Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.03.023

The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. O

Full text of DOI:10.1016/j.bmcl.2009.03.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2099–2102
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun
N-terminal kinase (JNK) inhibitors
*
*
Paul S. Humphries , Jennifer A. Lafontaine , Charles S. Agree, David Alexander, Ping Chen,
Quyen-Quyen T. Do, Lilian Y. Li, Elizabeth A. Lunney, Ranjan J. Rajapakse, Karen Siegel,
Sergei L. Timofeevski, Tianlun Wang, David M. Wilhite
Pfizer Global R&D, 10770 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal
kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for
a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c,
whose kinase selectivity is reported.
Received 24 January 2009
Accepted 9 March 2009
Available online 13 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
JNK
c-Jun N-terminal kinase
Diabetes, pyrimidine, pyrazole
Kinase selectivity
Type 2 diabetes is a metabolic disorder that accounts for 120
million patients worldwide and the number is likely to grow to
greater than 366 million by the year 2030.1 Patients with type 2
diabetes are insulin resistant, a condition in which the body fails
to respond to insulin properly.
The c-Jun N-terminal protein kinases (JNKs) are a family of ser-
ine/threonine protein kinases and members of the mitogen-acti-
vated protein kinase (MAPK) family. JNK1 has recently emerged
as an attractive target for diabetes therapy, since JNK1 is believed
to play a key role in linking obesity and insulin resistance.2 JNK1
disrupts the insulin signaling cascade via phosphorylation of the
insulin receptor substrate (IRS-1) at serine307, which leads to the
degradation of IRS-1. JNK1ꢀ/ꢀ mice show marked reduction in both
plasma glucose and insulin concentrations relative to their wild-
type littermates, and thus are protected from diet-induced obes-
ity.2b In addition, JNK1 activity is elevated in adipocytes of type 2
diabetic patients.3 Inhibitors of JNK1 can potentially increase insu-
lin sensitivity, and therefore could be useful as therapeutics for the
treatment of type 2 diabetes. Therefore, considerable efforts have
been directed toward the identification of JNK1 inhibitors suitable
for clinical development.4
Figure 1. Initial hit from high-throughput screening.
competitive JNK1 inhibitor. This hit was extremely attractive based
on it’s high ligand efficiency,6 and promising selectivity against an
initial panel of kinases. JNK1 inhibitors need to be cell permeable
to reach the intracellular targets, therefore a phospho c-Jun cell-
based ELISA assay was also utilized to test the compounds cellular
potency.7 In this assay, compound 1 was moderately active with an
IC50 of 460 nM. The above properties motivated us to initiate hit-
to-lead chemistry to explore the activity of this class of compounds
as JNK1 inhibitors.
We wish to report here our efforts toward the identification of
4-substituted-2-aminopyrimidines as novel ATP competitive,
pan-JNK inhibitors.5 A high-throughput screening (HTS) of the Pfiz-
er compound collection identified compound 1 (Fig. 1) as an ATP-
Unfortunately, 1 was rapidly cleared in vitro (human liver
microsomes (HLM) Cl = 116 lL/min/kg). Metabolite ID studies re-
vealed that metabolism was occurring exclusively on the alkyl-
amino portion of the molecule. Our strategy was thus to
dramatically reduce the lipophilicity (ELogD = 4.42) of this hit,
* Corresponding authors. Tel.: +1 860 705 0559 (P.S.H.).
E-mail address: phumphri@gmail.com (P.S. Humphries).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.023

2100
P. S. Humphries et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2099–2102
Table 1
while maintaining or improving the ligand efficiency (LE = 0.44).
More specifically, we wished to expediently and efficiently vary
the alkylamino portion of the molecule.
