO4,
filtered, and concentrated to a solid. This was purified via silica
gel chromatography, eluting with 50:50 hexanes/ethyl acetate and
loading as a solution in hot ethyl acetate to give 34 mg (31%) of
1
1
in vacuo to give 7.2 mg (34%) of a white powder. H NMR (500
a white solid. H NMR (300 MHz, DMSO-d6) δ 10.29 (s, 1H),
MHz, DMSO-d6) δ 3.10 (s, 3H), 3.31 (s, 2H), 3.66 (s, 2H), 3.69
(d, J ) 5.49 Hz, 6H), 4.63 (t, J ) 5.80 Hz, 2H), 6.78-6.83 (m,
2H), 6.90 (d, J ) 8.54 Hz, 1H), 7.01 (s, 2H), 7.18 (s, 1H), 10.21
(s, 1H). MS (ESI) m/e ) 435 (M + H), 433 (M - H).
N-(4-Amino-6-butylsulfanyl-5-cyanopyridin-2-yl)acetamide (21).
This was prepared according to the procedure for 6a, substituting
5 for 2 and using a volume of CH2Cl2 equal to the amount of
pyridine. The reaction was run at -78 °C for 1 h. The product was
isolated as a pale-yellow solid (yield 100%). 1H NMR (300 MHz,
DMSO-d6) δ 10.13 (s, 1H), 7.27 (s, 1H), 6.91 (s, 2H), 3.19 (t, J )
7.1 Hz, 2H), 2.10 (s, 3H), 1.64-1.52 (m, 2H), 1.45-1.35 (m, 2H),
0.89 (t, J ) 7.1 Hz, 3H); MS (ESI) 265 (M + H)+, 263 (M - H)-.
Anal. (C12H16N4OS) C, H, N.
7.39 (s, 1H), 6.82 (s, 2H), 2.63 (m, 2H), 2.07 (m, 3H), 1.67 (m,
2H), 0.92 (t, 3H, J ) 7.5 Hz); MS (ESI) m/e 219 (M + H)+, 217
(M - H)-. Anal. (C11H14N4O) C, H, N.
N-(4-Amino-5-cyanopyridin-2-yl)-2-(2,5-dimethoxyphenyl)ac-
etamide (26). To a solution of 38 mg (0.097 mmol) of 17 in 3 mL
of DMF was added 1 mL of 1 M HCl. To this was added 50 mg
(0.81 mmol) of Zn dust. The mixture was stirred for 30 min and
then 1 mL of 1 M HCl was added. After an additional 30 min, the
mixture was decanted from excess Zn and was concentrated in
vacuo. The residue was taken up in 20 mL of warm ethyl acetate
and extracted with 1 M HCl (3 × 5 mL) and then brine (1 × 5
1
mL), dried, and concentrated to 5 mg (17%) of a white solid. H
NMR (300 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.21 (s, 1H), 7.46 (s,

3576 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
1H), 6.95 (s, 2H), 6.89 (d, J ) 8.8 Hz, 1H), 6.80 (m, 2H), 3.69 (m,
3H), 3.69 (s, 3H), 3.66 (s, 2H); MS (ESI) m/e ) 313 (M + H)+.
Anal. (C16H16N4O3‚0.35EtOAc‚0.47H2O) C, H, N.
from methanol gave 13 mg (43%) of a white solid. 1H NMR (300
MHz, DMSO-d6) δ 9.63 (s, 1H), 7.18 (s, 1H), 6.97 (s, 2H), 4.31
(q, J ) 7.1 Hz, 2H), 3.60 (m, 4H), 3.17 (s, 2H), 2.51 (m, 4H), 1.30
(t, J ) 7.0 Hz, 3H); MS (ESI) m/e ) 306 (M + H)+. Anal.
(C14H19N5O3‚0.17CH3OH) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-bromoaceta-
mide (27). To a solution of 1.03 g (5.77 mmol) of 1 in 12 mL of
pyridine and 12 mL of CH2Cl2 at -78 °C was added a solution of
1.41 g (6.99 mmol) of bromoacetyl bromide in 10 mL of CH2Cl2.
The mixture was stirred at -78 °C for 10 min. Then 5 mL of water
was added, and the mixture was allowed to warm until the ice had
melted. The mixture was diluted with 200 mL of water and then
extracted with ethyl acetate (3 × 30 mL). The combined ethyl
acetate layers were back-extracted with 1 M HCl(aq) (2 × 30 mL),
water (1 × 30 mL), and brine (1 × 30 mL), dried over MgSO4,
filtered, and concentrated in vacuo to 1.45 g (84%) of a white solid.
