Pyrazine-based Syk kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.02.087

A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines provided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed good inhibitory activity against Syk as well as remarkable activity in LAD2 cells degranulation assay.

Full text of DOI:10.1016/j.bmcl.2012.02.087

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2784–2788
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazine-based Syk kinase inhibitors
Pilar Forns b, Cristina Esteve a, Lorena Taboada a, Juan Antonio Alonso a, Adelina Orellana a,
Mónica Maldonado a, Cristina Carreño a, Isabel Ramis a, Manel López a, Montserrat Miralpeix a,
Bernat Vidal a,
⇑
a Almirall Research Center, Almirall, Ctra. Laureà Miró 408, E-08980 St. Feliu de Llobregat, Barcelona, Spain
b Almirall-Barcelona Science Park Unit, Barcelona Science Park, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells
degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines pro-
vided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed
good inhibitory activity against Syk as well as remarkable activity in LAD2 cells degranulation assay.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 25 January 2012
Revised 22 February 2012
Accepted 24 February 2012
Available online 2 March 2012
Keywords:
Syk
2-Aminopyrazine
LAD2
Spleen tyrosine kinase (Syk) is a cytosolic non-receptor protein
tyrosine kinase that plays an essential role in immunoreceptor sig-
naling, mainly in B cell receptor, Fc receptors, integrin and lectin
receptors.1 Syk is highly expressed in haematopoietic cells, includ-
ing B cells, mast cells, macrophages and neutrophils. Engagement
of these receptors with their ligands activates Syk, which orches-
trates several cellular responses (proliferation, differentiation, cell
adhesion, cytokine production, mast cell degranulation).1 There-
fore, Syk is considered a promising therapeutic target for the treat-
ment of immune-mediated disorders such as allergy, autoimmune
diseases and haematological malignancies.2 A number of ATP-com-
petitive Syk inhibitors have been described3 and it has been re-
ported that pharmacological inhibition of Syk kinase activity
modulates mast cell degranulation, leukocyte immune function
and suppress inflammation in vivo.4,5 Interestingly, since Syk is
positioned upstream in the cell signaling pathway of several
immunoreceptors, therapies with Syk inhibitors may be more
advantageous than drugs that inhibit a single downstream event.
To date, several compounds have entered into clinical develop-
ment. R-788 is currently in phase II for rheumatoid arthritis,6 other
autoimmune diseases and lymphoma. Few molecules are in early
phases of clinical studies, including R-343 for asthma7 and
PRT062607 for autoimmune diseases.
O
O
S
HN
R1
R2
N
N
N
O
NH2
N
NH2
NH2
1; IC50Syk: 10 µM
2
Figure 1. Aminopyrazine hit compound and proposed scaffold for expansion.
H-bonding motifs in the hinge region of the kinase and initial
SAR optimization work on the pyrazine scaffold was focused at
replacing the aniline group by a substituted carboxamide 2 to al-
low planarity of the molecule and SAR expansion to pick key inter-
actions in the ATP binding pocket.
The analogs described were prepared following the general syn-
thetic route described in Scheme 1. Functional group transforma-
tions from commercially available aminopyrazine 3 provide key
intermediate 4 that upon activation with HATU provides amide
intermediate 5. Final step of Suzuki reaction allows access to 2.
This straightforward synthetic pathway allowed rapid exploration
of the SAR in R1 and R2.
Random screening of our internal compound library identified
compound 1 as an ATP competitive Syk inhibitor (Fig. 1). After
docking studies we envisaged the 2-aminopyrazine moiety as key
The methylsulfonamidophenyl group that we speculated that
was pointing towards the solvent exposed area was left for later
optimization. Exploration of amide substituents (R1) included ali-
phatic aminoalcohols and aminopiperidines.
⇑
Corresponding author. Tel.: +34 93 291 3996; fax: +34 93 312 8635.
E-mail addresses: bernat.vidal@almirall.com, bvidal@almirall.es (B. Vidal).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.087

