Discovery of 2-(α-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS

Article abstract of DOI:10.1016/j.bmcl.2008.12.073

A series of 2-(α-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Full text of DOI:10.1016/j.bmcl.2008.12.073

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1206–1209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-(a-methylbenzylamino) pyrazines as potent Type II inhibitors
of FMS
*
Christopher J. Burns, Michael F. Harte , Xianyong Bu, Emmanuelle Fantino, Marilena Giarrusso, Max Joffe,
Margarita Kurek, Fiona S. Legge, Pasquale Razzino, Stephen Su, Herbert Treutlein, Soo San Wan,
Jun Zeng, Andrew F. Wilks
Cytopia Research Pty Ltd, 576 Swan Street, Richmond, VIC. 3121, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-(a-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine
receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are
reported.
Received 14 November 2008
Revised 17 December 2008
Accepted 17 December 2008
Available online 25 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
FMS
CSF-1R
M-CSF-1
CSF-1
Kinase inhibitor
Macrophage
Macrophage or monocyte colony stimulating factor (CSF-1)
interacts with cells through its one specific trans-membrane
receptor FMS (also known as CSF-1R)—a receptor tyrosine kinase.
Signal transduction through the CSF-1/FMS ligand–receptor com-
plex leads to the differentiation and proliferation of cells of the
monocyte/macrophage lineage.1 Over-expression of CSF-1 and/or
FMS has been implicated in a number of disease states including
the growth and metastasis of particular cancers,2 in promoting
osteoclast proliferation in bone osteolysis,3 in inflammatory dis-
eases such as rheumatoid arthritis,4 atherosclerosis,5 and Crohn’s
disease6 and in renal allograft rejection.7 Strong evidence supports
the major role that tumor associated macrophages (TAM) play in
the microtumor environment.8,9 A monoclonal antibody to CSF-1
developed by Pfizer, PD-0360324, has recently entered Phase 1
clinical trials for rheumatoid arthritis.10 Therefore, small molecule
FMS inhibitors are expected to be valuable therapeutic tools tar-
geting various cancer and inflammatory diseases.
attention to the potential use of thiazolyl bisamides as FMS inhib-
itors in the treatment of cancer.18
Focused screening of compounds from Cytopia’s internal small
molecule library identified the racemic a-methylbenzylamino pyr-
azine derivative 1 (Fig. 1) as a potent (IC50 22 nM) inhibitor of
FMS.19
As well as being a potent inhibitor of FMS in biochemical assays,
compound 1 also completely blocked CSF-1 induced survival in pri-
mary murine bone marrow-derived macrophages (BMM).19
Subsequently, we found that the S enantiomer of 1 was over 35
times more potent in the enzyme assay compared with its anti-
pode (S enantiomer IC50 11 nM, R enantiomer IC50 416 nM).20 We
therefore focused all subsequent SAR investigations on the S enan-
tiomeric series.
Initially, we discovered that replacement of the aromatic ring
directly attached to the pyrazine ring of 1 by a simple halide al-
Several chemotypes have been identified as displaying highly
potent FMS inhibitory activity including 2,4-diaminopyrimi-
dines,11 aminoindazoles,12 dimethoxyquinazolines,13 quinoline ur-
eas,4 2-quinolones,14 pyridopyrimidinones,15 2,4-disubstituted aryl
amides,16 and anilinoquinolines.17 A recent paper has also drawn
OMe
HO
H
N
N
N
NH
O
N
1
* Corresponding author. Tel.: +61 3 9208 4259; fax: +61 3 9208 4299.
E-mail address: michael.harte@cytopia.com.au (M.F. Harte).
Figure 1. Initial screening hit 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.073

