Highly potent aminopyridines as Syk kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.07.045

A novel class of potent Syk inhibitors has been developed from rational design. Highly potent aminopyridine derivatives bearing a 4-trifluoromethyl-2- pyridyl motif and represented by compound 13b IC₅₀: 0.6 nM were identified. Substitution by a 2-pyrazinyl motif and SAR expansion in position 4 of the central core provided diverse potent non-cytotoxic Syk inhibitors showing nanomolar activity inhibiting human mast cell line LAD2 degranulation.

Full text of DOI:10.1016/j.bmcl.2012.07.045

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5419–5423
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Highly potent aminopyridines as Syk kinase inhibitors
Marcos Castillo b, Pilar Forns b, Montse Erra a, Marta Mir a, Manel López a, Mónica Maldonado a,
Adelina Orellana a, Cristina Carreño a, Isabel Ramis a, Montserrat Miralpeix a, Bernat Vidal a,
⇑
a Almirall Research Center, Almirall, Ctra. Laureà Miró 408, 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 novel class of potent Syk inhibitors has been developed from rational design. Highly potent aminopyr-
idine derivatives bearing a 4-trifluoromethyl-2-pyridyl motif and represented by compound 13b IC50
0.6 nM were identified. Substitution by a 2-pyrazinyl motif and SAR expansion in position 4 of the central
core provided diverse potent non-cytotoxic Syk inhibitors showing nanomolar activity inhibiting human
mast cell line LAD2 degranulation.
Received 16 May 2012
Revised 9 July 2012
Accepted 11 July 2012
Available online 20 July 2012
:
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Syk kinase
Human mast cell line LAD2
Aminopyridines
Spleen tyrosine kinase (Syk) is a cytosolic non-receptor protein
tyrosine kinase essential for the signalling of several immunore-
ceptors including B-cell receptor, Fce and Fcg receptors. Upon
receptor engagement by allergen, antigen or immune complex
Syk mediates cellular responses (proliferation, differentiation,
adhesion, cytokine production, mast cell degranulation) that are
important for both allergic and antibody-mediated diseases.1 Thus,
pharmacological inhibition of Syk activity can modulate the
inflammatory response in allergic and autoimmune diseases.2 Fur-
thermore, as Syk is positioned upstream in the cell signalling path-
way, therapies with Syk inhibitors may be more advantageous
relative to drugs that inhibit a single downstream event.
In addition to these adaptive immune response functions, re-
cent studies have described new functions for Syk in innate im-
mune response by coupling activation of lectins and integrins
receptors to cellular responses.3 Syk is currently considered an
interesting biological target and the possibility to treat allergic,
autoimmune diseases and malignancies has prompted the devel-
opment of Syk inhibitors.4
As part of ongoing efforts toward the discovery of novel Syk
inhibitors,9 and based on previous knowledge10 we designed and
identified compound 1 ( Fig. 1) as an interesting starting point
where the nitrogen atom (1) of the picoline group and the attached
NH (2) could act as hinge binding motifs with the backbone of
Ala451. We also hypothesized that and the presence of nitrogen
atom 3 could help to keep the planarity between rings to allow
the right positioning for substitution in the upper pyridine ring
and in position 4.
We envisaged two areas of further optimization: exploration of
different rings and substitutions in the upper pyridine area and
systematic exploration of the picoline motif.
Initial SAR efforts were directed towards replacement and sub-
stitution of upper pyridyl motif to improve potency by capturing
new interactions with the protein. In accordance with docking
studies we envisaged that substitution in the meta position should
allow to establish a salt bridge interaction with Asp512. Thus, a
To date, few compounds are into clinical development. Fosta-
matinib currently in phase II for rheumatoid arthritis, other auto-
immune diseases and lymphoma has shown promising results.5
Other molecules are in early phases of clinical studies, including
PRT0626076 for autoimmune diseases and R343 for asthma.7 Re-
cently R343 has shown activity in early and late asthmatic re-
sponse in a phase I study in mild asthmatic patients by inhaled
route.8
N
Planarity between rings
3
N
Compound 1
Syk IC50: 2.7 µM
2
4
N
H
N
H
1
O
N
Ala451
⇑
Corresponding author. Tel.: +34 93 291 3996; fax: +34 93 312 8615.