P. Forns et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2784–2788
2785
R'
R
O
R1
O
O
N
R2
N
a, b, c
Br
N
d
e
N
Br
N
OH
NH2
OH
NH2
O
N
N
N
NH2
N
NH2
3
4
5
2
Scheme 1. Reagents and conditions: (a) H2SO4, MeOH, 80%; (b) Br2, AcOH, 70%; (c) NaOH, EtOH, 90%; (d) HATU, DIPEA, RR0NH, DMF, 50–95%; (e) R2-B(OH)2, PdCl2 dppf. DCM,
Cs2CO3, dioxane, 90 °C, 20–80%.
As exemplified in Table 1, a series of piperidines bearing diverse
linkers in order to capture an eventual salt bridge interaction with
Asp512 were explored. Interestingly, aminomethylpiperidine 11
provided the best Syk inhibitory activity probably because it is best
orientated to achieve an interaction with Asp512. We also explored
the introduction of aliphatic amines in R1. trans Aminocyclohexa-
nol derivative 12 provided some hints of potency. We then ex-
plored the introduction of adamantane motifs (that were
reported to fill a similar space in a series of JAK inhibitors).9 Inter-
estingly, adamantane as well as cis or trans adamantanol deriva-
tives (examples 13–15) showed good potency (see Table 1).
Tertiary amide 16 was not tolerated probably because of the wrong
orientation of the hydroxypiperidine modif. Thus a key element in
this SAR was the primary amide, which proved to be critical for
binding potency.
Further SAR exploration was focused on variations of the
substituent in position 5 keeping the best substitutions identified
in Table 1. We also explored the activity of most potent Syk inhib-
itors in a degranulation assay in the human LAD2 mast cell line.10,11
As evident from Table 2 an increase in enzymatic potency was
Table 1
Syk inhibitory activity8 of selected aminopyrazine derivativesa
NHSO2Me
R1
Table 2
Inhibition of Syk activitya and LAD2 cells degranulationb of aminopyrazine derivatives
N
O
H
N
NH2
N
R2
N
NH
O
Compound
R1
Syk IC50
2.4
(lM)
H
N
N
NH2
6
NH
Compound R2
Syk IC50 LAD2 IC50 (lM) or % of
(
lM)
inhibition at 1
lM
NH
H
N
7
8
0.67
1.4
H
N
H2N
11a
0.64
—
S
O
O
NH
NH
11b
0.34
0.06
0.1
1.96
1.1
H
N
9
1.9
O
F
F
F
NH
NH
11c
11d
11e
H
N
10
0.38
O
O
N
10%
0.86
H
N
N
11
12
0.12
2.6
H
H
N
0.085
N
H
OH
OH
11f
0.042
0.034
48%
65%
NH
13
14
0.65
0.38
N
H
N
O
OH
11g
N
NH
11h
11i
0.028
0.067
0.56
0.55
H2N
N
15
16
0.49
>50
H
N
OH
N
N
N
a,b
IC50 and percentage of inhibition values are an average of two or more
determinations.
a
IC50 values are an average of two or more determinations.

2786
P. Forns et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2784–2788
Table 3
Table 3 (continued)
Inhibition of Syk activitya and LAD2 cells degranulationb of aminopyrazine derivatives
Compound R2
Syk IC50
M)
LAD2 IC50
M)
OH
(
l
(l
H
R2
N
13n
0.08
0.06
1
N
N
H2N
N
O
N
NH2
13o
43%
N
Compound R2
Syk IC50
M)
LAD2 IC50
M)
HO
N
(
l
(l
O
F
F
13a
0.4
0.1
—
F
O
13p
0.04
0.06
0.16
0.18
N
H2N
N
N
13b
0.12
0.18
O
N
H
13q
N
13c
13d
0.11
0.05
O
N
H2N
N
a,b
IC50 and percentage of inhibition values are an average of two or more
determinations.
0.1
HN
HN
achieved with m-trifluoromethoxy analog 11c as well as indazolyl
compounds 11e and 11f. We felt there might be additional oppor-
tunities to explore the solvent exposed region using C4 substitution
trying to capture a new interaction with Lys458. Indeed biaryl
substituted compounds 11g, 11h and 11i turned out to be very po-
tent. Unfortunately all compounds from this subseries failed to give
acceptable potency in the LAD2 functional assay probably due to
low permeability (PAPP in CaCo-2 cell of compound 11h: AB: 0;
BA: 2.38 (cm/s) 10ꢀ6). The presence of the ionizable piperidine mo-
tif could also be detrimental for activity in LAD2 cells.
We then turned our attention to the non-basic substituents at
the amide motif. Since adamantane derivative 15 was cytotoxic in
CHO cells we decided to explore the SAR using non-cytotoxic cis
and trans adamantanol analogs 13 and 14. As depicted in Table 3,
3-(4-pyrazolyl)phenyl were the best substituents in R2 to achieve
best Syk inhibitory activities ranging between 30–80 nM. In terms
of functional activity in LAD2, compounds 13d, 13f, and 13i were
the most potent analogs with activities around 100 nM. Diverse
substitutions at the pyrazole ring were tolerated such as a basic
group (aminoethyl in compound 13n), polar groups such as
hydroxyethyl 13m, carboxamides 13p and even carboxylic acids
13o. However, cell versus enzyme IC50 results for the matched pairs
13m/13n and 13o/13p suggest that detrimental effect on cell po-
tency is more likely to be due to the presence of an ionisable group.
Similar SAR was observed with trans-adamantanol derivatives
(Table 4) with Syk inhibitory activities down to 20 nM and a max-
imum cellular potency of 120 nM (14a).
N
13e
13f
0.34
0.04
N
N
0.09
0.15
N
13g
13h
0.03
0.05
N
N
O
0.14
N
N
13i
13j
0.03
0.05
0.11
0.14
N
N
N
F
F
N
F
X-Ray structures of compounds 7 and 14b bound to Syk (Figs. 2
and 3) were obtained which confirmed our postulated binding
modes.12 From Figs. 2 and 3, the key H-bond interactions from
the aminopyrazole motif with the hinge region are maintained
(Glu449 and Ala451). In compound 7 a salt bridge interaction with
Asp512 and H-bond with Asn499 are observed whereas 14b cap-
tures an H-bond between Asp512 and the hydroxyl group of the
adamantanol motif. As expected, in both cases the R2 group is pro-
jecting towards the solvent exposed region, and in compound 14b
the pyrazole motif is capturing an H-bond interaction with Lys458.
Compound 13f is not cytotoxic in CHO cells, shows good perme-
ability in CaCo-2 cell (PAPP in (cm/s) 10ꢀ6 AB: 28, BA: 13) and in
13k
13l
0.04
0.04
0.06
0.17
0.22
0.12
N
N
N
N
13m
N
HO
N