Enzymatic and cellular activity of JNK inhibitors 1 and 7
Compound
R
JNK1 Ki (nM) Pc-Jun IC50 (nM) HLM (lL/min/kg)
The final list of compounds for synthesis was chosen by taking a
list of ꢁ1500 potential virtual compounds (potential products uti-
2b
7b
7c
1
Me
2060
48
151
26
NT
81
106
96
116
156
iPr
920
840
460
1100
c-C3H5
c-C5H9
c-C6H11
lizing commercially available primary amines) and applying the
0
following filters–cLogD < 3, PSA < 140 ÅA2, MW < 450, Pass Ro58
and predicted to be stable in HLM (calculation based on in-house
HLM data). The resulting ꢁ200 potential virtual compounds were
then docked (utilizing proprietary in-house software) into a co-
crystal structure of compound 1 with JNK1 and the top 50 com-
pounds chosen for synthesis.
7d
27
7e
7f
315
NT
NT
<6
14
1570
The initial route to these targets was via the 2-methylsulfonyl-
pyrimidine intermediate 6.9 This intermediate was accessed in a
straight forward fashion following the four step protocol below
(Scheme 1). 2-Mercapto-4-methyl pyrimidine 2 was alkylated with
iodomethane to afford 2-mercapto-4-methyl pyrimidine 3.10
Deprotonation and reaction of the anion of 3 with methyl 4-chlo-
robenzoate 4 gave ketone 5 in good yield.10 Ketone 5 was treated
with dimethylformamide dimethylacetal followed by hydrazine
hydrate yielding the required pyrazole.11 Oxidation of the methyl-
sulfide intermediate to the required methylsulfonylpyrimidine 6
was achieved in a straightforward fashion.12
Allbiological data aremeanvalues ofmultiple experimentsand thus shows thereader
that these have a higher accuracy than just providing data from one experiment.
NT, not tested.
crease the reactivity of the pyrimidine electrophile by choosing
to access these final targets via the 2-chloropyrimidine intermedi-
ate, which was accessed via a similar synthetic sequence as before.
This route provided access to the majority of the remaining ꢁ50
compounds assigned for synthesis.
The final SNAr step was optimized under a variety of conditions,
utilizing cyclopentylamine as our partner of choice.13 Initially it
was found that performing this reaction in dioxane under micro-
wave irradiation (137 °C, 10 min) afforded the required product
(7, where R = cyclopentyl) in a 77% yield. Unfortunately, when
using this protocol with a small test set of amines and amine
hydrochlorides, it was immediately apparent that heterogeneity
hindered the reaction progress in some instances. Further optimi-
zation showed that this reaction could be performed in 2-propanol
(175 °C, 10 min) with no diminution in yield. Due to the enhanced
solubility of the reagents and the higher reaction temperature, this
optimized protocol showed greater generality over a more diverse
set of amine monomers.
All the compounds were tested in our JNK1 enzymatic inhibi-
tion assay as well as a cell-based assay measuring inhibition of
TNFa
stimulated phosphorylation of c-Jun in HepG2 cells.7b The ef-
Table 2
Enzymatic and cellular activity of JNK inhibitors 8
Unfortunately, a number of amine monomers failed to give any
pure final target, due to either their lack of reactivity (presumably
due to steric hindrance and/or unfavorable electronics) or instabil-
ity under the reaction conditions. We therefore attempted to in-
Compound
R
JNK1 Ki
(nM)
Pc-Jun IC50
(nM)
HLM (
kg)
lL/min/
8a
H
2730
381
NT
21
21
8b
3500
8c
43
357
814
34
18
8d
169
8e
530
NT
14
8f
101
162
221
12
61
8g
6020
Scheme 1. Reagents and conditions: (a) NaOH, H2O, EtOH, MeI, rt, 16 h, 95%; (b)
LiHMDS, THF, 0 °C, 2 h, 89%; (c) (MeO)2CHNMe2, PhMe, reflux, 16 h then
Allbiological data aremeanvalues ofmultiple experimentsand thus shows thereader
that these have a higher accuracy than just providing data from one experiment.
NT, not tested.
i
H2NNH2ꢂH2O, EtOH, rt, 2 h, 84%; (d) mCPBA, CH2Cl2, rt, 16 h, 84%; (e) RNH2, PrOH,
lW, 175 °C, 30 min.