1H NMR (300 MHz, DMSO-d6) δ 10.50 (s, 1H), 7.16 (s, 1H), 6.99
(s, 2H), 4.33 (q, J ) 7.1 Hz, 2H), 4.11 (s, 2H), 1.30 (t, J ) 7.1 Hz,
3H); MS (ESI) m/e ) 299 (M + H)+, 301 (M + H)+.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-diethylaminoac-
etamide (28a). To a solution of 30 mg (0.10 mmol) of 27 in 0.5
mL of DMF was added 16 µL (0.15 mmol) of N,N-diethylamine
and then 52 µL (0.3 mmol) of N,N-diisopropylethylamine. The
mixture was stirred for 1 h and then concentrated in vacuo. The
residue was recrystallized from methanol to give 15 mg (51%) of
a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.50 (s, 1H), 7.18
(s, 1H), 6.99 (s, 2H), 4.25-4.32 (q, J ) 7.1 Hz, 2H), 3.17 (s, 2H),
2.56-2.62 (q, J ) 7.1 Hz, 4H), 1.27-1.31 (t, J ) 7.1 Hz, 3H),
0.97-1.02 (t, J ) 7.1 Hz, 6H); MS (ESI) m/e ) 292 (M + H)+,
290 (M - H)-. Anal. (C14H21N5O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-dibutylaminoac-
etamide (28b). To a solution of 30 mg (0.10 mmol) of N-(4-amino-
5-cyano-6-ethoxypyridin-2-yl)-2-bromoacetamide in 0.5 mL of
DMF was added 25 µL (0.15 mmol) of N,N-dibutylamine and then
52 µL (0.3 mmol) of N,N-diisopropylethylamine. The mixture was
stirred for 1 h and then diluted with H2O and filtered. The residue
was recrystallized from methanol to give 16 mg (60%) of a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.16 (s, 1H),
7.00 (s, 2H), 4.25-4.32 (q, J ) 6.8 Hz, 2H), 3.16 (s, 2H), 2.48-
2.52 (m, 4H), 1.32-1.41 (m, 8H), 1.27-1.31 (t, J ) 6.8 Hz, 3H),
0.84-0.89 (t, J ) 7.1 Hz, 6H); MS (ESI) m/e ) 348(M + H)+,
346 (M - H)-. Anal. (C18H29N5O2) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(4-hydroxypip-
eridin-1-yl)acetamide (31). 31 was prepared according to the
procedure for 28a, substituting 4-hydroxypiperidine for N,N-
diethylamine. Recrystallization from methanol gave 12 mg (38%)
1
of a white solid. H NMR (300 MHz, DMSO-d6) δ 9.52 (s, 1H),
7.18 (s, 1H), 6.98 (s, 2H), 4.60-4.61 (d, J ) 4.07 Hz, 1H), 4.27-
4.34 (q, J ) 7.12 Hz, J ) 7.12 Hz, 2H), 3.44-3.51 (m, 1H), 3.11
(s, 2H), 3.11 (s, 2H), 2.70-2.76 (m, 2H), 2.21-2.29 (m, 2H), 1.71-
1.75 (m, 2H), 1.36-1.48 (m, 2H), 1.27-1.32 (t, J ) 7.12 Hz, 3H);
MS (ESI) m/e ) 320 (M + H)+, 318 (M - H)-. Anal. (C15H21N5O3)
C, H, N.
1-[(4-Amino-5-cyano-6-ethoxypyridi-2-ylcarbamoyl)methyl]-
piperidine-4-caroxylic Acid (32). To a solution of 2.0 g (6.7 mmol)
of 27 in 20 mL of DMF was added 1.8 mL (13.3 mmol) of methyl
isonipecotate and 3.5 mL (20.0 mmol) of N,N-diisopropylethyl-
amine. The mixture was stirred at ambient temperature for 2 h. It
was concentrated under high vacuum with mild heating, and the
resulting solid was recrystallized from MeOH to give 1.9 g (77%)
1
of a pale-yellow solid. H NMR (300 MHz, DMSO-d6) δ 9.53 (s,
1H), 7.18 (s, 1H), 6.97 (s, 2H), 4.27-4.34 (q, J ) 7.12 Hz, 2H),
3.61 (s, 3H), 3.14 (s, 2H), 2.78-2.82 (m, 2H), 2.20-2.31 (m, 3H),
1.81-1.86 (m, 2H), 1.58-1.66 (m, 2H), 1.27-1.32 (t, J ) 7.12
Hz, 3H); MS (ESI) m/e ) 362 (M + H)+, 360 (M - H)-.
The solid was taken up in MeOH (40 mL). Then a solution of
1.4 g (10.5 mmol) of K2CO3 in 10 mL of H2O was added. The
hydrolysis mixture was heated at 50 °C for 2 h. The solvents were
removed in vacuo. Then the residue was taken up in H2O and
acidified with 1 M HCl to pH 5. The precipitate was filtered, rinsed
with H2O, and dried on the filter to give 1.1 g (51%) of a white
1
solid. H NMR (300 MHz, DMSO-d6) δ 12.17 (s, 1H), 9.51 (s,
1H), 7.19 (s, 1H), 6.96 (s, 2H), 4.27-4.34 (q, J ) 7.12 Hz, 2H),
3.13 (s, 2H), 2.78-2.82 (m, 2H), 2.19-2.26 (m, 3H), 1.79-1.84
(m, 2H), 1.51-1.64 (m, 2H), 1.27-1.32 (t, J ) 7.12 Hz, 3H); MS
(ESI) m/e ) 348 (M + H)+, 346 (M - H)-.
