Highly selective 2,4-diaminopyrimidine-5-carboxamide inhibitors of Sky kinase

Article abstract of DOI:10.1016/j.bmcl.2012.12.013

We report the SAR around a series of 2,4-diaminopyrimidine-5-carboxamide inhibitors of Sky kinase. 2-Aminophenethyl analogs demonstrate excellent potency but moderate kinase selectivity, while 2-aminobenzyl analogs that fill the Ala571 subpocket exhibit good inhibition activity and excellent kinase selectivity.

Full text of DOI:10.1016/j.bmcl.2012.12.013

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1046–1050
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly selective 2,4-diaminopyrimidine-5-carboxamide inhibitors
of Sky kinase
à
§
–
⇑
Noel A. Powell , Jennifer K. Hoffman , Fred L. Ciske , Michael D. Kaufman , Jeffrey T. Kohrt ,
John Quin III, Derek J. Sheehan k, Amy Delaney, Sangita M. Baxi , Cornel Catana, Patrick McConnell,
Jeff Ohren, Lisa A. Perrin, Jeremy J. Edmunds àà
Pfizer Global Research & Development, Michigan Laboratories, Ann Arbor, MI 48105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 20 December 2012
We report the SAR around a series of 2,4-diaminopyrimidine-5-carboxamide inhibitors of Sky kinase.
2-Aminophenethyl analogs demonstrate excellent potency but moderate kinase selectivity, while 2-ami-
nobenzyl analogs that fill the Ala571 subpocket exhibit good inhibition activity and excellent kinase
selectivity.
Keywords:
Kinase Inhibitor
Anti-platelet therapy
Hit-to-lead
Ó 2012 Elsevier Ltd. All rights reserved.
Coronary heart disease is the leading cause of mortality and
morbidity in the United States.1 There are an estimated 40 million
people in the US at risk for thrombotic events due to atherosclero-
sis or prior history of cardiovascular disease or stroke. The current
front-line treatment for arterial thrombosis is clopidogrel
(PlavixÒ), which has significant limitations in terms of efficacy
without aspirin, and is also a pro-drug that must be metabolized
by liver enzymes to present a pharmacodynamic effect. About
one quarter to one third of patients do not adequately metabolize
clopidogrel and are resistant to its effect.2 In addition, the active
drug binds irreversibly to platelets, resulting in a high risk for in-
creased bleeding. Thus, there is a need for safer, more effective
anti-platelet agents useful to a broad patient population.
thrombosis.4,5 Mechanistically, Gas6 appears to play a critical role
as a platelet response amplifier, enhancing platelet aggregation
and granule secretion to known endogenous agonists. Gas6 is a li-
gand for three tyrosine kinase receptors designated Sky (Tyro3/
Rse), Axl, and Mer that are expressed on human and mouse plate-
lets,5 and induces intracellular signaling through the PI3K and Akt
pathways.6 Gas6-neutralizing antibodies inhibit platelet aggrega-
tion, without increased bleeding in mice.5 In addition, Gas6 knock-
out mice do not have any significant difference in bleeding time
compared to wild type animals.4 Gas6 receptor knockout (Skyꢀ/ꢀ
,
Axlꢀ/ꢀ, or Merꢀ/ꢀ) mice also exhibit protection against thrombo-
embolism with normal bleeding times.7 Previous studies from
our laboratories have demonstrated that Sky specific antibodies in-
hibit human platelet degranulation and aggregation to the same
extent as Gas6 inhibition, and result in comparable efficacy to clop-
idogrel treatment in a mouse model of thrombosis with no signif-
icant increase in bleeding time.5 Thus, inhibition of Sky potentially
represents the first antithrombotic therapy that is not associated
with bleeding side effects.
Gas6 (growth arrest specific gene 6) is a member of the Vitamin
K dependent family of proteins which have all been shown to have
a role in thrombosis and hemostasis.3 Deficiency or inhibition of
Gas6 causes platelet dysfunction and protects mice against
⇑
Corresponding author at present address: Eisai Inc., 4 Corporate Drive, Andover,
MA 01810, USA. Tel.: +1 978 837 4683.
We recently described the discovery of a spiroindoline-based
series of Sky kinase inhibitors that exhibited excellent selectivity
against a panel of 30 kinases designed to broadly represent the ki-
nome.8 However, that series suffered from poor oral bioavailability
and CYP2D6 inhibition. Consequently we were interested in iden-
tifying additional chemical matter with improved properties. Com-
pound 1 was identified from a HTS of the Pfizer compound library
as a potential lead that exhibited no inhibition of dofetilide binding
in the hERG K+ channel. In addition, although 2,4-diaminopyrimi-
dine-5-carboxyamides are well precedented kinase inhibitor tem-
plates, the 3-aminopropyl-butyrolactam sidechain appeared to
E-mail address: napowell@comcast.net (N.A. Powell).
Current address: Hamilton Company, 4970 Energy Way, Reno, NV 89502, USA.
Current address: Cayman Chemical, 1180 East Ellsworth Road, Ann Arbor, MI
à
48108, USA.
§
Current address: Deciphera Pharmaceuticals, LLC 643 Massachusetts, Ste 200,
Lawrence, KS 66044, USA.
–
Current address: Pfizer Worldwide Research & Development, Eastern Point Road,
Groton, CT 06340, USA.
k
Current address: Pfizer Animal Health, Kalamazoo, MI 49007, USA.
Current address: Pfizer Worldwide Research
& Development, 10646 Science
Center Drive, San Diego, CA 92121, USA.
àà
Current address: Abbott Labs, 381 Plantation Street, Worcester, MA 01605, USA.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.013