E-mail address: bernat.vidal@almirall.com (B. Vidal).
Figure 1. Knowledge based designed starting point.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.045

5420
M. Castillo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5419–5423
R1
Br
c
1
4a R1 = NH2
R = NHCH2CH2NH2
4d
4e
b
N
d
1
4b R1 = CN
R = CH2NH2
N
R1
e
N
H
N
4f R = CH2CH2NH2
4c R = CH2CH2NHBOC
3
N
H
N
a
4
B
HO
OH
a
H2N
N
N
R1
N
F
F
N
Br
b
f, e
NHR4R5
N
N
B
F
N
Br
N
N
2
N
H
N
N
H
N
Br
HO
OH
7a-g
6
5
Scheme 1. Reagents and conditions: (a) t-BuOK, benzene, 80 °C, 67%; (b) PdCl2dppfÁDCM, Na2CO3, EtOH-toluene, 90 °C, 34–85%; (c) (i) BOCNHCH2CHO, NaBH3CN, MeOH-
AcOH, r.t.; (ii) TFA, CH2Cl2, r.t. 40% two steps; (d) Pd/C, H2, MeOH-aq. HCl, rt, 75%; (e) TFA, CH2Cl2, r.t. 65%; (f) K2CO3, DMSO, 100 °C, sealed tube, 54–84%.
Table 1
Table 2
Syk inhibitory activity11 of selected aminopyridine derivativesa
Syk inhibitory activity of selected piperazine derivativesa
X
R1
NH
X
N
N
N
N
H
N
R2
N
H
Compound
X
R1
Syk IC50 (nM)
R2
Syk IC50 (nM)
NH2
Compound
X
4a
4d
4e
CH
CH
CH
3242
1671
160
CF3
NH2
NH2
13a
N
1.8
H
N
N
CF3
4f
CH
91
NH2
H
N
7a
7b
7c
N
N
N
200
1872
528
13b
CH
0.6
NH2
OH
H
N
N
N
H
N
NH2
13c
13d
N
62
43
N
N
NH
7d
7e
N
N
14
N
N
CH
N
NH2
NH2
450
a
IC50 values are an average of two or more determinations.
7f
N
N
114
27
Treatment of commercially available 2,6-dibromopyridine 2 with
2-aminopicoline followed by Suzuki-type coupling reaction with
different phenylboronic acids afforded the corresponding amino-
pyridines 4a–c. Functional group transformations provided final
compounds 4d–f. For a pyridine as the upper ring, treatment of
(2) with 2-fluoro-4-pyridylboronic provided key intermediate (5).
Pd catalyzed N-arylation with 2-aminopicoline followed by heating
pyridyl fluoride with the appropriate amines provided compounds
7a–g that were submitted (except for 7b) to a deprotection of the
BOC group with TFA.
N
N
NH
7g
a
IC50 values are an average of two or more determinations.
convergent synthesis was devised in order to rapidly access mole-
cules containing different substituents at this position (Scheme 1).

M. Castillo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5419–5423
5421
N
F
N
B
8
F
a
BOC
e, f
NH
b
N
N
O
O
X
N
X
N
NH2
9
N
N
R2
BOC
N
N
H
NH2
N
12a-b
13a-d
I
a
c, d
12a X = N
12b X = CH
B
O
O
Br
10
11
Scheme 2. Reagents and conditions: (a) 2-amino-6-bromopyridine, PdCl2dppf.DCM, Na2CO3, EtOH-toluene, 90 °C, 62–63%; (b) K2CO3, DMSO, 100 °C, sealed tube, 54–84%; (c)
Kt-BuO, Pd2(dba)3, BINAP, toluene, 100 °C, 53%; (d) bis(pinacolato)diboron, PdCl2dppf, dppf, KOAc, 80 °C, 90%; (e) 2-bromo-4-(trifluoromethyl)pyridine (for 13a–b) or 2-
chloropyrazine (for 13c–d), Cs2CO3, Pd2(dba)3, Xantphos, dioxane, 95 °C, 65–82%; (f) TFA, CH2Cl2, rt 83–98%.