1-[(4-Amino-5-cyano-6-ethoxypyridin-2-ylcarbamoyl)methyl]-
piperidine-4-carboxylic Acid Butylamide (33). To a solution of
32 (30 mg, 0.086 mmol) in DMF (0.4 mL) was added butylamine
(9 µL, 0.086 mmol), triethylamine (24 µL, 0.17 mmol), and
O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate
(TBTU) (41 mg, 0.13 mmol). After being stirred for 1 h at ambient
temperature, the mixture was diluted with H2O. Then the precipitate
was filtered. The crude solid was recrystallized from MeOH to give
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-butylaminoac-
etamide (28c). 28c was prepared according to the procecdure for
28a, substituting n-butylamine for N,N-diethylamine. The crude
product was recrystallized from methanol to give 15 mg (51%) of
a white solid. 1H NMR (300 MHz, DMSO-d6) δ 7.19 (s, 1H), 6.96
(s, 2H), 4.26-4.33 (q, J ) 6.78z, J ) 7.12 Hz, 2H), 3.32 (s, 2H),
3.25 (s, 2H), 1.35-1.41 (m, 4H), 1.27-1.31 (t, J ) 7.12 Hz, 3H),
0.85-0.90 (t, J ) 7.12 Hz, 3H); MS (ESI) m/e ) 292 (M + H)+,
290 (M - H)-. Anal. (C14H21N5O2‚0.22CH3OH) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-[(2-hydroxyeth-
yl)propylamino]acetamide (28d). 28d was prepared according to
the procedure for 28b, substituting N-(2-hydroxyethyl)-N-propyl-
amine for N,N-diethylamine. The crude product was recrystallized
1
20 mg (57%) of a white solid. H NMR (300 MHz, DMSO-d6) δ
9.47 (s, 1H), 7.68-7.72 (t, J ) 5.42 Hz, 1H), 7.19 (s, 1H), 6.97 (s,
2H), 4.27-4.34 (q, J ) 7.12 Hz, 2H), 3.12 (s, 2H), 2.99-3.05 (m,
2H), 2.83-2.89 (m, 2H), 2.05-2.19 (m, 3H), 1.60-1.64 (m, 4H),
1.22-1.39 (m, 7H), 0.83-0.88 (t, J ) 7.12 Hz, 3H); MS (ESI)
m/e ) 403 (M + H)+, 401 (M - H)-. Anal. (C20H30N6O3‚0.24CH3-
OH) C, H, N.
1
from methanol to give 10 mg (38%) of a white solid. H NMR
(300 MHz, DMSO-d6) δ 9.76 (s, 1H), 7.19 (s, 1H), 6.96 (s, 1H),
4.62 (s, 1H), 4.26-4.30 (q, J ) 7.01 Hz, 2H), 3.48 (s, 2H), 3.25
(s, 2H), 2.60-2.62 (t, J ) 5.80 Hz, 2H), 1.40-1.44 (q, J ) 7.01
Hz, 2H), 1.27-1.29 (t, J ) 7.02 Hz, 3H), 0.87-0.9 (t, J ) 7.32
Hz, 3H); MS (ESI) m/e ) 322 (M + H)+, 320 (M - H)-. Anal.
(C15H23N5O3) C, H, N.
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-piperidin-1-ylac-
etamide (29). 29 was prepared according to the procedure for 28a,
substituting piperidine for N,N-diethylamine. Recrystallization from
methanol gave 15 mg (49%) of a white solid. 1H NMR (300 MHz,
DMSO-d6) δ 9.51 (s, 1H), 7.18 (s, 1H), 6.98 (s, 2H), 4.30 (q, J )
7.1 Hz, 2H), 3.09 (s, 2H), 2.46 (m, 4H), 1.53 (m, 4H), 1.41 (m,
2H), 1.30 (t, J ) 7.0 Hz, 3H); MS (ESI) m/e ) 304 (M + H)+.
Anal. (C15H21N5O2‚0.26CH3OH) C, H, N.