N. A. Powell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1046–1050
1047
Table 1
confer some kinase selectivity, as 1 exhibited little activity in sev-
SAR of 2,4-diaminopyrimidine-5-carboxamide series
eral other internal kinase high throughput screens (data not
shown). As selectivity against the broader kinome is a paramount
concern for a non-oncology kinase inhibitor, we chose to pursue
further structure–activity relationship (SAR) studies around this
template.
O
HN
N
N
O
R2
R1
O
N
H
N
N
H
HN
N
R1-NH
R2-NH
Sky IC50
1.9
(lM)
a
ID
N
O
HN
HN
N
HN
N
NH
1
N
N
HN
HN
5
4.2
1
N
Sky IC50 =1.90 µM
Dofetilide Binding = 6% @ 10 µM
HN
HN
HN
HN
HN
HN
HN
6
7
8
7.2
N
Analogs were prepared as described in Scheme 1. Condensation of
N-(3-aminopropyl)-butyrolactam with acid chloride 29 provided
the amide 3. The chloride was displaced by an amine (R1NH2) in
hot CH3CN to afford 4. After m-CPBA oxidation of the thiomethyl
group to the methyl sulfone, displacement of the sulfone by a sec-
ond primary amine (R2NH2) in refluxing 1,4-dioxane yielded the
desired 2,4-diaminopyrimidine analogs.
Our initial SAR efforts began by investigating the role of the
nitrogen atom in the pyridine ring of the R2 sidechain (Table 1).
The 3- and 2-pyridine rings exhibited two and threefold less Sky
1.1
0.81
HN
HN
9
1.1
2.0
HN
inhibition activity (5 and 6, IC50 = 4.2 and 7.2 lM, respectively),
10
indicating that the 4-pyridine 1 makes a positive hydrogen-bond
accepting interaction. Recognizing that the aniline moiety in the
R1 sidechain represented a potential toxicophore, we were pleased
to find that replacement of the aniline ring with a cyclopentyl-
amine resulted in a slight increase in inhibition activity (7,
HN
HN
HN
HN
11
12
0.26
0.11
IC50 = 1.1
nitrogen atom made a positive hydrogen-bond acceptor interac-
tion, phenethyl group resulted in similar activity (8,
IC50 = 0.811 M). Addition of an R-methyl substituent at the ben-
zylic position of the phenethyl group resulted in similar activity
(9, IC50 = 1.1 M), while a S-methyl substituted afforded a twofold
lM). While our initial SAR indicated that the 4-pyridine
F
a
HN
HN
HN
l
13
14
15
0.18
0.17
0.36
Cl
l
Cl
loss of activity (10, IC50 = 1.95). Constraining the conformational
flexible phenethyl moiety into a 2-aminoindane gave a substantial
improvement in the Sky inhibition activity (11, IC50 = 0.26 lM).
Likewise, addition of electron withdrawing substituents on the
phenyl ring also led to improved Sky inhibition (12–15). The
importance of a potential hydrogen-bond acceptor on the phenyl
ring was also illustrated by the improved inhibition activity exhib-
HN
HN
HN
Cl
Cl
HN
HN
Cl
ited by the 4-methoxyphenethyl analog 16 (IC50 = 0.20 lM) and
16
17
18
0.20
OMe
OMe
HN
HN
HN
HN
O
0.037
0.27
O
HN
Cl
N
a
N
Cl
OMe
OMe
N
O
MeS
N
2
Cl
N
3
MeS
N
O
HN
HN
HN
HN
19
20
0.068
0.035
HN
N
OH
OMe
b
c, d
O
5-32
4
MeS
N
NH
R1
OH
a
IC50 data is an average of duplicate runs using an ELISA format assay with ATP
concentration of 60 M.
Scheme 1. (a) N-(3-Aminopropyl)-butyrolactam, Et3N, DMAP, CH2Cl2; (b) R1NH2,
Et3N, CH3CN, 80 °C; (c) m-CPBA, CH2Cl2, 0 °C; (d) R2NH2, Et3N, 1,4-dioxane, reflux.
l