P. S. Humphries et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2099–2102
2101
Table 4
fect of different alkylamino substitution was studied first, and
Kinase selectivity profile of JNK inhibitor 9c
these results are summarized in Table 1. Attempts to reduce the
microsomal clearance by reducing MW either led to lower enzy-
matic potency (e.g., 7a) or no significant reduction in clearance
(e.g., 7b). Alternatively, reduction in lipophilicity led to improved
microsomal clearance, but at the expense of enzymatic potency
(e.g., 7e and 7f), reduced in vitro permeability (e.g., 7e–CaCo-2
AB = 0.3 and BA = 13.2 ꢃ 10ꢀ6 cm/s) or increased hERG affinity
(e.g., 7f–Dofetilide Ki = 1020 nM). It is also interesting to note that
a drop-off in potency was observed in moving from the JNK1 enzy-
matic assay to the cellular c-Jun assay. In some cases (e.g., 7c–
CaCo-2 AB = 28.5 and BA = 15.0 ꢃ 10ꢀ6 cm/s) this was not due to
poor in vitro permeability.
The above SAR indicated that the 4-chlorophenyl moiety was so
lipophilic that charged and/or extremely polar aminoalkyl substit-
uents were required to obtain reasonable LogD values, thus com-
promising permeability or increasing hERG affinity. It was thus
decided that alternatives to the initial 4-chlorophenyl moiety were
required, which was achieved using synthetic schemes similar to
the one shown in Scheme 1.
As summarized in Table 2, successful replacements for the 4-
chlorophenyl moiety were obtained. Dramatic reduction in MW
and lipophilicity led to a large reduction in enzymatic potency
(e.g., 8a), but with much improved microsomal clearance. Of the
tetrahydropyran isomers tested, 8c was the most potent, and
microsomal clearance was still good. Interestingly, sulfonamide
8f displayed moderate enzymatic potency, only a small drop-off
in cellular potency and low clearance.
Kinase
IC50
(
l
M)
Kinase
IC50
(
l
M)
Kinase
IC50 (lM)
JNK1
JNK2
JNK3
CDK2/cyclinA
MKK7b
MKK4
0.012a
0.017a
0.016a
0.59
0.86
0.92
GSK3b
SAPK3
IKKb
MAPK2
MKK6
SAPK2b
AMPK
1.9
6.2
13
13
15
IR
>30
>30
>30
>30
>30
>30
>30
MAPKAP-K2
MEK1
p70S6K
PDK1
15
>30
PKCH
SAPK4
1.2
SAPK2a
a
Values are inhibitor constant Ki.
substituents led to good enzymatic potency (e.g., 9a and 9b), but
increased microsomal clearance. Cyclohexanol moieties provided
good enzymatic and cellular potency along with reduced clearance
(e.g., 9c). Cyclohexylamine substituents also provided a good bal-
ance of enzymatic and cellular potency with low clearance (e.g.,
9e).
The selectivity data of 9c against a panel of protein and lipid ki-
nases are listed in Table 4. This compound is a pan-JNK inhibitor
with an estimated margin of at least 100-fold over other MAP ki-
nases (MKK1, MKK4, MKK7b and p38MAPKacd), >27-fold over gly-
cogen synthase kinase-3b, and 65-fold over cyclin-dependent
kinase-2.