C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1206–1209
1207
Table 2
lowed us to retain sub-micromolar activity in biochemical as-
says.20 We therefore investigated the SAR of the aryl amide bond
using the 2-chloropyrazine scaffold. We confirmed that both the
presence of the amide bond proton and carbonyl were required
for potency (Table 1).
Alterations to the terminal position of the amide bond
H
Cl
N
N
N
NH
To probe the range of terminal moieties tolerated on the amide
bond, we prepared a series of derivatives, selected examples of
which are displayed in Table 2.
O
R
Compound
R
FMS IC50 (nM)
857
Substitution of the pyridyl ring meta to the amide bond (Table 2
8–10) greatly increased potency in the biochemical assay. We also
found that the pyridyl ring could be exchanged for a mono- (12, 14,
and 15) or disubstituted (13, 16) phenyl ring without losing po-
tency, thus removing potentially deleterious interactions with
metabolizing enzymes. Replacement of the pyridyl ring with other
heteroaromatic or heterocyclic rings (17, 18) led to a loss of
potency.
Whereas both 9 and 15 had high predicted clearance values
based on human microsomal data, 15 proved to have a reasonable
in vivo rat PK profile when dosed iv at 5 mg/kg: t1/2 5.9 h, blood
CLTOT 12.7 mL/min/kg and Vz 30.2 L/kg.
2
N
7
8
9
40
15
5
N
N
N
N
Counterscreening of 15 across a diverse panel of more than 300
kinases showed high potency against a limited number of en-
zymes, including the closely related Type III receptor tyrosine ki-
nases shown in Table 3, DDR1 and FRK.21 A Kd of greater than
Br
10
11
12
4
40
10
10 lM was seen against most other kinases. Compound 15 also
inhibited FMS dependent proliferation of the murine cell line MNFS
60 and CSF-1 dependent growth of murine BMM, as shown in Table
4, without showing general cellular cytotoxicity against human
kidney and liver cell lines HEK293 and HepG2, respectively.22
We prepared compounds of the type shown in Tables 1 and 2
via the procedure shown in Scheme 1. Thus, a catalytic Leuckart–
Wallach type reductive amination23 of commercially available
3-nitroacetophenone 19 gave the racemic amine. Resolution with
CF3
Cl
F
13
14
15
16
17
18
9
9
L-tartaric acid provided the m-nitro-substituted S-a-methylbenzyl-
amine 20. Following reduction of the nitro group and base-
promoted coupling to 2,6-dichloropyrazine, we generated a series
of amides via conventional coupling reactions with suitable
carboxylic acids.24
Homology models of the FMS kinase, based on a crystal struc-
ture of its most closely structurally related kinase cKit (PDB code
1T46) allowed us to perform docking studies on this series.26 Ini-
tially, docking experiments involving 9 indicated that the com-
pound interacted with the DFG-out (inactive) conformation of
2
2
F
S
2000
5000
N
N
Table 1
Alterations to the amide bond
Table 3
Kinase selectivity of 15
H
Cl
N
N
N
X
R
H
Cl
N
N
N
NH
Compound
X
R
FMS IC50 (nM)
O
2
3
4
NHCO
NH2
857
>5000
>5000
N
Enzyme
Kd (nM)
Enzyme
Kd (nM)
—
FMS
cKit
PDGFRb
1.9
2.2
2.3
3
5.7
7.8
9.1
12
RET
Src
95
1300
N(Me)CO
AURKB
EGFR
FGFR1,2,3
Flt3
JAK3
bRAF
TIE2
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
N
N
N
PDGFR
a
VEGFR-1
DDR1
VEGFR-2
FRK
5
6
NHCH2
NHSO2
>5000
>5000
VEGFR-3
27

1208
C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1206–1209
Table 4
Cellular activity of 15
To assess the validity of the two binding modes, we modified
compound 9 as shown in Table 5. Thus, when the pyridine N of 9
was exchanged with a CH to give compound 15 there was no
change in inhibitory activity on FMS. When we replaced the pyra-
zine ring of 9 with a 3,5-disubstituted pyridine (compound 21)
there was again minimal change in activity. However, when we re-
placed the pyrazine with a 2,6-disubstituted pyridine, 22, we lost
all inhibitory activity. This observation confirmed binding mode
B (Fig. 2): loss of the pyridine N from 9 does not affect activity
while loss of the pyrazine 4-N in this series does.
Cell line
MNFS 60
211
BMM
107
HEK293
>20,000
HepG2
IC50 (nM)
>20,000
a,b,c
NO2
d
O
H2N
H2N
NO2
NH2
19
20
Hence, as shown in Figure 3, the chloropyrazine moiety of 15
interacts with the adenine-hinge binding region of the ATP pocket
while the amide bond sits over the DFG motif in the ‘allosteric site’.
e
f
H
N
H
Cl
N
N
Cl
N
N
NH2
NH
N
O
15
Table 5
Inhibitory activity of modified pyridine and pyrazine ring compounds in FMS
biochemical assays
Scheme 1. Reagents and conditions: (a) ammonium formate, [RhCp*Cl2]2, MeOH,
70 °C, 5 h; (b) chiral resolution with
-tartaric acid25; (c) NaOH (aq); 25% over 3
L
steps; (d) H2, Pd/C, EtOH, 30 psi, 30 min, 100%; (e) 2,6-dichloropyrazine, dioxane,
K2CO3, 120 °C, 72 h, 85%; (f) m-toluic acid, EDAC, 4-pyrrolidinopyridine, NEt3,
CH2Cl2, rt, 48 h, 83%.
H
N
R
NH
O
X
the enzyme in two possible binding modes as shown in Figure 2. In
binding mode A the pyridine N of 9 is involved in a hinge binding
interaction with Cys666 whereas in binding mode B it is the 4-N
of the pyrazine ring that initiates this contact. In both cases there
are interactions with the DFG motif which is in the ‘out’
configuration.
Compound
R
X
FMS IC50 (nM)
5
Cl
N
9
N
N
N
Cl
Br
15
21
22
CH
N
2
5
N
N
N
Br
N
>5000
Figure 3. Model of 15 complexed with FMS in the DFG-out conformation.26
Proposed hydrogen bonds in black are shown between 4-N of the pyrazine and
hinge (Cys666 backbone), between the secondary amine and gatekeeper (Thr663
hydroxy), between the amide carbonyl and DFG motif (Asp796 backbone) and
Figure 2. Two potential binding modes of 9 in the ATP binding pocket of FMS.
Hinge interactions are with Cys666 and Thr663 (gatekeeper residue). In binding
mode A, hinge interactions are postulated for the pyridine N and amide carbonyl. In
binding mode B, hinge interactions are postulated for 4-N of the pyrazine and the
secondary amine. Putative hydrogen bonds are indicated by a dashed line.
between the amide NH and
a-helix (Glu633 carboxylate). The 3-methylbenzamide
moiety extends into the allosteric site.