1-[(4-Amino-5-cyano-6-ethoxypyridin-2ylcarbamoyl)methyl]-
piperidine-4-carboxylic Acid Isopropyl Amide (34). 34 was
prepared according to the procedure for 33, substituting isopropyl-
amine for n-butylamine. The crude solid was recrystallized from
MeOH to give 17 mg (51%) of a white solid. 1H NMR (300 MHz,
DMSO-d6) δ 9.47 (s, 1H), 7.58 (d, J ) 7.80 Hz, 1H), 7.19 (s, 1H),
6.97 (s, 2H), 4.27-4.34 (q, J ) 7.12 Hz, 2H), 3.77-3.84 (m, 1H),
3.12 (s, 2H), 2.84-2.89 (m, 2H), 2.11-2.18 (m, 2H), 2.02-2.08
(m, 1H), 1.56-1.64 (m, 4H), 1.28-1.32 (t, J ) 7.12 Hz, 3H), 1.02
(d, J ) 6.78 Hz, 6H); MS (ESI) m/e ) 389 (M + H)+, 387 (M -
H)-. Anal. (C19H28N6O3) C, H, N.
N-(4-Amino-5-chloro-6-ethoxypyridin-2-yl)-2-(4-methane-
sulfonyl-2,5-dimethoxyphenyl)acetamide (35). To a solution of
50 mg of crude 38 in 1 mL of CH2Cl2 and 0.3 mL of pyridine at
-78 °C was added a solution of 53 mg (0.18 mmol) of acid chloride
11 in 1 mL of CH2Cl2. The mixture was stirred at -78 °C for 20
min. Then 2 mL of water was added, and the mixture was allowed
N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-morpholin-4-
ylacetamide (30). 30 was prepared according to the procedure for
28a, substituting morpholine for N,N-diethylamine. Recrystallization

Aminopyridine-Based JNK inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3577
to warm until the ice melted. The solvents were removed in vacuo,
and then the residue was taken up in 20 mL of diethyl ether. The
solution was extracted with water (1 × 5 mL), 1 M HCl(aq) (2 × 5
mL), saturated NaHCO3(aq) (2 × 5 mL), and brine (1 × 5 mL),
dried over MgSO4, filtered, and concentrated to a solid. This was
recrystallized from methanol to give 30 mg (38%) of a white solid.
1H NMR (300 MHz, DMSO-d6) δ 9.98 (s, 1H), 7.30 (s, 1H), 7.26
(s, 1H), 7.14 (s, 1H), 6.20 (s, 2H), 4.31 (q, 2H, J ) 7.1 Hz), 3.89
(s, 3H), 3.79 (s, 2H), 3.77 (s, 3H), 3.24 (s, 3H), 2.50 (t, 3H, J )
7.1 Hz); MS (ESI) m/e ) 444 (M + H)+, 442 (M - H)-. Anal.
(C18H22ClN3O6S) C, H, N.
was washed with 1 M HCl(aq) (1 × 30 mL), saturated NaHCO3(aq)
(1 × 30 mL), and brine (2 × 30 mL), dried over MgSO4, filtered,
and concentrated to give 22 mg (100%) of the amide as a yellow
1
solid. H NMR (300 MHz, DMSO-d6) δ 10.62 (s, 1H), 7.69 (s,
1H), 4.42 (q, J ) 7.1 Hz, 2H), 2.41 (s, 3H), 2.12 (s, 3H), 1.34 (t,
J ) 7.1 Hz, 3H); MS (ESI) m/e ) 220 (M + H)+. Anal.
(C11H13N3O2‚0.15EtOAc) C, H, N.
Pentanoic Acid (4-Amino-3-chloro-5-cyano-6-ethoxypyridin-
2-yl)amide (41). To a solution of 20 mg (0.076 mmol) of 6b in 1
mL of CH2Cl2 was added 100 µL (0.10 mmol) of SO2Cl2 in CH2-
Cl2. The mixture was stirred at ambient temperature for 10 min.
Then 1 mL of 5% NaHSO3(aq) was added, and the CH2Cl2 was
removed in vacuo. The residue was taken up in ethyl acetate (10
mL), extracted with water (1 × 5 mL) and then brine (1 × 5 mL),
dried over MgSO4, filtered, and concentrated. The product was
purified via reversed-phase HPLC (5-95% CH3CN in NH4OAc
4-Amino-6-ethoxypyridine-2-carboxylic Acid Methyl Ester
(37). To 1.08 g (6.6 mmol) of 4-amino-2,6-dichloropyridine (36)
in 1.5 mL of EtOH was added 2.7 mL (6.6 mmol) of 2.5 M EtONa
in EtOH. The mixture was heated in a sealed tube at 150 °C with
an oil bath for 6 h. Water was added, and the mixture was extracted
with EtOAc. The extracts were concentrated and purified via MPLC,
eluting with a gradient of 10-30% ethyl acetate in hexanes to give
1
gradient) to give 12 mg (53%) of a white solid. H NMR (300
MHz, DMSO-d6) δ 9.85 (s, 1H), 7.17 (s, 2H), 4.30 (q, J ) 7.0 Hz,
2H), 2.37 (t, J ) 7.5 Hz, 2H), 1.45-1.68 (m, 2H), 1.21-1.44 (m,
5H), 0.88 (t, J ) 7.3 Hz, 3H); MS (ESI) m/e ) 297 (M + H)+.Anal.