P. Forns et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2784–2788
2787
Table 4
Inhibition of Syk activitya and LAD2 cells degranulationb of selected aminopyrazine
derivatives
OH
HN
R2
N
O
N
NH2
Compound R2
Syk IC50
M)
LAD2 IC50
M)
(
l
(l
N
14a
0.18
0.02
0.4
N
H
14b
14c
14d
0.12
N
N
Figure 3. X-ray of crystal structure of 14b in Syk.10
0.02
0.06
0.18
0.14
N
N
be excluded some contribution of off-target activities to the cell
activity observed.
In conclusion, we have shown that a hit compound (1) has been
successfully modified to provide potent Syk inhibitors with good
cellular potencies. Placement of cis and trans adamantanol motifs
in R2 were key structural elements to achieve good cellular po-
tency. Introduction of 3-(4-pyrazolyl)phenyl groups in R1 resulted
in potent compounds 13d, 13f, 13i, 14b and 14d with improved
cellular potencies. The inhibitor 13f shows acceptable selectivity
profile and good permeability.
N
HO
N
a,b
IC50 values are an average of two or more determinations.
Acknowledgments
The authors acknowledge the contribution of Drs. A. Kirshen-
baum and Y. Wu (NIH, Bethesda) and the US Public Health Services
for providing the LAD2 cell line and for the technical assistance and
Mr. Josep Maria Huerta for his help with NMR experiments.
References and notes
1. Mócsai, A.; Ruland, J.; Tybulewicz, V. L. J. Nat. Rev. Immunol. 2010, 10, 387.
2. Singh, R.; Matsuda, E. S. Annu. Rep. Med. Chem. 2007, 42, 379.
3. Riccaboni, M.; Bianch, I.; Petrillo, P. Drug Discovery Today 2010, 15, 517.
4. Braselmann, S.; Taylor, V.; Zhao, H.; Wang, S.; Sylvain, C.; Baluom M.; Kunbin,
Q; Herlaar, E.; Lau, A.; Young, C.; Wong, B. R.; Lovell, S.; Sun, T.; Park, G.;
Argade,, A.; Jurcevic, S.; Pine, P.; Singh, R.; Grossbard, E. B.; Payan, D. G.;
Masuda, E. S. J. Pharmacol. Exp. Ther. 2006, 319, 998.
5. Coffey, G.; DeGuzman, F.; Inagaki, M.; Pak, Y.; Delanye, M. S.; Ives, D.; Betz, A.;
Jia, Z. J.; Pandley, A. P.; Baker, D.; Hollenbach, S. J.; Phillips, D. R.; Sinha, U. J.
Pharmacol. Exp. Ther. 2012, 340, 350.
6. Weinblatt, M. E.; Kavanaugh, A.; Genovese, M. C.; Musser, T. K.; Grosbard, E. B.;
Magilavy, D. B. N. Eng. J. Med. 2010, 3636, 1303.
7. Singh, R.; Masuda, E. S.; Payan P. G. J. Med. Chem. 2012, ASAP (doi:10.1021/
jm201271b).
8. Syk kinase inhibition assay: Recombinant human full-length Syk was supplied
by Millipore. The activity of Syk kinase was assessed using a radioactive
filtration kinase assay. The kinase assay was performed in buffer (50 mM Tris–
HCl (pH 7.5), 0.1 mM EGTA, 4 mM Mg(CH3COO)2, 0.1 mM Na3VO4, 0.1% (v/v) b-
Figure 2. X-ray of crystal structure of 7 in Syk.10
mercaptoethanol, 0.133 mg/ml BSA(bovine serum albumin).
substrate poly-GT (100 g/mL final) and 4 l of Syk (1 nM final) were added
over 1.5 l of various concentrations of compound or DMSO vehicle (5% final).
Then, the reaction was initiated by the addition of 14.5 L of 0.36 Ci [
33P]
ATP (10 mCi/ml, Perkin–Elmer) and 15 M of ATP. The reaction mixture took
place in a half-area NBS plate (Corning). After 40 min at room temperature,
15 L of reaction mixture was transferred to a filter plate (Millipore) previously
pre-wetted with a solution of 75 mM phosphoric acid. The filter plate was
washed three times with 200 L phosphoric acid per wash on a vacuum
manifold (Millipore cat # MSVMHTS00). Finally, 30 L Optiphase™ Supermix
(Perkin–Elmer) was added to each test well and allowed to incubate for at least
A 10 lL of
terms of physicochemical properties, 13f shows moderate solubil-
l
l
l
ity (aqueous solubility pH 7.4: 25
1.5.
lg/mL) with a measured logD7.4:
l
l
c-
l
In terms of selectivity 13f did show less than 50% inhibition at
M in 20 kinases in a 42 kinase panel,13 being the kinases most
l
1
l
potently inhibited (>90%) Aurora A, Fgfr3, Fms, TrakA and Yes.
Lyn kinase was also inhibited with an IC50 value of 325 nM. Consid-
l
l
ering the role of Lyn in the signaling of Fce
RI receptor14 it can not