JNK inhibitor 9c attenuated the effects of elevated fatty acids on
induction of insulin resistance both in vitro and in vivo and the re-
sults of this have been reported elsewhere.7b
In summary, we have identified a series of 4-substituted-2-
aminopyrimidines as potent JNK inhibitors with good cellular
activity. Optimization of in vitro clearance was achieved by a
reduction in lipophilicity and also by more evenly distributing
polarity throughout the molecule. This exercise led to the identifi-
cation of 9c, which showed good selectivity across a panel of di-
verse protein and lipid kinases. Further work demonstrating the
utility of this tool compound for treating diabetes and/or obesity
has been reported previously.7b
At this point, we chose to fix the 3-tetrahydropyranyl moiety
(as seen in 8c), due to it’s improved rat microsomal (RLM) clear-
ance vs sulfonamide 8f (RLM Cl = 18 lL/min/mg for 8c and 52 lL/
min/mg for 8f). The effect of different alkylamino substitution
was once again studied with this new less lipophilic 3-tetrahydro-
pyran moiety, and these results are summarized in Table 3. Alkyl
Table 3
Enzymatic and cellular activity of JNK inhibitors 8c and 9
Acknowledgments
The authors gratefully acknowledge all our colleagues in the
JNK1 program for their technical support in the evaluation of the
compounds presented in this Letter. We also thank John Tatlock
and Joseph Warmus for stimulating discussions and feedback on
this Letter.
References and notes
Compound
R
JNK1 Ki
(nM)
Pc-Jun IC50
(nM)
HLM (
kg)
lL/min/
1. Wild, S.; Roglic, G.; Green, A.; Sicree, R.; King, H. Diabetes Care 2004, 27, 1047.
2. (a) Aguirre, V.; Uchida, T.; Yenush, L.; Davis, R.; White, M. F. J. Biol. Chem. 2000,
275, 9047; (b) Hirosumi, J.; Tuncman, G.; Chang, L.; Görgün, C. Z.; Uysal, K. T.;
Maeda, K.; Karin, M.; Hotamisligil, G. S. Nature 2002, 420, 333; (c) Bennett, B. L.;
Satoh, Y.; Lewis, A. J. Curr. Opin. Pharmacol. 2003, 3, 420; (d) Wellen, K. E.;
Hotamisligil, G. S. J. Clin. Invest. 2005, 115, 1111.
8c
9b
iPr
c-C5H9
43
12
357
245
40
89
9c
12
98
<15
3. Waeber, G.; Delplanque, J.; Bonny, C.; Mooser, V.; Steinmann, M.; Widmann, C.;
Maillard, A.; Miklossy, J.; Dina, C.; Hani, E. H.; Vionnet, N.; Nicod, P.; Boutin, P.;
Froguel, P. Nat. Genet. 2000, 24, 291.
4. (a) Rckle, T.; Biamonte, M.; Grippi-Valloton, T.; Arkinstall, S.; Cambet, Y.;
Camps, M.; Chabert, C.; Church, D. J.; Halazy, S.; Jiang, X.; Martinou, I.; Nichols,
A.; Sauer, W.; Gotteland, J.-P. J. Med. Chem. 2004, 47, 6921; (b) Gaillard, P.;
Jeanclaude-Etter, I.; Ardissone, V.; Arkinstall, S.; Cambet, Y.; Camps, M.;
Chabert, C.; Church, D.; Cirillo, R.; Gretener, D.; Halazy, S.; Nichols, A.;
Szyndralewiez, C.; Vitte, P.-A.; Gotteland, J.-P. J. Med. Chem. 2005, 48, 4596;
(c) Stocks, M. J.; Barber, S.; Ford, R.; Leroux, F.; St-Gallay, S.; Teague, S.; Xue, Y.
Bioorg. Med. Chem. Lett. 2005, 15, 3459; (d) Szczepankiewicz, B. G.; Kosogof, C.;
Nelson, L. T. J.; Liu, G.; Liu, B.; Zhao, H.; Serby, M. D.; Xin, Z.; Liu, M.; Gum, R. J.;
Haasch, D. L.; Stashko, M. A.; Olejniczak, E. T.; Sun, C.; Dorwin, S. A.; Haskins, K.;
Abad-Zapatero, C.; Fry, E. H.; Hutchins, C. W.; Sham, H. L.; Rondinone, C. M.;
Trevillyan, J. M. J. Med. Chem. 2006, 49, 3563; (e) Liu, M.; Xin, Z.; Clampit, J. E.;
Wang, S.; Gum, R. J.; Haasch, D. L.; Trevillyan, J. M.; Abad-Zapatero, C.; Fry, E.