(C13H17ClN4O2‚0.2H2O) C, H, N.
1
1.0 g (88%) of the ethyl ether. H NMR (300 MHz, DMSO-d6) δ
6.24 (broad s, 2H), 6.19 (s, 1H), 5.76 (s, 1H), 4.13 (q, J ) 7.2 Hz,
2H), 1.23 (t, J ) 7.2 Hz, 3H). To a solution of 3.0 g (17 mmol) of
the ethyl ether in 20 mL of MeOH was added 360 mg (0.44 mmol)
of PdCl2(CH2Cl2)dppf and then 4.9 mL (35 mmol) of Et3N. The
mixture was heated at 100 °C under 100 psi of CO for 6 h. The
solvent was removed in vacuo. Then the crude product was purified
via MPLC, eluting with a gradient of 10-40% ethyl acetate in
Pentanoic Acid (4-Amino-5-cyano-6-ethoxypyridin-2-yl)meth-
ylamide (42). To a solution of 25 mg (0.095 mmol) of 6b in 1 mL
of THF was added 4 mg (0.10 mmol) of 60% NaH in mineral oil.
The mixture was stirred at ambient temperature for 5 min, and then
12 µL (0.19 mmol) of methyl iodide was added. The mixture was
stirred at ambient temperature for 18 h and then concentrated in
vacuo. The residue was taken up in 10 mL of diethyl ether and
then extracted with water (2 × 3 mL) and brine (1 × 3 mL), dried
over MgSO4, filtered, and concentrated to a solid. The product was
purified via silica gel chromatography, eluting with 40% ethyl
acetate in hexanes to give 6.7 mg (26%) of a white solid. 1H NMR
(300 MHz, DMSO-d6) δ 7.06 (s, 2H), 6.39 (s, 1H), 4.30 (q, J )
7.1 Hz, 2H), 3.17 (s, 3H), 2.39 (t, J ) 7.6 Hz, 2H), 1.42-1.56 (m,
2H), 1.16-1.36 (m, 5H), 0.83 (t, J ) 7.3 Hz, 3H); MS (ESI)
m/e ) 277 (M + H)+. Anal. (C14H20N4O2) C, H, N.
1
hexanes to give 2.4 g (72%) of methyl ester 37. H NMR (300
MHz, DMSO-d6) δ 6.96 (s, 1H), 6.22 (broad s, 2H), 5.95 (s, 1H),
4.21 (q, J ) 7.2 Hz, 2H), 3.78 (s, 3H), 1.26 (t, J ) 7.2 Hz, 3H).
To a solution of 400 mg (2.0 mmol) of 37 in 4 mL of DMF was
added N-chlorosuccinimide (267 mg, 2.0 mmol). The mixture was
stirred at ambient temperature for 3 days and then diluted with
water. The precipitate was collected and purified via MPLC, eluting
with a gradient of 10-30% ethyl acetate in hexanes to give 220
mg (48%) of 4-amino-5-chloro-6-ethoxypyridine-2-carboxylic acid
methyl ester. 1H NMR (300 MHz, DMSO-d6) δ 7.17 (s, 1H), 6.55
(broad s, 2H), 4.33 (q, J ) 7.2 Hz, 2H), 3.80 (s, 3H), 1.31 (t, J )
7.2 Hz, 3H).
N-(6-Amino-5-cyano-4-ethoxypyridin-2-yl)acetamide (43). To
a solution of 210 mg (0.893 mmol) of nicotinamide 45 in 2.5 mL
of pyridine was added 61 mg (0.89 mmol) of imidazole. The
mixture was cooled with an ice bath. Then 170 µL (1.8 mmol) of
POCl3 was added, and stirring was continued for 1 h. The solvent
was removed in vacuo, and then the residue was taken up in 10
mL of 1 M HCl(aq) and extracted with diethyl ether (3 × 5 mL).
The combined ether layers were back-extracted with H2O (1 × 5
mL) and then brine (1 × 5 mL), dried over MgSO4, filtered, and
concentrated in vacuo to a solid. This was purified via MPLC,
eluting with 30% ethyl acetate in hexanes to give 99 mg (51%) of
2,6-dichloro-4-ethoxynicotinonitrile as a solid. 1H NMR (300 MHz,
DMSO-d6) δ 7.61 (s, 1H), 4.39 (q, J ) 7.1 Hz, 2H), 1.38 (t, J )
7.1 Hz, 3H). To 150 mg (0.69 mmol) of the nitrile in 1 mL of
dioxane was added 15 mL of 28% NH4OH. The mixture was heated
in an autoclave at 130 °C for 24 h. The solvents were removed in
vacuo. Then the residue was taken up in 10 mL of ethyl acetate
and extracted with H2O (1 × 3 mL) and then brine (1 × 3 mL),
dried over MgSO4, filtered, and concentrated in vacuo. The product
was purified via MPLC, eluting with a 60-100% ethyl acetate/
5-Chloro-6-ethoxypyridine-2,4-diamine (38). To 231 mg (1.00
mmol) of the methyl ester was added 5 mL of 7 M NH3 in CH3-
OH. The solution was stirred at ambient temperature for 5 h and
then concentrated in vacuo to give the amide as a solid (215 mg,
100%). To a suspension of 186 mg (0.866 mmol) of the amide in
3 mL of 2-propanol was added 2.6 mL (5.2 mmol) of 2 M NaOH(aq)
and then 2 mL (1.64 mmol) of 0.82 M NaOCl(aq). The suspension
cleared upon addition of NaOCl, and stirring was continued for 2
h at ambient temperature. The solvents were removed in vacuo.
