Discovery of novel FMS kinase inhibitors as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2008.01.059

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC₅₀ = 0.0008 μM) served as a proof-of-concept candidate in a collagen-induced model of arthri

Full text of DOI:10.1016/j.bmcl.2008.01.059

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1642–1648
Discovery of novel FMS kinase inhibitors
as anti-inflammatory agents
Carl R. Illig, Jinsheng Chen, Mark J. Wall, Kenneth J. Wilson, Shelley K. Ballentine,
M. Jonathan Rudolph, Renee L. DesJarlais, Yanmin Chen, Carsten Schubert,
Ioanna Petrounia, Carl S. Crysler, Christopher J. Molloy, Margery A. Chaikin,
Carl L. Manthey, Mark R. Player, Bruce E. Tomczuk and Sanath K. Meegalla*
Johnson & Johnson Pharmaceutical Research & Development, Welsh & McKean Roads, Spring House, PA 19477, USA
Received 14 December 2007; revised 14 January 2008; accepted 15 January 2008
Available online 19 January 2008
Abstract—The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted aryla-
mides. Compound 8 (FMS kinase IC50 = 0.0008 lM) served as a proof-of-concept candidate in a collagen-induced model of arthritis
in mice.
Ó 2008 Elsevier Ltd. All rights reserved.
The colony stimulating factor-1 receptor (CSF-1R, also
known as M-CSFR, or FMS) is the exclusive receptor
for its ligand, colony stimulating factor-1 (CSF-1 or
macrophage colony stimulating factor, M-CSF). The
binding of CSF-1 to the extracellular domain of FMS
induces the dimerization and trans-autophosphorylation
of several cytoplasmic tyrosine residues. These phos-
phorylated sites function as binding sites for Src homol-
ogy-2 (SH2) domain-containing signaling proteins
which promote gene expression and proliferation. High
expression of FMS is limited principally to monocyte/
macrophages, oocytes, trophoblasts, mammary epithe-
lium (during lactation), and to cells of the macrophage
lineage, while CSF is the predominant growth factor
for macrophage lineage cells including osteoclasts.1,2
Recent studies have demonstrated a direct correlation
between tumor-associated macrophage numbers and tu-
mor progression3 and between synovial macrophage
numbers and disease severity in rheumatoid arthritis.4
Further, osteoclasts mediate bone erosions leading to
pain and fracture in metastatic bone disease and defor-
mity in rheumatoid arthritis.
Hence the inhibition of FMS appears to be of therapeu-
tic value in treating diseases such as rheumatoid arthritis
and metastatic cancer to the bone where osteoclasts and
macrophages are pathogenic. This hypothesis is also
well-supported by the biological studies conducted with
CSF-1 deficient mice.5–7
A recent publication8 describes the identification of
arylamides (1 and 2) as FMS inhibitors. Herein we de-
scribe the results of further investigation of the SAR
of this chemotype and the identification of a potent
FMS inhibitor suitable for in vivo studies (See Fig. 1).
In addition to the lead-like properties of the 5-hydroxy-
methyl compound 2, it also stabilized an engineered
form of the FMS kinase domain9 and thereby provided
X-ray crystallographic information to guide optimiza-
tion efforts. It was immediately apparent from the
N
H
N
N
H
N
CN
N
3
4
O
O
O
6
O
OH
Keywords: FMS; CSF-1R; M-CSF; Colony stimulating factor-1;
Macrophages; Anti-inflammatory activity.
IC50 = 0.4 μM
IC50 = 0.024 μM
2
1
*
Figure 1. FMS high-throughput screening hit
compound 2.
1 and the lead
Corresponding author. Tel.: +1 610 458 8959; fax +1 610 458 8249;
e-mail: smeegall@prdus.jnj.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.059