Keeping the biaryl amine motif fixed, we proceeded to examine
the effects of various substituents in R1 in Syk inhibitory activity
(Table 1).11 Increasing the chain length resulted in a rapid increase
in potency with an optimal chain length of 4 atoms (see 4f and 7a
Gatekeeper Met448
vs 4e and 7c). Replacement of the free amine in the chain by a hy-
droxyl group results in a drop of activity (7a vs 7b) probably be-
cause the ammonium motif is involved in
a salt bridge
interaction with Asp514 of the Syk protein. Interestingly the pres-
ence of a cyclic amine substituent in R1 such as piperazine or
homopiperazine (7d and 7g) provided the best Syk inhibitory
activities while nitrogen extrusion as in the aminopyrrolidine
and aminopiperidine derivatives 7e and 7f resulted less active.
We then explored replacement of the picoline motif by 4-tri-
fluoromethyl-2-pyridyl and 2-pyrazinyl derivatives keeping the
piperazine motif fixed. As observed in Table 2, outstanding poten-
cies were observed with trifluoromethyl analogues 13a and 13b
whereas similar potencies were observed for 13c and 13d with a
4.4-fold drop in potency when 13c is compared to the picoline
analogue 7d.
Ala451
Asp512
Asn499
Figure 2. X-ray co-crystal structure of compound 13b in the ATP binding site of Syk
kinase.
Boc
N
NH
Boc
N
N
N
N
N
Cl
N
B
N
N
N
N
c
d, e
+
N
N
N
N
O2N
N
H
N
N
R3
17
15
HO
OH
b
H2N
N
H
N
H
N
H
a
18
19a-l
Cl
N
F
N
O2N
Cl
Br
14
16
Scheme 3. Reagents and conditions: (a) 2-aminopyrazine, Cs2CO3, Pd2(dba)3, Xantphos, dioxane, 95 °C, 63%; (b) (i) tert-butyl piperazine-1-carboxylate, K2CO3, DMSO, 100 °C,
sealed tube, 78%; (ii) bis(pinacolato)diboron, PdCl2dppf, dppf, KOAc, dioxane, 80 °C, 100%; (c) (i) PdCl2dppfÁDCM, Na2CO3, EtOH-toluene, 90 °C, 42%; (ii) Fe, aq. NH4Cl/EtOH,
70 °C, 45%; (d) for 19b–c: R4CHO, NaBH3CN, MeOH-AcOH, r.t., 54–85%; for 19d–j: R4COOH, EDC.HCl, Et3N, HOBt, CAN, rt or R4COOH, HATU, DIEA, DMF, or R4COCl, DIPEA, DCM,
À78 °C, 60–100%; for 19k–l: (i) MeOOCR4COOH, EDC.HCl, Et3N, HOBt, r.t., 56–74%; (ii) LiOH, THF/H2O, rt 33–75% (e) TFA, CH2Cl2, rt 60–98%.

5422
M. Castillo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5419–5423
Table 3
Piperazinyl derivatives 13a–d were prepared by the synthetic
Inhibition of Syk activity and LAD2 cells degranulation14 of 4,6-diaminopyridine
derivativesa,b
route described in Scheme 2. Suzuki coupling of boronate 8 with
2-amino-6-bromopyridine provided intermediate 9 that by substi-
tution with a conveniently protected piperazine led to intermedi-
ate 12a. Commercially available 1-bromo-3-iodobenzene (10)
was converted to boronate 11 which upon Suzuki coupling with
2-amino-6-bomopyridine provided intermediate 12b. Palladium
catalyzed N-arylation of 12a-b with the corresponding 2-halohet-
erocycles followed by removal of the BOC protecting group yielded
compounds 13a–d.
X-ray structure of compound 13b bound to Syk (Fig. 2) con-
firmed our postulated binding mode.12 The pyridine N1 atom and
2-amino group form bidentate hydrogen bonds with the backbone
of the hinge residue Ala451. These position the trifluoromethyl
moiety of the inhibitor in the hydrophobic area close to the gate-
keeper residue Met448. Probably this hydrophobic interaction con-
tributes to the high potency observed. In addition hydrogen
bonding and salt bridge interactions are observed between the ion-
ized nitrogen of the piperidine and Asn499 and Asp512
‘respectively’.