Then the residue was taken up in 5 mL of water and filtered through
a short plug of diatomaceous earth. The filter pad was washed with
water (3 × 1 mL). Then 1 M HCl was added to the combined
filtrate and washings to adjust to pH 1. The pH was brought to
>13 with 2 M NaOH(aq), and then the aqueous suspension was
extracted with ethyl acetate (3 × 5 mL). The combined organic
layers were back-extracted with brine (1 × 5 mL), dried over
MgSO4, filtered, and concentrated to 93 mg of an impure solid.
This was used without further purification for subsequent acylations.
1H NMR (300 MHz, DMSO-d6) δ 5.38 (s, 2H), 5.23 (d, 1H, J )
1.7 Hz), 5.18 (s, 2H), 5.13 (d, 1H, J ) 1.7 Hz), 4.05 (q, 2H, J )
7.1 Hz), 1.19 (t, 3H, J ) 7.1 Hz); MS (ESI) m/e ) 154 (M + H)+.
N-(5-Cyano-6-ethoxy-4-methylpyridin-2-yl)acetamide (39).
6-Amino-2-chloro-4-methylnicotinonitrile (40)33 (84 mg, 0.50
mmol) was subjected to the procedure described for the preparation
of 2 to provide 58 mg (65%) of 6-amino-2-ethoxy-4-methylnico-
tinonitrile. 1H NMR (300 MHz, DMSO-d6) δ 6.88 (s, 2H), 5.95 (s,
1H), 4.30 (q, J ) 7.1 Hz, 2H), 2.20 (s, 3H), 1.28 (t, J ) 7.1 Hz,
3H); MS (ESI) m/e ) 178 (M + H)+. To a solution of 18 mg
(0.10 mmol) of 6-amino-2-ethoxy-4-methylnicotinonitrile in 0.5 mL
of CH2Cl2 was added 0.2 mL of pyridine and then 21 µL (0.3 mmol)
of acetyl chloride. The mixture was stirred at ambient temperature
for 15 min. Then brine (20 mL) was added, and the mixture was
extracted with ethyl acetate (1 × 50 mL). The ethyl acetate layer
1
hexanes gradient to give 37 mg (29%) of a white solid. H NMR
(300 MHz, DMSO-d6) δ 6.35 (s, 2H), 6.07 (s, 2H), 5.39 (s, 1H),
4.01 (q, J ) 7.1 Hz, 2H), 1.31 (t, J ) 7.1 Hz, 3H); MS (ESI)
m/e ) 235 (M + H)+. To 10 mg (0.056 mmol) of 2,6-diamino-4-
ethoxynicotinonitrile in 0.2 mL of pyridine was added 3 drops of
acetyl chloride via pipet, while cooling with an ice bath. The mixture
was stirred for 10 min and then diluted with 1 mL of H2O to give
a white precipitate. The solvents were decanted, and the solid was
washed with 2 × 1 mL of H2O, dried, and then recrystallized from
methanol to give 3 mg (24%) of a white solid. 1H NMR (500 MHz,
DMSO-d6) δ 10.25 (s, 1H), 7.20 (s, 1H), 6.47 (s, 2H), 4.11 (q, 2
H, J ) 7.1 Hz), 2.07 (s, 3H), 1.33 (t, 3H, J ) 7.1 Hz); MS (ESI)
m/e ) 221 (M + H)+. Anal. (C10H12N4O2‚0.15CH3OH) C,H,N.
2,6-Dichloro-4-ethoxynicotinamide (45). To 1.82 g (10 0.0
mmol) of 2,4,6-trichloropyridine (44)34 was added 6 mL of ethanol

3578 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Szczepankiewicz et al.
and then 4 mL (10.2 mmol) of 2.54 M KOH in ethanol. The mixture
was stirred at reflux for 30 min and then concentrated in vacuo.