H.; Sham, H. L.; Liu, G. Bioorg. Med. Chem. Lett. 2006, 16, 2590; (f) Liu, G.; Zhao,
H.; Liu, B.; Xin, Z.; Liu, M.; Kosogof, C.; Szczepankiewicz, B. G.; Wang, S.;
9d
9e
9f
36
9
209
114
299
15
11
<8
28
All biological dataaremean valuesof multipleexperiments and thusshows the reader
that these have a higher accuracy than just providing data from one experiment.
NT, not tested.

2102
P. S. Humphries et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2099–2102
Clampit, J. E.; Gum, R. J.; Haasch, D. L.; Trevillyan, J. M.; Sham, H. L. Bioorg. Chem.
Lett. 2006, 16, 5723; (g) Liu, M.; Wang, S.; Clampit, J. E.; Gum, R. J.; Haasch, D. L.;
Yu, X.-H.; McDonnell, S. R.; Chen, P.; Yie, J.; Ogilvie, K. M.; Fraser, J.; Briscoe, C. P.
Am. J. Physiol. 2008, 295, E1142.
Rondinone, C. M.; Trevillyan, J. M.; Abad-Zapatero, C.; Fry, E. H.; Sham, H. L.;
Liu, G. Bioorg. Med. Chem. Lett. 2007, 17, 668; (h) Alam, M.; Beevers, R. E.; Ceska,
T.; Davenport, R. J.; Dickson, K. M.; Fortunato, M.; Gowers, L.; Haughan, A. F.;
James, L. A.; Jones, M. W.; Kinsella, N.; Lowe, C.; Meissner, J. W. G.; Nicolas, A.-
L.; Perry, B. G.; Phillips, D. J.; Pitt, W. R.; Platt, A.; Ratcliffe, A. J.; Sharpe, A.; Tait,
L. J. Bioorg. Med. Chem. Lett. 2007, 17, 3463; (i) Jiang, R.; Duckett, D.; Chen, W.;
Habel, W.; Habel, J.; Ling, Y. Y.; LoGrasso, P.; Kamenecka, T. M. Bioorg. Med.
Chem. Lett. 2007, 17, 6378.
8. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Del. Rev.
1997, 23, 3.
9. Humphries, P. S.; Do, Q.-Q. T.; Wilhite, D. M. Tetrahedron Lett., doi:10.1016/
j.tetlet.2009.03.073.
10. Almansa Rosales, C.; Virgili Bernado, M.; Grima Poveda, P. M. WO 2006/013095
A2, 2006; Chem. Abstr. 2006, 144, 212773.
11. Garcia-Echeverria, C. WO 2005/068452 A1, 2005; Chem. Abstr. 2005, 143,
172889.
5. Chen, P.; Hong, Y.; Humphries, P. S.; Johnson, T. O.; Lafontaine, J. A.; Liu, S.;
Lunney, E. A. WO 2007/129195 A2, 2007; Chem. Abstr. 2007, 147, 541894.
6. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
7. (a) Gupta, S.; Campbell, D.; Derijard, B.; Davis, R. J. Sci. 1995, 267, 389; (b) Cho,
H.; Black, S. C.; Looper, D.; Shi, M.; Kelly-Sullivan, D.; Timofeevski, S.; Siegel, K.;
12. Uehling, D. E.; Stevens, K. L.; Dickerson, S. H.; Waterson, A. G.; Harris, P. A.;
Sammond, D. M.; Hubbard, R. D.; Emerson, H. K.; Wilson, J. W. WO 2006/
068826 A2, 2006; Chem. Abstr. 2006, 145, 103711.
13. Tavares, F. X.; Boucheron, J. A.; Dickerson, S. H.; Griffin, R. J.; Preugschat, F.;
Thomson, S. A.; Wang, T. Y.; Zhou, H.-Q. J. Med. Chem. 2004, 47, 4716.