1048
N. A. Powell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1046–1050
10
Figure 1. X-ray crystal structure of 7 (yellow stick) bound in the ATP active site of Sky (pdb ID code: 4FEQ).
Dotted lines denote hydrogen bonds formed with protein
residues. The glycine-rich loop has been removed for clarity.
the 3-methoxyphenethyl analog 17 (IC50 = 0.037
nation of the 3,4-dimethoxyphenethyl proved to be less active than
the single substituents (18, IC50 = 0.27 M). Since methoxy ethers
are potential sites of metabolism, we examined the potential phe-
nol metabolite and found that the 4-hydroxyphenethyl analog 19
exhibited potent Sky inhibition activity (IC50 = 0.068 lM). Unlike
the 3,4-dimethoxyphenethyl analog 18, combination of the 4-phe-
nol and 3-methoxy substituents led to potent inhibition activity
(20, IC50 = 0.035 lM).
We obtained a crystal structure of 7 bound in the ATP binding
site of Sky (Fig. 1). The N-(3-aminopropyl)-butyrolactam sidechain
is oriented towards solvent, while the 2-aminopyrimidine-5-car-
boxamide core forms three hydrogen bond contacts with the
l
M). The combi-
Pro594 carbonyl and the Met596 amide NH and carbonyl in the
hinge region. The hydrogen bond between the 5-carboxamide NH
and the Met596 carbonyl is enforced by an intramolecular hydro-
gen bond between the 4-amino NH and amide carbonyl. In our pre-
viously reported Sky crystal structure (3QUP),8 we observed an
orienting interaction of Lys540 and Asp663 with an inhibitor bind-
ing directly to Asp663. In this structure, however, the 4-pyridineth-
yl group is positioned to interrupt the charge–charge contact
between Lys540 and Asp663. This interaction is presumably key
for the potent inhibitory activity of the 3-methoxyphenyl analog
17 and the phenolic analogs 19 and 20. The 4-pyridinethyl group
of 7 does not occupy the Ala571 subpocket of the Sky ATP binding
site. Previous reports from our group have demonstrated that fill-
l
Table 3
Table 2
SAR of 2-benzylamino analogs
Heatmap of kinase selectivity for selected compoundsa
O
HN
N
X
N
O
HN
N
NH
R
a
Analog
R
X
Sky IC50
(l
M)
HLM t1/2 (min)
21
22
23
24
25
26
27
28
29
30
31
32
H
4-Cl
4-OCH3
4-CH3
3-Cl
3-CF3
3-CH3
2-Cl
2-OCH3
2-OCH2CH3
2,5-Cl2
2,5-Cl2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
2.1
9.5
11.2
11.2
0.850
9.7
11
NT
NT
NT
1.3
NT
8
4.6
14
6
3
1.58
0.400
0.215
0.355
0.070
6
2
a
Fold selectivity (kinase IC50/Sky IC50) colored by green = >100-fold, yellow = 10
a
Values are means of duplicate experiments, (NT = not tested) with ATP conc-
entation of 60 M.
to 100-fold, red = <10-fold. IC50 data for individual kinases can be found in the
Supplementary data available on-line.
l