NH
N
N
N
N
R3
N
H
N
H
N
Compound
R3
Syk IC50 (nM)
LAD2 IC50 (nM)
19a
19b
H
Me
4
4
118
188
19c
8
585
HO
HN
O
19d
4
14%@1
1000
lM
Analysis of the crystal structure of 13b suggested that large sub-
stituents in position 4 of the central pyridine ring would be accom-
modated, and allow exploration of additional interaction sites or
modulation of physicochemical properties since it should be point-
ing towards the solvent exposed area.
Unfortunately, most potent analogues identified (7d, 13a–b) suf-
fered from cytotoxicity in CHO cells in the range of 1–5 lM level.
O
19e
5
N
Although the cause of the high cytotoxicity of this compounds is un-
clear, we hypothesized that a decrease in lipophilicity (c log P of 7d,
13a–b: 3.7, 4 and 4.6) could be beneficial to identify less cytotoxic
compounds. Indeed, pyrazine substituted analogues 13c–d (c log P:
1.9 and 2.5) did show cytotoxicity values in CHO cells greater than15
microM. We decided to go further by exploring SAR in position 4
keeping the pyrazine motif in the hinge binding area as well as the
piperazinyl substituted pyridine in the upper region.
Diaminopyridine derivatives 19a–l were prepared following the
synthetic route described in Scheme 3. Key intermediate 18 was
prepared from Suzuki coupling of chloropyridine 15 and boronic
acid 17. Direct removal of BOC group yields compound 19a
whereas reductive amination or standard coupling conditions for
amide formation with intermediate 18 followed by BOC removal
provided compounds 19b–l.
O
O
19f
28
6
362
122
O
19g
N
N
O
19h
9
331
N
O
As can be observed from Table 3, the placement of a primary
amine at position 4 (compound 19a) resulted in more than 10-fold
increase in enzymatic potency compared to analogue 13c. With
single digit nanomolar potency achieved we began to profile com-
pounds in a degranulation assay in the human mast cell line LAD2
cells.13 Methyl or benzyl substitution in R3 (19b–c) did not result in
significant impact on Syk enzymatic potency compared to 19a with
a slight drop in cellular potency for 19b and 5-fold decrease for 19c
‘respectively’. When amides in R3 were explored, 4-pyperidinyl
analogues 19d–e produced comparable potency to 19a although
a dramatic drop of activity in LAD2 was observed. The presence
of an ionisable group in 19d may be the reason for the low activity
whereas 19e kept some cellular activity. Aryl, heteroaryl and het-
eroarylmethyl amides (19f–j) were also well tolerated with single
digit nanomolar activities inhibiting Syk (except for 19f) and LAD2
potency in a range of IC50 = 122–362 nM. Zwitterionic compounds
19k–l were also examined, keeping both enzymatic and cellular
potencies. Compounds 19a–l did not show cytotoxicity in CHO
19i
19j
7
4
169
352
N
N
O
N
O
O
19k
19l
8
140
204
HOOC
HOOC
N
10
a,b
IC50 and percentage of inhibition values are an average of two or more
determinations.
cells (IC50 >20 lM).
Compound 19a was tested in a kinase selectivity panel,15 being
the kinases most potently inhibited (>90%) Fgr, Flt3, Lyn and Yes.
In conclusion, a novel class of Syk inhibitors has been discov-
ered from a structure-based design. Subnanomolar compound
13b was co-crystalized bound to Syk enzyme. This allowed the
identification of an area for SAR expansion that led to highly potent
Considering the role of Lyn in the signaling of Fce
RI receptor16 it
can not be excluded some contribution of off-target activities to
the cell activity observed.

