Synthesis and evaluation of novel 3,4,6-substituted 2-quinolones as FMS kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.01.088

A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-qui

Full text of DOI:10.1016/j.bmcl.2008.01.088

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2097–2102
Synthesis and evaluation of novel 3,4,6-substituted
2-quinolones as FMS kinase inhibitors
Mark J. Wall, Jinsheng Chen, Sanath Meegalla, Shelley K. Ballentine, Kenneth J. Wilson,
Renee L. DesJarlais, Carsten Schubert, Margery A. Chaikin, Carl Crysler,
Ioanna P. Petrounia, Robert R. Donatelli, Edward J. Yurkow, Lisa Boczon,
Marie Mazzulla, Mark R. Player, Raymond J. Patch, Carl L. Manthey,
Christopher Molloy, Bruce Tomczuk and Carl R. Illig*
Johnson & Johnson Pharmaceutical Research & Development, Welsh & McKean Roads, Spring House, PA 19477, USA
Received 14 December 2007; revised 21 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of mac-
rophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-
yl)-2-quinolone-6-carbonitrile 21b, has an IC50 of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage
cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.
Ó 2008 Elsevier Ltd. All rights reserved.
The macrophage colony-stimulating factor-1 receptor
(FMS) is the cell surface receptor for colony-stimulating
factor-1 (CSF-1), which controls growth and differentia-
tion of the monocyte/macrophage lineage.1 FMS is a
member of the type III receptor tyrosine kinases that
also includes FLT-3, PDGFR, and KIT. Macrophages
are thought to play an important role in several diseases,
including cancer and inflammation. For example, of the
several leukocyte lineages present in the rheumatoid
arthritis synovium, increased levels of macrophages best
correlate with disease severity and response to therapy.2
In addition, expression of FMS in breast cancer has
been linked to poor survivability and increased tumor
size, where presumably the receptor is involved in local
invasion and metastasis.3–5 Consequently there is signif-
icant interest in modulating the CSF-1 pathway and sev-
eral structural classes of small-molecule inhibitors of
FMS have been reported.6–11
inhibitors.11 In this paper we describe the synthesis and
optimization of one of these classes of compounds, the
2-quinolones, shown in Figure 1.
A high-throughput screen using ThermofluorÒ technol-
ogy12 produced quinolone 1, with an IC50 of 1 lM for
FMS. Initial chemistry efforts began by synthesizing
analogues with substitution in the 3- and 6-positions
of the quinolone. These compounds could be readily
prepared by condensation of 2-aminophenyl ketones 2
with a-substituted acetic acids in phosphorus oxychlo-
ride to give the corresponding 2-chloroquinolines 3 that
are subsequently hydrolyzed to the corresponding quin-
olones 4 in aqueous acetic acid according to the proce-
dures outlined in Scheme 1. The aminophenyl ketones
We have recently published an X-ray crystal structure of
the FMS kinase domain in complex with two classes of
O
4
Cl
FMS
O
6
3
IC50 = 1 μM
N
H
O
Keywords: FMS; cFMS; Colony-stimulating factor-1 receptor; CSF-1;
M-CSF; 2-Quinolones.
1
*
Corresponding author. Tel.: +1 610 458 6057; fax: +1 610 458
8249; e-mail: cillig@prdus.jnj.com
Figure 1. ThermofluorÒ HTS screening hit.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.088

2098
M. J. Wall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2097–2102
O
R2
HO
R1
R1
R2
Cl
R1
70-80%
R2
O
a
b
O
10-80%
NH2
N
N
H
2
3
4a-m
Scheme 1. Reagents and conditions: (a) Phosphorus oxychloride, 105 °C; (b) 80% aqueous acetic acid, ammonium acetate, 110 °C.
and acetic acids used were commercially available, with
the exception of the 5-cyano analogue of 2 that was pre-
pared from the 5-bromo compound by reaction with
Table 1. FMS kinase activity of compounds 4a–m13
CuCN in refluxing DMF. Initial SAR studies probed
substituent effects at the 3-position of the quinolone.