2788
P. Forns et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2784–2788
1 h before counting. Radioactivity counting was performed in
a
Wallac
After the activation, supernatants were removed and transferred to another
well for b-hexosaminidase determination. Cells were lyzed in two cycles of
freeze-thaw and they were incubated for 90 min at 37°C with 1 mM b-
hexosaminidase substrate (p-nitrophenyl-N-acetyl-D-glucosamide, in citric
buffer, pH 4.5). Reaction was stopped with 0.4 M glycine solution (pH 10.0)
and absorbance was read at 405 nm.
MicroBetaÒ, and the tyrosine phosphorylation activity by Syk was calculated.
9. Takayuki, I.; Akira; T.; Kazuo, N.; Hiroshi, S.; Fumie, T.; Shoei, S.; Keiko, H.;
Yutaka, N.; Koichiro, M.; Hisao, H.; Shigeki, K.; Yasuyu, H. WO2007077949.
10. Kirshenbaum, A. S.; Akin, C.; Wu, Y.; Rottem, M.; Goff, J. P.; Beaven, M. A.; Rao,
K.; Metcalfe, D. D. Leuk. Res. 2003, 27, 677.
11. LAD2 cells degranulation assay: the human mast cell line LAD2, established at
the National Institutes of Health (NIH) was provided by Dr. Arnold
Kirshenbaum through a biological materials license agreement. Cells were
sensitized in its normal growth media (Complete StemPro-34 SFM, containing
StemPro-34 nutrient supplement; 2 mM glutamine and 100 ng/ml SCF) by
adding 100 ng/ml of biotin-labeled IgE overnight (37°, 5% CO2) in a humidified
atmosphere (90%). Sensitized LAD2 cells were washed with release buffer
(137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 5.6 mM glucose, 1.8 mM CaCl2,
1.3 mM MgSO4, 0.025% BSA) and adjusted to a density of 0.2 ꢁ 106/ml. Cells
were transferred to the assay plate (10,000 cells/well) and pre-incubated with
drugs for 30 min, and then activated with 125 ng/ml of streptavidin for 30 min.
12. The coordinates of 7 and 14b bound to Syk have been deposited in the Protein
Databank PDB ID code 4DFL for 7 and PDB ID code 4DFN for 14b.
13. Kinases with <50% inhibition at 1
FAK(h), GSK3b(h), IKKb(h), Itk(h), JNK3(h), Met(h), MKK6(h), PDK1(h),
PKCbII(h), PKC (h), PKC (h), PRAK(h), Pyk2(h), ROCK-II(h), TAK1(h), ZAP-
70(h); kinases with >50% inhibition at 1 M: ALK(h), Aurora-A(h), Axl(h),
CDK1/cyclinB(h), cSRC(h), FGFR3(h), Fgr(h), Flt1(h), Flt3(h), Fms(h), Fyn(h),
GSK3 (h), Hck(h), JAK2(h), JAK3(h), Lck(h), Lyn(h), Pim-1(h), Ret(h), Rsk4(h),
lM: BTK(h), CaMKIId(h), CHK1(h), CK1d(h),
c
e
l
a
TrkA(h), Yes(h).
14. Galli, S. J.; Kalesnikoff, J.; Grimbaldeston, M. A.; Piliponsky, A. M.; Williams, C.
M. M.; Tsai, M. Annu. Rev. Immunol. 2005, 23, 749.