C. R. Illig et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1642–1648
1643
disubstituted arylamide chemotype. The initial focus
was the introduction of amine substituents at the 2-
and 4-positions as the ease of sequential SNAr reactions
on dihalonitrobenzenes permitted rapid analoging for
SAR development. Two consecutive SNAr reactions of
2,4-dichloronitrobenzene 3 (X = Cl) with 4-methylpiper-
idine and a primary or secondary amine in refluxing
THF, respectively, installed the desired C-2 and C-4
substituents in high yields. The reduction of the nitro
group to the aniline followed by standard amide bond
formation with 5-cyanofuran-2-carbonyl chloride affor-
ded the desired final product 7. Compound 16 was pre-
pared using thiomorpholine as the second nucleophile
followed by oxidation prior to acid chloride coupling.
Compounds 15 and 20 were prepared employing
methylamine as the second nucleophile followed by acyl-
ation and sulfonylation prior to the amide bond forma-
tion. Compound 19 was made in the same fashion using
NH3 in place of methylamine. Compound 21 was made
substituting amine R1R2NH with sodium ethoxide.
Compound 22 was synthesized by the Sonogashira cou-
pling of propargyl alcohol with intermediate 4 where
X = Br, followed by Pd-catalyzed hydrogenation, mesy-
lation and substitution of the mesylate with methylethyl-
amine followed by acylation of the resulting aniline.
N
N
Cl
NO
NO
2
2
NO
a
b
2
R
1
X
N
X
R
2
3
4
5
CN
c
d
N
N
O
H
N
NH
2
R
1
R
O
1
N
R
N
R
2
2
6
7
Scheme 1. Synthesis of 2,4-disubstituted arylamides. Reagents and
conditions: (a) 4-methylpiperidine/ rt; (b) R1R2NH, THF, reflux 2 h;
(c) H2, Pd/C, EtOH, rt, 1 h; (d) 5-cyanofuran-2-carbonyl chloride,
DCM, Et3N, rt, 3 h.
co-crystal structure of FMS and 2 that there was insuf-
ficient space at the 6-position of the benzene ring to al-
low substitution without significant disruption of key
binding interactions. Initial efforts geared toward
exploring the C-5 position did not lead to further
improvement in potency of the lead compound (data
not shown). The co-crystal structure of the kinase do-
main of FMS and 28 revealed the C-4 position to be a
promising site for further elaboration, and optimization
efforts began at this site.
First, the SAR at the C-4 position (Table 1, A) was ex-
plored with a variety of N-substitutions, including both
saturated and unsaturated N-containing heterocycles.
As we have previously reported that both 5-cyanofuran
The synthesis of compound 7 in Scheme 1 illustrates the
general chemistry involved in the synthesis of the 2,4-
Table 1. FMS enzyme inhibitory activity10 for 8–23
B
H
N
C
O
A
a
Compound
A
B
C
FMS IC50 (lM)
N
N
CN
CN
CN
8
0.0008
HN
N
Me
N
N
N
N
N
9
10
11
12
0.0010
0.0007
0.0009
0.0006
HN
HN
O
Me
Me
Me
Me
N
N
HN
O
N
N
MeO S
2
CN
CN
N
O
O
N
HO
N
N
13
0.0110
O
Me
CN
(continued on next page)

1644
C. R. Illig et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1642–1648
Table 1 (continued)
a
Compound
A
B
C
FMS IC50 (lM)
N
N
N
N
N
N
N
N
N
N
N
N
14
15
16
17
18
19
20
21
22
23
O
O
O
O
O
O
O
O
O
O
0.0170
0.0830
0.0007
0.0640
0.0030
0.0710
0.0250
0.0210
0.0520
0.0630
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
O
N
N
Me
Me
N
O S
2
Me
N
N
N
N
MeO SN
2
H
MeO SN
2
Me
EtO
Me
N
Et
F
aFor assay information see Ref. 10.
and 4-cyanopyrrole analogues8 at the C-1 position
exhibited essentially equal potency, a fair activity com-
parison can be made between cyanofuran and cyanopyr-
role analogues.
only the need for a basic group at the 4-position but a
steric requirement that these smaller groups do not
fulfill.
The chemistry shown in Scheme 1 was utilized to per-
form a limited exploration of six-membered N-contain-
ing heterocycles at the C-2 position (Table 2, B). The
compounds were evaluated in an in vitro FMS assay
as well as in a cell-based FMS activity assay (bone-mar-
row derived macrophage (BMDM) proliferation as-
say).10 The data in the Table suggest that small
lipophilic groups are tolerated on the piperidine (24–
27). The replacement of piperidine with morpholine 29
and piperidinone 28 showed a significant decrease in
activity. As reported previously8 both 5-cyanofuran
and 4-cyanopyrrole analogues at the C-1 position (24
vs 25) exhibited essentially equal potency.
The biological activities of the analogues revealed that
the piperazine is a preferred substituent at the C-4 posi-
tion (8–12). The addition of a methyl group to the
4-piperazine (9 vs. 8) had no effect. Introduction of elec-
tron-withdrawing substituents to attenuate the basicity
of the piperazine nitrogen also did not have a significant
effect on the activity as observed in compounds (10–12).
Replacement of basic piperazine at C-4 was detrimental,
as seen in morpholine 13, piperidine 14, and pyrazole 17.
The replacement of piperazine with imidazole 18 re-
tained activity, though 3-fold less potent than the piper-
azine analogue 8. The insertion of an amide linker in
between the central core and piperazine 15 also exerted
a negative effect in the FMS inhibitory activity. The
introduction of halides, alkyl and alkoxy substituents
and sulfonamides (22, 21, 23, 19 and 20, respectively) re-
sulted in a loss of potency. These results indicate not
The SAR development in the central ring was carried
out by the replacement of the benzenoid ring with vari-
ous azaheteroaromatic rings. The synthetic routes em-
ployed in the preparation of these analogues are