Ethyl, methyl, and isopropyl esters for R2 were found
to be equipotent, whereas activity diminished with more
1
2
sterically demanding esters. The direct 3-ethylamido
analogue (R2 = CONHEt) was inactive, as was the cor-
responding free carboxylic acid (data not shown). How-
ever, a variety of five-membered aromatic heterocycles
were identified as suitable replacements for the ethyl car-
boxylate (Table 1). Of these, 3-methylisoxazol-5-yl was
the most potent providing a 7-fold improvement over
the original hit compound (compare 4a to 1). More ba-
sic aromatic heterocycles such as imidazole 4k also affor-
ded a significant (5-fold) increase in potency relative to
1.
R
R
N
H
O
Compound
R1
Cl
R2
IC50 (lM)
O
N
4a
0.14
CH
3
O
N
N
N
N
N
N
N
4b
4c
4d
4e
4f
CN
F
0.09
0.25
0.34
>5
CH
3
O
O
O
O
The importance of substitution at the 6-position of the
quinolone system was demonstrated in early series ana-
logues. A dramatic loss of activity (>50-fold) was ob-
served with the des-chloro analogue of 1; the 7- and 8-
positional isomers were also found to be inactive (data
not shown). Additional studies probing substituent ef-
fects at the 6-position were carried out on 3-isoxazol-
5-yl analogues (Table 1). In this case, chloro and cyano
groups (4a and 4b, respectively) imparted the highest
levels of activity.
CH
3
3
3
3
NO2
NH2
H
CH
CH
CH
>5
The SAR of the central core of the quinolone scaffold
was examined next by preparing the 1,8-, 1,7-, 1,6-,
and 1,5-naphthyridin-2-one analogues (9a–d). The syn-
thesis of these compounds required aminopyridyl ke-
tones 5–8 that were synthesized according to the
procedures outlined in Scheme 2. Compounds 5–7 were
prepared according to the procedure of Turner14 by
ortho lithiation of 2-, 3-, and 4-(pivaloylamino)pyridines
followed by quenching with the Weinreb amide15 of ben-
zoic acid and acid-catalyzed removal of the pivaloyl
group. The 3-aminopyridyl-2-phenyl ketone 8 was pre-
pared by lithium-bromine exchange of 2-bromo-3-(piv-
aloylamino)pyridine followed by quenching with the
Weinreb amide and deprotection. The aminopyridyl ke-
tones 5–8 were then condensed with 3-methylisoxazole-
5-acetic acid according to the procedures in Scheme 1
to give compounds 9a–d.
O
O
4g
4h
4i
Cl
0.30
0.32
0.36
0.83
0.18
0.28
0.55
i-Pr
Cl
NH
2
H
N
Cl
H
N
N
N
4j
Cl
CH
3
4k
4l
Cl
N
H
N
N
CN
CN
N
H
The most potent compound was the 1,7-naphthyridin-2-
one 9b (Table 2), which was 5-fold less potent than the
analogous quinolone compound 4a. The 1,8-naphthyri-
din-2-one 9a also had some activity (IC50 = 1 lM), but,
CH
3
4m
N

M. J. Wall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2097–2102
2099
O
in general, these compounds were less active and this
scaffold was not further investigated.
a
b,c
N
N
O
N
NH2
N
H
NH2
O
The synthetic route used to optimize the 4-substituent is
shown in Scheme 3. Since few aminophenylketones 2
were commercially available, an alternate synthetic
route to our earlier method (Scheme 1) was required.
Thus, 5-chloroisatoic anhydride 10 was first converted
to the dibenzyl-protected aminobenzoate 11, which
was then coupled to 3-methylisoxazole-5-acetic acid
using neat phosphorus oxychloride. Cyclization with
NaH, followed by reaction with triflic anhydride gave
the triflate 13. The triflate was then coupled to boronic
acids using a Suzuki–Miyaura reaction, followed by
benzyl deprotection with neat triflic acid to yield com-
pounds 14a–j.
5
Cl
Cl
d,c Cl
a
O
A
A
A
B
NH2
B
N
H
B
NH2
6 A = CH, B = N
7 A = N, B = CH
O
N
Br
N
Br
N
b,c
a
O
NH2
N
H
NH2
8
Scheme 2. Reagents and conditions: (a) Pivaloyl chloride, NEt3,
DCM; (b) n-BuLi, THF, À78 °C, 3 h, then PhCONMeOMe; (c) 3 N
HCl, 100 °C; (d) t-BuLi, THF, À78 °C, 3 h, then PhCONMeOMe.