The residue was taken up in 10 mL of water and extracted with
diethyl ether (2 × 10 mL). The combined ether layers were back-
extracted with brine (1 × 10 mL), dried over MgSO4, filtered, and
concentrated to an oil. This was purified via silica gel chromatog-
raphy, eluting with 2.5% ethyl acetate/hexanes to recover the less
polar 2-ethoxy isomer and then with 5% ethyl acetate/hexanes to
give 750 mg (39%) of 2,6-dichloro-4-ethoxypyridine. 1H NMR (300
MHz, DMSO-d6) δ 7.17 (s, 2H), 4.20 (q, J ) 7.0 Hz, 2H), 1.32 (t,
J ) 7.0 Hz, 3H); MS (ESI) m/e ) 191 (M + H)+. To a solution of
450 mg (2.34 mmol) of 2,6-dichloro-4-ethoxypyridine in 8 mL of
THF at -78 °C under N2 was added 2 mL of 1.6 M n-butyllithium
in hexanes. The mixture was stirred at -78 °C for 10 min, and
then CO2 gas was introduced into the mixture while warming to
ambient temperature. The solvent was removed in vacuo, and then
the residue was taken up in 10 mL of water and extracted with
diethyl ether (2 × 10 mL). The aqueous layer was made acidic by
addition of 5 mL of 1 M HCl(aq). Then it was extracted with ethyl
acetate (3 × 10 mL). The combined ethyl acetate layers were back-
extracted with brine (1 × 10 mL), dried over MgSO4, filtered, and
concentrated to an oil. (The oil contained a mixture of 2,6-dichloro-
4-ethoxynicotinic acid and 2,6-dichloro-4-ethoxypyridine-3,5-di-
carboxylic acid, which was used without purification.) The oil was
taken up in 2 mL of thionyl chloride, and the mixture was heated
at reflux for 30 min. Then the excess thionyl chloride was removed
in vacuo. The residue was taken up in 2 mL of THF, and then 5
mL of 28% NH4OH(aq) was added. The mixture was stirred at
ambient temperature for 30 min, and then 10 mL of water was
added. The suspension was extracted with ethyl acetate (3 × 10
mL). Then the combined ethyl acetate layers were back-extracted
with brine (1 × 10 mL), dried over MgSO4, filtered, and
concentrated to an oily solid. This was suspended in ethyl acetate
and filtered to remove the insoluble diamide. Concentration of the
filtrate gave 210 mg (38%) of nicotinamide 45 as a white solid. 1H
NMR (300 MHz, DMSO-d6) δ 7.91 (s, 1H), 7.69 (s, 1H), 7.34 (s,
1H), 4.22 (q, J ) 7.1 Hz, 2H), 1.30 (t, J ) 7.1 Hz, 3H); MS (ESI)
m/e ) 235 (M + H)+.
ATF-2 Phosphorylation Assay. JNK-1, JNK-2, and JNK-3 were
purchased from Upstate Cell Signaling Solutions (formerly Upstate
Biotechnology).47 Kinase reactions were performed in a 50 µL
volume containing 10 ng/well JNK-1 (4.4 nM), 10 ng/well JNK-2
(4.1 nM), or 10 ng/well (3.8 nM) JNK-3, 1 µM BT-GST-ATF-2
substrate, and γ- [33P]-ATP (5 µM, 400 µCi/µmol) in a buffer
containing 20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl2,
0.1% Triton X-100, and 1 mM dTT in a 96-well polypropylene
plate. Reactions were carried out at 15-20 °C and stopped after
60 min with the addition of EDTA, giving a final concentration of
50 mM. The 30 µL aliquots of the quenched reactions were
transferred to a Streptavidin FlashPlate containing 170 µL of PBS.
The plate was incubated at ambient temperature for 30 min. The
plate was then washed 4 times with PBS. The plate was then
counted in a Perkin-Elmer Micro-Beta plate counter, 1 min per well.
Kinase Selectivity Assays. Recombinant protein kinases were
either commercially obtained or expressed using the FastBac
bacculovirus expression system (GIBCO BRL, Gaithersburg, MD)
and purified using either nickel (his-tag) or glutathione (GST)
affinity chromatography. Kinase assays were conducted in 24 µL
volumes on 384-well microplates using TECAN liquid-handling
automation. Ser/Thr-kinase assays were performed using a radioac-
tive FlashPlate-based assay platform.48 In this format, biotinylated
substrate peptide (2 µM), γ-[33P]-ATP (5 µM, 2 mCi/µmol),
inhibitors (3-10000 nM in 2% DMSO), and enzyme were
incubated for 1 h in buffer containing 25 mM Hepes, pH 7.5, 1
mM DTT, 10 mM MgCl2, 100 µM Na3VO4, and 0.075 mg/mL
Triton X-100, stopped with 80 µL of stop buffer containing 100
mM EDTA and 4 M NaCl, transferred to streptavidin-coated 384-
well FlashPlates (Perkin-Elmer, Boston, MA), which were then
washed 3 times and read using a TopCount microplate reader
(Perkin-Elmer). Tyr kinase reactions were performed using a time-
resolved fluorescence (HTRF) platform. In this format, biotinylated
substrate peptide (0.5 µM), ATP (10 µM to 1 mM), inhibitors (3-
10000 nM in 2% DMSO), and enzyme were incubated for 1 h in
buffer containing 50 mM Hepes, pH 7.4, 1 mM DTT, 10 mM
MgCl2, 2 mM MnCl2, 100 µM Na3VO4, and 0.01% BSA. Reactions
were stopped with 50 µL of revelation buffer containing (final
concentrations of) Eu-conjugated anti-pY-PT66 antibody (0.05 µg/
mL) (CisBio), PycolLink Streptaviding-APC (0.001 µg/mL)
(Prozyme), and 60 mM EDTA in a buffer containing 25 mM Hepes,
pH 7.4, 250 mM KF, 0.005% Tween-20, and 0.05% BSA.