C. R. Illig et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1642–1648
Table 2. FMS inhibitory activity for 24–29
1645
B
H
N
C
O
N
N
a
b
Compound
B
C
FMS IC50 (lM)
BMDM IC50 (lM)
24
0.0008
0.0017
0.0010
0.005
N
O
CN
H
N
25
26
0.002
0.002
N
N
CN
CN
CH3
O
O
O
O
F
27
28
29
0.0063
>0.060
0.080
ND
>10
2.8
N
CN
CN
CN
O
N
N
O
a For assay information see Ref. 10.
b BMDM- bone marrow derived macrophage proliferation assay. For assay information, see Ref. 8.
shown in Scheme 2. The common synthetic features in-
clude two nucleophilic substitutions employing 4-meth-
ylpiperidine and 4-methylpiperazine as the nucleophiles
with various dichloronitroazaheterocycles (30, 36, 41,
and 46) and conversion of the nitro functions to amines
followed by acylation with 5-cyanofuran-2-carbonyl
chloride. The synthesis of the pyrazine analogue 35
was carried out by mono-substitution of 2,6-dichloro-
pyrazine with 4-methylpiperidine followed by nitration
of the resulting product to obtain the intermediate 32.
The yield of the formation of this compound was poor
(8%) due to the rapid formation of bisnitro compound.
Although the preparation of two pyridyl analogues (40
and 51) and the pyrimidine analogue 45 was more
straightforward, the synthesis of compound 51 required
the preparation of 3,5-dichloro-2-nitropyridine 47. This
compound was prepared in 60% yield by the oxidation
of the corresponding amine 46 with Caro’s acid,
K2S2O8, in concd H2SO4. Finally the displacement of
the 3-chloro substituent of 47 with 4-methylpiperidine
was complicated by the competitive displacement of
the 2-nitro group.
B
B
B
Cl
B
B
H
N
NO
NH
NO
2
2
2
e
N
N
b
2
N
C
a
a
c
d
N
N
N
N
N
N
N
N
N
Cl
A
A
O
Cl
Cl
A
(33)
(32)
(34)
(30)
(31)
(35)
B
B
B
Cl
B
H
N
NO
2
NH
NO
NO
C
2
2
d
e
c
N
N
N
N
N
O
A
X
A
X
A
X
Cl
X
Cl
X
(39)
(38)
(40)
X = C-H (36)
X = N (41)
(37)
(44)
(43)
(45)
(42)
B
B
B
Cl
Cl
B
H
N
NO
NH
2
NO
NH
NO
2
2
2
2
C
a
f
c
e
d
N
N
N
N
N
N
O
Cl
A
A
Cl
Cl
A
(49)
(48)
(50)
(46)
(47)
(51)
Scheme 2. Synthesis of heterocyclic core analogues. Reagents and conditions: (a) 4-Methylpiperidine/THF (b) NO2BF4, CH3CN; (c) N-
methylpiperazine, THF; (d) Pd/C, EtOH, H2 (10 psi); (e) 5-cyanofuran-2-carbonyl chloride, Et3N, DCM; (f) H2SO4, K2S2O8, 0 °C ! RT, 2 h.