The data in Table 3 show that only a modest 1.5-fold
improvement in potency over the unsubstituted phenyl
group was obtained with a 4-ethylphenyl group 14b or
with a cycloheptene ring 14g. With the former, ethyl
substitution appears to be optimal; methyl and isopro-
pyl substitution resulted in diminished activity. Within
the cycloalkene series, the cycloheptene 14g was more
potent than the cyclohexene 14f (>2-fold), and satura-
tion to the cyclohexane 14h further reduced activity.
We attempted to introduce polar functionality in this
portion of the molecule to improve the solubility and
potency of the series. Hydroxy and methoxy substitu-
ents (14d and 14e, respectively), while tolerated, afforded
no increase in inhibitory potency. Conversely, incorpo-
ration of a pyridyl ring (14i) significantly attenuated
activity.
Table 2. FMS kinase activity of napthyridones 9a–d
Compound
Structure
IC50 (lM)
N
Ph
O
Cl
9a
1.0
N
N
H
O
O
N
Ph
Cl
N
9b
9c
9d
0.72
8%a
>20
N
H
O
N
In the course of these analogue studies, an X-ray co-
crystal structure of quinolone 4a bound to the FMS ki-
nase domain was obtained.11 Figure 2 shows compound
4a bound in the nucleotide-binding pocket of FMS in
the inactive form. Both the N-1 and O-2 of the quino-
lone ring, in the keto tautomeric form, make hydrogen
bonds with the backbone of the hinge residues Cys666
Ph
O
N
N
H
O
O
˚
˚
(2.90 A) and Glu664 (3.32 A). The hydrophobic phenyl
ring occupies the ribose-binding pocket, and the chloro-
aryl ring is located in the adenine pocket. The 3-methy-
lisoxazole is rotationally disordered. Two positions are
seen which differ by a 180-degree rotation of the bond
with the quinolone core. Specific interactions between
the 3-methylisoxazole and the protein are not observed.
N
Ph
N
N
H
O
a Percent inhibition at 5 lM.
O
O
O
Cl
Cl
a
Cl
b
OBn
O
OBn
Bn
O
N
N
O
N
H
NHBn
12
R3
10
11
O
N
N
O
OTf
O
O
Cl
Cl
c
d, e
N
H
O
N
Bn
14a-j
13
Scheme 3. Reagents and conditions: (a) Benzyl bromide, Na2CO3, DMF; (b) 3-methylisoxazole-5-acetic acid, phosphorus oxychloride; (c) NaH;
triflic anhydride, DMF; (d) R3B(OH)2, Pd(PPh3)4, Na2CO3, toluene, ethanol, 80 °C; (e) triflic acid.

2100
M. J. Wall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2097–2102
Table 3. FMS kinase activity of compounds 14a–j
R3
O
O
N
Cl
N
H
Compound
R3
IC50 (lM)
14a
0.11
14b
14c
14d
14e
14f
14g
14h
14i
0.09
0.20
0.17
0.14
0.22
0.09
0.34
2.1
Figure 2. Crystal structure of 4a bound to the FMS kinase domain.
˚
FMS residues within 5 A of the quinolone are shown with gray
carbons and the quinolone is shown with green carbons. The green
dotted lines represent hydrogen bonds made by the quinolone core.
The black dotted line is a close contact between two oxygens in the
structure and predicted hydrogen bonds for the imidazoles 21a and
21b. (PDB Id: 2i0v).
SEM
N
SEM
N
H
N
O
c, d, e
a,b
O
HO
N
N
N
17
14j
0.24
15
16
g, h
R
I
R
I
f
R
NH2
N
N
O
In light of this structural information, we re-visited
quinolone analogues substituted at the 3-position with
imidazole systems, as these generally tended to exhibit
improved solubility characteristics as compared with
their isoxazole counterparts. Specifically, we targeted
2-methylimidazole derivatives, since these were expected
to occupy the same spatial region as the 3-methylisoxaz-
ole system in this protein-binding mode.