M. Castillo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5419–5423
5423
over 1.5
Then, the reaction was initiated by the addition of 14.5
ATP (10 mCi/ml, PerkinElmer) 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
(PerkinElmer) was added to each test well and allowed to incubate for at least
l
l of various concentrations of compound or DMSO vehicle (5% final).
and diverse non-cytotoxic derivatives showing good activity in
LAD2 degranulation assay.
l
L of 0.36 Ci [
l
c-
33P]
l
l
Acknowledgements
l
The authors acknowledge the contribution of Dr. A. Kirshen-
baum and Dr. Y. Wu (NIH, Bethesda) and the US Public Health Ser-
vices for providing the LAD2 cell line.
l
1 h before counting. Radioactivity counting was performed in
a Wallac
MicroBetaÒ, and the tyrosine phosphorylation activity by Syk was calculated.
12. The coordinates of 13b bound to Syk have been deposited in the Protein
Databank PDB ID code 4F4P.
References and notes
13. 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.
1. Turner, M.; Schweighoffer, E.; Colucci, F.; Di Santo, J. P.; Tybulewicz, V. L.
Immunol. Today 2000, 21, 148.
14. 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 Â 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.
After the activation, supernatants were removed and transferred to another
well for b-hexosaminidase determination. Cells were lyzed in 2 cycles of
freeze-thaw and they were incubated for 90 min at 37 °C with 1 mM b-
2. Singh, R.; Matsuda, E. S. Annu. Rep. Med. Chem. 2007, 42, 379.
3. Mócsai, A.; Ruland, J.; Tybulewicz, V. L. J. Nat. Rev. Immunol. 2010, 10, 387.
4. Riccaboni, M.; Bianch, I.; Petrillo, P. Drug Discovery Today 2010, 15, 517.
5. Weinblatt, M. E.; Kavanaugh, A.; Genovese, M. C.; Musser, T. K.; Grosbard, E. B.;
Magilavy, D. B. N. Eng. J. Med. 2010, 3636, 1303.
6. 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.
7. Singh, R.; Masuda, E. S.; Payan, D. G. J. Med. Chem. 2012, 55, 3614.
8. Allan, R.; Jones, I.; Bartley, M.; Wilbraham, D.; Singh, D. Am. J. Respir. Crit. Care
Med. 2012, 185, A2774.
9. (a) Forns, P.; Esteve, C.; Taboada, L.; Alonso, J. A.; Orellana, A.; Maldonado, M.;
Carreño, C.; Ramis, I.; López, M.; Miralpeix, M.; Vidal, B. Bioorg. Med. Chem. Lett.
2012, 22, 2784; (b) Vidal, B.; Forns, M. P.; Castillo, M.; Erra, M. Mir, M.; PCT Int.
Appl. WO2012041476, 2012.
10. Kodama, I.; Noji, S.; Imamura, K.; Mizojiri, R.; Aoki, K.; Takagi, H.; Naka, Y.; Ito,
G.; Shinoda, K.; Fujiwara, A.; Kurihara, K.; Tanaka, M.; PCT Int. Appl.
WO2006093247, 2006.
11. 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-
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.
15. Kinases with < 50% inhibition at 1 lM: Alk(h), Aurora-A(h), Axl(h), CaMKIId(h),
CDK1/cyclinB(h), CHK1(h), CK1d(h), FAK(h), FGFR3(h), Flt1(h), Fms(h), Fyn(h),
GSK3b(h), IKKb(h), JNK3(h), Met(h), MKK6(h), PDK1(h), Pim-1(h), PKCbII(h),
PKC
ZAP-70(h); kinases with >50% inhibition at 1
Flt3(h), GSK3 (h), Hck(h), Itk(h), Lck(h), Lyn(h), Ret(h), Yes(h).
c
(h), PKC
e
(h), PRAK(h), Pyk2(h), ROCK-II(h), Rsk4(h),TAK1(h), TrkA(h),
lM: BTK(h), cSRC(h), Fgr(h),
a
16. Galli, S. J.; Kalesnikoff, J.; Grimbaldeston, M. A.; Piliponsky, A. M.; Williams, C.
mercaptoethanol, 0.133 mg/ml BSA(bovine serum albumin).
A 10 lL of
M. M.; Tsai, M. Annu. Rev. Immunol. 2005, 23, 749.
substrate poly-GT (100 g/mL final) and 4 l of Syk (1 nM final) were added
l
l