1646
C. R. Illig et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1642–1648
Table 3. FMS inhibitory activity10 for heterocyclic core analogues
Glu664
N
Y
H
N
CN
X
O
2.8
2.6
Z
O
Cys666
3.2
N
Thr663
Lys666
N
Compound
% Inhibition
at 2 lM
FMS IC50
(lM)a
26
35
40
45
51
X = Y = Z = CAH,
100
8
0.001
—
Glu633
X = Z = N, Y = CAH,
X = N, Y = Z = CAH,
X = Y = N, Z = CAH,
X = Y = CAH, Z = N
Leu785
97
2
0.01
—
69
0.72
a For assay information see Ref. 10.
The FMS inhibition data10 for these aza analogues are
shown in Table 3. All the heterocyclic core analogues
were less active than the corresponding benzenoid coun-
terpart 26. The presence of a single N in the ring was
better tolerated than two and the preferred position of
the N appeared be the 3-position between the two exo-
cyclic nitrogens. However, the most active compound
40 was 10-fold less active than the corresponding benze-
noid compound while its regioisomer 51 exhibited 700-
fold less potency. The pyrazine and pyrimidine ana-
logues 35 and 45 showed negligible inhibition of FMS
at 2 lM.
Binding models for the interaction of FMS kinase with
4-substituted analogues could readily be built from the
published crystal structure of FMS with 2, PDB ID
2i0y.9 Figure 2 shows the model of 26 with FMS. Key
interactions include a hydrogen bond between the amide
carbonyl and the backbone NH of Cys666 of FMS, an
internal hydrogen bond between the amide NH and
the O of the furan, and hydrophobic interactions for
both the 2- and 4-substituents. The 2-substituent binds
in a hydrophobic region made up of the glycine-rich
loop on one wall and the side chain of Leu785 on the
other. The 4-substituent binds in a relatively hydropho-
bic region extending out from the ATP-binding site to-
ward solution. The major interaction with the protein
is a hydrophobic interaction along the left wall of the
site in the lower graphic of Figure 2. The SAR supports
this binding mode. We believe that the equivalent po-
tency of the furan and pyrrole groups can be explained
by a trade-off of interactions. The furan can accept an
internal hydrogen bond from the amide NH, stabilizing
the necessary planar conformation, while the pyrrole in-
stead can make an additional hydrogen bond between
the pyrrole NH and the backbone carbonyl of FMS res-
idue Glu664. The 2-position has a tight steric fit with
only a small amount of room at the 40 site, explaining
the loss in activity for substitution at the 20- or 30-posi-
tions. Hydrophilic substitution at the 40-position is also
not tolerated. At the 4-position, hydrophilic groups
placed close to the central ring such as the amide 15,
pyrazole 17, imidazole 18 or sulfone (19 and 20) all dra-
matically decrease activity. However, polarity is well-
tolerated at the 40-position of the C-4 substituent. This
Figure 2. Model of compound 26 (green carbons) bound to the FMS
kinase (white carbons). The top graphic shows a cutaway view of the
complex, heavy atoms only, with the hydrogen bonds displayed as a
magenta dotted line and a potential hydrogen bond displayed as a cyan
˚
dotted line. Distances are in A. The bottom view shows an end-on view
of the complex with all atomic molecular surfaces displayed.11
is quite solvent exposed and does not interfere with
the hydrophobic interactions of the ring. Acyclic substit-
uents at C-4 are also less active, presumably because
they cannot adequately fill the site.
Having successfully achieved good in vitro potency, the
pharmacokinetic profiles of several of the more potent
compounds (24, 26, and 8) were evaluated. The pharma-
cokinetic parameters for these compounds are shown in
Table 4. All compounds showed good oral bioavailabil-
ity in mice ranging from 32% to 45%. However, cyano-
pyrrole 8 was clearly superior having a longer t1/2 of
244 min, lower clearance, and moderate volume of dis-
tribution; thus it was chosen for further study.
Table 5 shows the kinase selectivity of 8. Although this
compound was highly potent against FMS, it also inhib-
ited the receptor tyrosine kinases Kit, Axl, TrkA, and
Flt-3 and IRK-b at concentrations less than 0.1 lM.
However, all other 89 kinases tested were inhibited only
at much higher concentrations.
The therapeutic potential of 8 was then evaluated in a
murine collagen-induced model of arthritis (CIA). Oral