18a,b
19a,b
NH2
20a,b
N
i, j, k
20a,b + 17
R
N
H
N
H
O
The synthesis of the required 2-methylimidazole-5-acetic
acid 17 is shown in Scheme 4. 2-Methylimidazole was
protected with a SEM group, which directs the metalla-
tion with tert-butyl lithium to imidazole C-5. The anion
was quenched with N,N-dimethylformamide to yield the
aldehyde 16, which was subsequently homologated to
the acetic acid ethyl ester using methyl (methyl-
thio)methyl sulfoxide followed by ethanolic HCl.16 The
ester was then hydrolyzed to the acid using KOH to give
17. We then prepared aminoketones 20a,b to incorpo-
rate the optimized groups in the C-4 and C-6 positions
of the quinolone scaffold. This was accomplished from
21a R = Cl
21b R = CN
Scheme 4. Reagents and conditions: (a) SEMCl, NaH, DMF; (b) t-
BuLi, DMF, THF, À78 °C; (c) MeSCH2SOMe, NaH, THF; (d) 3 N
HCl (g)/EtOH; (e) 2 N KOH, ethanol; (f) DMF dimethyl acetal,
toluene, 100 °C; (g) i-PrMgCl, 4-EtPhCONMeOMe; (h) 3 N aq HCl,
THF; (i) POCl3, DCM, 25 °C; (j) piperidine, toluene, 110 °C; (k) 6 N
HCl (aqueous), ethanol (1:1), 80 °C.
the 4-chloro- or 4-cyano-2-iodoaniline by first protect-
ing the amino group as the amidine followed by forma-
tion of the Grignard reagent with isopropylmagnesium

M. J. Wall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2097–2102
2101
Table 4. IC50 data for compounds 21a,b for cellular BMDM FMS
assay and for in vitro FMS, FLT-3, KIT, and PDGFR-b assays
12.00
10.00
8.00
6.00
4.00
2.00
0.00
Compound BMDM FMS FLT-3 KIT PDGFR-b
(lM) (lM) (lM)
0.067 >1
0.170 >1
(lM)
(lM)
21a
21b
0.028
0.005
0.0021 0.034
0.0025 0.047
Table 5. Pharmacokinetic parameters for 21b in rat; 2 mg/kg IV and
10 mg/kg PO
Compound t1/2 (IV) Cmax
(min)
VZ
Cl
(ng/ml) (ml/kg) (ml/min/kg) (%)
F
CSF-1
21b mg/kg
-
+
0
+
5
+
+
21b
64
317 8600 93 56
0
20
50
Figure 3. Pharmacodynamic activity of 21b at FMS. B6C3F1 mice
were dosed orally with 21b in 20% 2-hydroxypropyl-b-cyclodextrin and
6 h later given an iv injection containing 1.0 lg recombinant CSF-1.
Fifteen minutes later, the mice were euthanized and c-fos mRNA was
measured in spleen lysates.
chloride according to the method of Knochel.17,18 The
magnesium species was then reacted with the Weinreb
amide of 4-ethylbenzoic acid followed by amidine
deprotection to give 20a,b. The aminophenylketones
20a,b were then coupled to the imidazole acetic acid
17 with phosphorus oxychloride in dichloromethane fol-
lowed by cyclization with piperidine in refluxing toluene.
Finally, SEM group removal was effected with 6 N HCl
in ethanol to give 21a,b.
to baseline by 30 min. Furthermore, this induced expres-
sion was shown to be dose-dependent and specific for
CSF-1 as it was blocked completely in mice pre-dosed
with anti-CSF-1 (data not shown). Figure 3 shows the
FMS inhibitory effect of an oral dose of 5, 20, and
50 mg/kg of 21b after 6 h. In the group treated with
50 mg/kg of 21b, c-fos mRNA expression was reduced
to control levels showing that FMS signaling is com-
pletely inhibited.
The data in Table 4 show that introduction of a 2-
methyl group on the imidazole has a profound effect
on the activity of the series. In the enzymatic assay, both
compounds were in the 2 nM range and, in the cellular
assay that measures proliferation of bone marrow-de-
rived macrophages (BMDM)10 in response to CSF-1,
the cyano 21b was 5 nM, 5-fold better than the chloro
21a. It appears that the imidazole can make an addi-
tional hydrogen bond with the FMS kinase (see
Fig. 2). This may be the reason for the improved activity
of the imidazole compared to the isoxazole. There is no
clear structural explanation for the large gain in activity
with the addition of the methyl group.
In conclusion, we have identified a novel and potent
class of FMS kinase inhibitors based on a quinolone
core. The compounds have good selectivity versus highly
homologous kinases and good oral bioavailability. Inhi-
bition of FMS signaling in vivo was also demonstrated
in a mouse pharmacodynamic model.