Following 60 min of incubation with revelation buffer, the reactions
were read using an Envision fluorescence microplate reader (Perkin-
Elmer) with 615 nm excitation and 665 nm emission. IC50 values
were determined from six compound titration curves, and corre-
sponding Ki values were calculated using the Cheng-Prusoff
equation Ki ) IC50/(1 + ([ATP]/Km)).49
Cellular Assay (c-Jun Phosphorylation). HepG2 human hepato-
ma cells (ATCC) were cultured in low glucose MEM supplemented
with 1× NEAA, 1× sodium pyruvate, and 10% FBS (all from
Invitrogen, “complete media”). For P-c-Jun assays, cells were plated
at 5 × 104 cells/well in 500 µL of complete media on 24-well
collagen-coated plates and incubated overnight. Serial compound
dilutions were made in DMSO at 100×, and then 5 µL was added
directly to the media on the cells to provide the final inhibitor
concentrations (final DMSO concentration in media was 1%). After
1 h, cells were stimulated with vehicle control or TNFR for 30
min and harvested in 70 µL of lysis buffer (TBS (54 mM Tris-
HCl, pH 7.6, 150 mM NaCl, Sigma), 1% TritonX-100 (Sigma),
0.5% Nonidet P-40 (Sigma), 0.25% sodium deoxycholate (Sigma),
1 mM EDTA, 1 mM EGTA (Sigma), 0.5 mM sodium fluoride
(Sigma), 1 mM pervanadate (Sigma), 1 µM microcystin (Calbio-
chem), 1 mM AEBSF (Roche), 1 tablet of complete EDTA Free-
Mini inhibitor cocktail (Roche)) and frozen at -80 °C prior to use
in the P-c-Jun assay. P-c-Jun ELISAs were performed as described
by the manufacturer (Cell Signaling Technology) using 50 µL of
cell extract.
X-ray Crystallography. Purified JNK-1-R1 isoform50 with a
C-His-tag extension was incubated with a 5× molar excess of the
JIP-1 peptide.36 The crystallization experiments were in the hanging
drop vapor diffusion format at 4 °C. Hanging drops consisted of 2
µL of the protein/peptide solution plus 2 µL of well solution over
1 mL of reservoir. The reservoir contained 3 M (NH4)SO4 (2.8-
3.1 M range) and 10-14% glycerol at a pH between 5.8 and 7.0.
Cocrystallization with compound 6t was achieved by dissolving
DMSO stock solutions of 6t (100 or 50 mM) into the protein/peptide
solution in a 3-fold molar excess. The protein/JIP-1/compound 6t
complex was allowed to incubate for at least an hour on ice and
was later set up to crystallize. Crystals grew in 4-7 days. Large
crystals of the complex had the morphology of hexagonal bipyra-
mids.
The structure was solved by molecular replacement using a model
of residues 63-373 of the PDB entry 1jnk, corresponding to the
structure of JNK-3. Data were collected at the IMCA-CAT facility
using a Quantum210 detector and consisted of 100 1° frames with
an exposure per frame of 5 s. Data were collected with the standard
station software, processed with HKL2000, and the structure
solution and refinement were done using CNX and QUANTA. The
coordinates for the structure of the refined JNK-1/JIP-1/compound
6t complex have been deposited with the RCSB collaborative (PDB)
with entry code 2GMX.
Acknowledgment. We thank David Beno and the R4EK
staff for pharmacokinetic studies, the R46Y protein expression/
purification group for providing the JNK-1R used in these
studies, and Peter Dandliker, Candace Black-Shaefer, and Linda
Chovan for membrane permeability and microsomal incubation
studies. We also thank Kent Stewart for helpful discussions
regarding kinase inhibition modes and selectivity.

Aminopyridine-Based JNK inhibitors
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Supporting Information Available: Substrates, assay formats,
and constructs for kinases in Table 5; elemental analysis data for
compounds 6a-u, 7, 8, 10, 17, 18a,b, 19a-e, 20a,b, 21-26, 28a-
d, 29-31, 33-35, 39, and 41-43. This material is available free
of charge via the Internet at http://pubs.acs.org.
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