C. J. Burns et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1206–1209
1209
16. Illig, C. R.; Chen, J.; Wall, M. J.; Wilson, K. J.; Ballentine, S. K.; Rudolph, M. J.;
DesJarlais, R. L.; Chen, Y.; Schubert, C.; Petrounia, I.; Crysler, C. S.; Molloy, C. J.;
Chaikin, M. A.; Manthey, C. L.; Player, M. R.; Tomczuk, B. E.; Meegalla, S. K.
Bioorg. Med. Chem. Lett. 2008, 18, 1642.
17. Smalley, T. L., Jr.; Chamberlain, S. D.; Mills, W. Y.; Musso, D. L.; Randhawa,
S. A.; Ray, J. A.; Samano, V.; Frick, L. Bioorg. Med. Chem. Lett. 2007, 17,
6257.
18. Scott, D. A.; Aquila, B. M.; Bebernitz, G. A.; Cook, D. J.; Dakin, L. A.; Deegan, T. L.;
Hattersley, M. M.; Ioannidis, S.; Lyne, P. D.; Omer, C. A.; Ye, M.; Zheng, X. Bioorg.
Med. Chem. Lett. 2008, 18, 4794.
19. Irvine, K. M.; Burns, C. J.; Wilks, A. F.; Su, S.; Hume, D. A.; Sweet, M. J. FASEB J.
2006, 20, 1921.
The 3-methylbenzamide moiety of 15 further extends into the
hydrophobic region of the allosteric site. Overlay of our homology
model with a subsequently published crystallographic structure of
FMS, PDB code 3BEA,15 produced an RMSD of 1.5 Å over C
a atoms,
validating our FMS modeling approach. Compounds that bind to
the inactive conformation of kinases in this manner are commonly
referred to as Type II kinase inhibitors.27
In summary, we have developed a new series of Type II FMS
inhibitors with excellent biochemical and cellular potency. Our
ongoing efforts to improve the PK profile of this series will be re-
ported in due course.
20. For a description of biochemical assay conditions see: Burns, C. J.; Harte, M. F.;
Palmer, J. T. Inhibitors of kinase activity, WO2008058341.
21. KINOMEscanTM screen performed by AMBIT Biosciences, San Diego. See:
Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.;
Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.;
Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.;
Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. Nat. Biotechnol. 2008,
26, 127.
Acknowledgments
The authors would like to thank our colleague James T. Palmer
for useful discussions and help in the preparation of this
manuscript.
22. Bone marrow macrophages (BMM) were isolated from C57BL6 murine bone
marrow using Miltenyi Biotec MACS beads (CD11b). BMM (3 ꢀ 104 cells/well)
were cultured in RPMI 1640 medium supplemented with 10% FBS, 2-
mercaptoethanol (50 mM) and the test compound for 1 h at 37 °C in 5% CO2.
CSF-1 (20 ng/mL, R&D Systems Inc., Minneapolis, MN) was then added and the
system was incubated for a further 5 days. The inhibitory activity of test
compounds against M-CSF induced BMM growth was determined using
Alamar BlueTM (Serotec). MNFS 60 cells [ATCC, CRL-1838] (5 ꢀ 103 cells/well)
were similarly cultured with CSF-1 for 72 h before addition of Alamar Blue.
HEK293 (4 ꢀ 105 cells/mL) and HepG2 cells (2 ꢀ 105 cells/mL) were cultured in
microtest plates in RPMI 1640 medium supplemented with 10% FBS and the
test compound for 72 h at 37 °C in 5% CO2. Cell proliferation was assessed using
Alamar BlueTM (Serotec).
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