References and notes
The selectivity of 21a and 21b toward other kinases in
the type III receptor kinase family (FLT-3, KIT, and
PDGFR-b) was also evaluated (Table 4).13 Both com-
pounds showed excellent selectivity for FMS versus
PDGFR-b (>400-fold) and, versus KIT, good selectivity
was also achieved (70- and 30-fold for cyano and chloro
analogues 21b and 21a, respectively). Both compounds
exhibited modest selectivity for FMS versus FLT-3,
(15- to 20-fold).
1. Sherr, C. J. Biochim. Biophys. Acta 1988, 948, 225.
2. Moss, S. T.; Hamilton, J. A. Immunobiology 2000, 202, 18.
3. Kluger, H. M.; Dolled-Filhart, M.; Rodov, S.; Kacinski,
B. M.; Camp, R. L.; Rimm, D. L. Clin. Cancer Res. 2004,
10, 173.
4. Lin, E. Y.; Nguyen, A. V.; Russell, R. G.; Pollard, J. W. J.
Exp. Med. 2001, 193, 727.
5. Yee, L. D.; Liu, L. Anticancer Res. 2000, 20, 4379.
6. Conway, J. G.; McDonald, B.; Parham, J.; Keith, B.;
Rusnak, D. W.; Shaw, E.; Jansen, M.; Lin, P.; Payne, A.;
Crosby, R. M.; Johnson, J. H.; Frick, L.; Lin, M. H.;
Depee, S.; Tadepalli, S.; Votta, B.; James, I.; Fuller, K.;
Chambers, T. J.; Kull, F. C.; Chamberlain, S. D.;
Hutchins, J. T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
16078.
The pharmacokinetic profile of compound 21b in the rat
shows that the compound has good oral bioavailability
at 56% (Table 5), although the rate of clearance was
high.
7. Irvine, K. M.; Burns, C. J.; Wilks, A. F.; Su, S.; Hume, D.
A.; Sweet, M. J. FASEB J. 2006, 20, 1921.
8. Ohno, H.; Kubo, K.; Murooka, H.; Kobayashi, Y.;
Nishitoba, T.; Shibuya, M.; Yoneda, T.; Isoe, T. Mol.
Cancer Ther. 2006, 5, 2634.
9. 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.
Having obtained suitable pharmacokinetics for in vivo
testing, compound 21b was evaluated in a mouse phar-
macodynamic model that is based on the ability of
CSF-1 to induce c-fos mRNA in macrophages.19 Fol-
lowing tail vein injection of CSF-1, c-fos mRNA in-
creased more than 10-fold in the spleen. The induction
was robust within 15 min but was transient, returning

2102
M. J. Wall et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2097–2102
10. Patch, R. J.; Brandt, B. M.; Asgari, D.; Baindur, N.;
Chadha, N. K.; Georgiadis, T.; Cheung, W. S.; Petrounia,
I. P.; Donatelli, R. R.; Chaikin, M. A.; Player, M. R.
Bioorg. Med. Chem. Lett. 2007, 17, 6070.
11. Schubert, C.; Schalk-Hihi, C.; Struble, G. T.; Ma, H. C.;
Petrounia, I. P.; Brandt, B.; Deckman, I. C.; Patch, R. J.;
Player, M. R.; Spurlino, J. C.; Springer, B. A. J. Biol.
Chem. 2007, 282, 4094.
12. Carver, T. E.; Bordeau, B.; Cummings, M. D.; Petrella, E.
C.; Pucci, M. J.; Zawadzke, L. E.; Dougherty, B. A.;
Tredup, J. A.; Bryson, J. W.; Yanchunas, J., Jr.; Doyle,
M. L.; Witmer, M. R.; Nelen, M. I.; DesJarlais, R. L.;
Jaeger, E. P.; Devine, H.; Asel, E. D.; Springer, B. A.;
Bone, R.; Salemme, F. R.; Todd, M. J. J. Biol. Chem.
2005, 280, 11704.
KIT reactions contained 1 nM KIT, 50 lM ATP, and
100 lg/ml poly Glu4Tyr. PDGFR-b reactions contained
12 nM PDGFR-b, 10 lM ATP, and 10 lg/ml poly
Glu4Tyr. Typical between-run coefficient of variation
was less than 30%.