N. A. Powell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1046–1050
1049
Figure 2. X-ray crystal structure of 29 (yellow stick) bound in the ATP active site of Sky (pdb ID code: 4FF8).10 The glycine-rich loop has been removed for clarity.
ing the Ala571 pocket is key to achieving selectivity for Sky versus
a representative selectivity panel.8 As expected from the observed
binding mode in the crystal structure, these compounds also inhib-
ited off-target kinases as illustrated by analog 18 which exhibited
potent inhibition of Abl, FGFR, and Lck, as well as other members of
the HGFR family, Axl, and Mer (Table 2).11
To improve the kinase selectivity, we hypothesized that a short-
er and less conformationally constrained sidechain at the pyrimi-
dine 2-position would better fill the Ala571 subpocket.
Accordingly, we investigated the SAR of a series of 2-benzylamino
analogs (Table 3). The unsubstituted benzyl analog 21 exhibited
2.6-fold reduced inhibitory activity compared to the phenethyl
analog 8. Substitution at the para-position resulted in further
reduction in activity (analogs 22–24), indicating that steric con-
straints were overriding any electronic or lipophilic substituent
properties. The 3-Cl analog 25 exhibited improved Sky inhibition
that the primary metabolite was oxidative hydroxylation at the
-carbonyl C atom in the butyrolactam ring. To block this route
of metabolism, we converted the butyrolactam ring to an oxazoli-
din-2-one ring, which exhibited potent Sky inhibition activity (32,
IC50 = 0.070 lM), but no improvement in HLM stability.
In conclusion, we have discovered a series of Sky inhibitors
based on a 2,4-diaminopyrimidine-5-carboxamide template as po-
tential anti-platelet therapy. 2-Phenethylamino analogs with
hydrogen-bond accepting groups or phenols at the para- or meta-
positions of the phenyl ring exhibited potent inhibition of Sky,
but poor kinase selectivity. 2-Benzylamino analogs with ortho- or
meta-substituents exhibited good inhibition activity and excellent
kinase selectivity. These analogs suffer from poor HLM stability. Ef-
forts to improve the HLM stability of this series will be the subject
of the following article.
a
activity (IC50 = 0.85 lM), while the 3-CF3 and 3-CH3 substituents
were detrimental to activity (26 and 27, respectively). Electron-
Supplementary data
withdrawing groups were also not tolerated at the ortho-position
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.013.
(28, IC50 = 1.58
l
M), while the electron-donating 2-OCH3 group
sharp improvement in Sky inhibition (29,
M). Further extension of the ortho-substituent to a 2-
exhibited
IC50 = 0.40
a
l
OEt group resulted in further improved inhibition activity (30,
IC50 = 0.215 M). The combination of an ortho-Cl and meta-Cl sub-
stituents into a 2,5-disubstitution pattern also resulted in im-
l
References and notes
1. American Heart Association. Heart Disease and Stroke Statistics—2007 Update.
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2006, 36, 373.
4. Angelillo-Scherrer, A.; de Frutos, P. G.; Aparicio, C.; Melis, E.; Savi, P.; Lupu, F.;
Arnout, J.; Dewerchin, M.; Hoylaerts, M. F.; Herbert, J.-M.; Collen, D.; Dahlbäck,
B.; Carmeliet, P. Nat. Med. 2001, 7, 215.
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Perrin, L. A. J. Thromb. Haemost. 2005, 3, 733.
6. Nagata, K.; Ohashi, K.; Takano, T.; Arita, H.; Zong, C.; Hanafusa, H.; Mizuno, K. J.
Biol. Chem. 1996, 271, 30022.
7. Angelillo-Scherrer, A.; Burnier, L.; Flores, N.; Savi, P.; DeMol, M.; Schaeffer, P.;
Herbert, J.-M.; Lemke, G.; Goff, S. P.; Matsushima, G. K.; Earp, H. S.; Vesin, C.;
Hoylaerts, M. F.; Plaisance, S.; Collen, D.; Conway, E. M.; Wehrle-Haller, B.;
Carmeliet, P. J. Clin. Invest. 2005, 115, 237.
8. Powell, N. A.; Kohrt, J. T.; Filipski, K. J.; Kaufman, M.; Sheehan, D.; Edmunds,
Jeremy J.; Delaney, A.; Wang, Y.; Bourbonais, F.; Lee, D.-Y.; Schwende, F.; Sun,
proved activity relative to the monosubstituted chlorobenzyl
analogs (31, IC50 = 0.355 lM). A crystal structure of analog 29
was obtained that confirmed our hypothesis that a less conforma-
tionally mobile benzyl sidechain would fill the Ala571 subpocket
(Fig. 2).8 As expected from this binding mode, analogs 29 and 31
exhibited excellent kinase selectivity against a panel of 20 & 28
kinases, respectively (Table 2).
All compounds examined thus far exhibited low metabolic sta-
bility when incubated with human liver microsomes. While the
aromatic ring in the 2-benzylamine sidechain represented a poten-
tial site of metabolism, the presence of electron-withdrawing or
electron-donating substituents had no affect on the HLM stability.
Identification of the metabolites by LC–MS–MS methods indicated

1050
N. A. Powell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1046–1050
F.; Fauman, E.; McConnell, P.; Catana, C.; Chen, H.; Ohren, J.; Perrin, L. A. Bioorg.
Med. Chem. Lett. 2012, 22, 190.
9. Baughman, T. A.; Boyce, J. P.; Darwish, I. S.; Howbert, J. J.; Ihle, N. C.; Jackson, R.
W.; Jeffrey, S. C.; Maeda, D.; Yager, K. M. U.S. Patent 7,176,310, 2007.
10. Coordinates for the Sky/7 complex (4FEQ) and Sky/29 complex (4FF8) have
been deposited at www.rcsb.org.
11. Liu, J.; Yang, C.; Simpson, C.; DeRyckere, D.; Van Deusen, A.; Miley, M. J.;
Kireeve, D.; Norris-Drouin, J.; Sather, S.; Hunter, D.; Korboukh, V. K.; Patel, H. S.;
Janzen, W. P.; Machius, M.; Johnson, G. L.; Earp, H. S.; Graham, D. K.; Frye, S. V.;
Wang, X. ACS Med. Chem. Lett. 2012, 3, 129.