14. Turner, J. A. J. Org. Chem. 1990, 55, 4744.
15. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
16. Garuti, L.; Giovanninetti, G.; Bova, S.; Chiarini, A. Arch.
Pharm. 1988, 321, 377.
17. Varchi, G.; Jensen, A. E.; Dohle, W.; Ricci, A.; Cahiez,
G.; Knochel, P. Synlett 2001, 477.
18. Abarbri, M.; Thibonnet, J.; Berillon, L.; Dehmel, F.;
Rottlander, M.; Knochel, P. J. Org. Chem. 2000, 65,
4618.
19. Male B6C3F1 mice (Taconic Farms), 6 weeks of age, were
used to define the pharmacodynamic activity of FMS
inhibitors. Six hours following an oral dose of vehicle or
compound formulated in 20% 2-hydroxypropyl-b-cyclo-
dextrin (HPCD), groups of six mice were administered
saline or 1.0 lg CSF-1 (Cell Biosciences) by tail vein
injection. Fifteen minutes later, mice were euthanized.
Spleens were isolated and snap frozen on dry ice. The
frozen tissue was homogenized in 1 ml of Trizol (Invitro-
gen) per 50 mg of tissue using a hand-held Pellet Pestle
(Kontes). RNA was purified according to the Trizol
instructions and treated with 6.8 Kunitz units of RNase
free DNase (Qiagen) to degrade contaminating genomic
DNA. Sample RNA was purified further using RNeasy
columns (Qiagen). RT-PCRs were performed in a 25 lL
reaction volume using Reverse Transciptase qPCR Master
Mix (Eurogentec) and approximately 50 ng RNA based
on OD260. Applied Biosystems, Inc., was the source for the
primer probe set for mouse c-fos mRNA (part#
Mm00487425) and 18S rRNA (part# 4333760F). Ampli-
fication and detection were performed using the ABI
Prism 7700 Sequence Detection system with the following
profile: 48 °C for 30 min, 95 °C for 10 min and 40 cycles of
95 °C for 15 s and 60 °C for 1 min. Standard curves (six 4-
fold dilutions) were created for c-fos mRNA and for 18S
rRNA using RNA isolated from one vehicle-treated, CSF-
1-induced mouse. Relative expression levels in all other
samples were calculated based on the standard curves. c-
Fos mRNA values were divided by 18S rRNA values to
normalize for total RNA content.
13. FMS, FLT-3, KIT, and PDGFR-b kinase assays: The full
cytoplasmic regions of FMS (538–972) and FLT-3 (571–
993) encompassing the tyrosine kinase domains were
expressed and purified from a baculovirus system as
described (Schalk-Hihi, C.; Ma, H. C.; Struble, G. T.;
Bayoumy, S.; Williams, R.; Devine, E.; Petrounia, I. P.;
Mezzasalma, T.; Zeng, L.; Schubert, C.; Grasberger, B.;
Springer, B. A.; Deckman, I. C. J. Biol. Chem. 2007, 282,
4085). KIT and PDGFR-b was purchased from ProQin-
ase. FMS 555–568 peptide (SYEGNSYTFIDPTQ) was
synthesized and purified by AnaSpec. FMS was assayed
using a fluorescence polarization (FP) competition immu-
noassay that measured FMS phosphorylation of FMS
555–568 peptide at Y561. The reaction mixture (10 lL)
contained 100 mM Hepes, pH 7.5, 1 mM DTT, 0.01%
Tween 20 (v/v), 2% DMSO, 308 lM FMS 555–568
peptide, 1 mM ATP, 5 mM MgCl2, and 0.7 nM FMS.
The reaction was initiated with ATP, incubated 80 min at
room temperature, and quenched by the addition of
5.4 mM EDTA. Ten microliters of FP buffer/tracer/
phospho-Y antibody mix (Tyrosine kinase assay kit,
Green P2837, Invitrogen) was added to the quenched
reaction, and FP was measured after 30 min using an
Analyst reader (Molecular Devices) at excitation/emission
of 485/530 nm. FLT-3, KIT, and PDGFR-b were assayed
using the fluorescence polarization competition format as
described for FMS except that poly Glu4Tyr (Sigma) was
used as a universal substrate. FLT-3 reactions contained
10 nM FLT-3, 113 lM ATP, and 20 lg/ml poly Glu4Tyr.