C. R. Illig et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1642–1648
Table 4. Pharmacokinetic parameters for 24, 26 and 8
1647
Compound
t1/2 (IV) (min)
Cmax (ng/ml)
Vss (ml/kg)
Cl
F (%) (ml/min/kg)
24a
26b
8b
62
98
386
454
853
7970
11370
5635
107
42
32
39
45
244
16
Male CD-1 mice dosed 2 mg/kg iv and 10 mg/kg po.
a 10% DMSO in PEG 400.
b 20% HP-b-CD.
Table 5. Kinase selectivity of 8
The therapeutic potential of 8 was then evaluated in a
murine collagen-induced model of arthritis (CIA). Oral
twice-daily dosing of 30 and 5 mg/kg reduced the overall
progression of clinical scores by 65% and 35%, respec-
tively, as shown in Figure 3.
a
Kinase
IC50 (lM)
FMS
Kit
Axl
0.00078
0.0035
0.0064
0.011
TrkA
Flt3
Consistent with the clinical scores, histological examina-
tion (Table 6) revealed a reduction of pannus formation
and a reduction in the destruction of bone and cartilage.
Importantly, pannus growth and bone destruction were
reduced by 80%, a level of efficacy comparable to anti-
TNF strategies in CIA models.12 We were also able to
show that macrophages were a cellular target of 8 as
these cells were depleted from the joints of treated mice
(Fig. 4).
0.018
0.083
IRKb
a IC50 values were determined at the respective ATP Km of each
kinase.
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Vehicle po bid
(30 mg/kg) po bid
(5 mg/kg) po bid
In conclusion, optimization of the original lead 2 led
to the discovery of a series of potent, orally active,
and novel small-molecule FMS kinase inhibitors. One
of these, 8, was employed in an in vivo efficacy study
to demonstrate their potential benefit in the treatment
of arthritis. These data from the collagen-induced
arthritis model in mice showed that the evaluated
FMS inhibitor was efficacious in reducing bone erosion,
pannus invasion, cartilage damage and inflammation.
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Study Day
Figure 3. Clinical scores for suppression of CIA by 8.
In light of the literature precedent for the involvement of
macrophages and cells of the macrophage lineage in
inflammation, metastatic bone disease, and arthritis,
these agents may have good utility for the treatment
of these diseases.
twice-daily dosing of 30 and 5 mg/kg reduced the overall
progression of clinical scores by 65% and 35%, respec-
tively, as shown in Figure 3.
Table 6. Impact of 8 on histopathology of collagen-induced arthritis13
Inflammation mean (SE)
Cartilage damage mean (SE)
Pannus invasion mean (SE)
Bone erosion mean (SE)
Disease-free
0
0
0
0
CIA, Vehicle
CIA, 5 mg/kg
CIA, 30 mg/kg
3.28 (0.25)
2.01 (0.24)a
1.54 (0.22)a
3.16 (0.28)
1.87 (0.20)a
1.44 (0.21)a
1.82 (0.25)
0.75 (0.12)a
0.40(0.10)a
1.82 (0.25)
0.75 (0.12)a
0.37(0.11)a
a p < 0.05.
Figure 4. Selective depletion of macrophages in CIA model by 8. Paws were fixed, decalcified, and sectioned, and stained for F4/80 positive
macrophages. Note the markedly reduced level of F4/80 positive macrophages in the paw of the compound 8-treated mouse (30 mg/kg).

1648
C. R. Illig et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1642–1648
(Tyrosine kinase assay kit, Green P2837, Invitrogen) was
References and notes
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, Axl,
TrkA, and IRKb 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, incubated for
25 min. KIT reactions contained 1 nM KIT, 50 lM ATP,
and 100 lg/ml poly Glu4Tyr, incubated for 30 min. Axl
reactions contained 0.5 nM Axl, 20 lM ATP, and 25 lg/
ml poly Glu4Tyr, incubated for 11 min. TrkA reactions
contained 5 nM TrkA, 20 lM ATP, and 20 lg/ml poly
Glu4Tyr, incubated for 27 min. IRKb reactions contained
25 nM IRKb, 50 lM ATP, and 20 lg/ml poly Glu4Tyr,
incubated for 20 min. IC50s were calculated using Graph-
Pad PrismÒ software and a four-parameter logistics
equation. Reported IC50 values are means of at least
three experiments. Typical between-run coefficient of
variation was less than 20%. Inhibition of 89 other kinases
at 1 and 0.1 lM were conducted by Invitrogen and
Millipore kinase profiling services.
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13. The CIA model was performed by Bolder BioPath, Inc.,
Boulder, CO. Male B10RIII (Jackson Labs) were immu-
nized with type II collagen (Elastin Products), in Freund’s
complete adjuvant (with supplemental M. tuberculosis,
4 mg/ml, Difco) on Days 0 and 15. On Day 12, animals
were randomized by body weight into groups (n = 15), and
dosing was initiated and continued every day (BID, 12 h
intervals) for a total of 14 days. Clinical scores were given
for each of the paws (right front, left front, right rear, left
rear) as follows: 0 = normal; 1 = 1 hind or fore paw joint
affected or minimal diffuse erythema and swelling; 2 = 2
hind or fore paw joints affected or mild diffuse erythema
and swelling; 3 = 3 hind or fore paw joints affected or
moderate diffuse erythema and swelling; 4 = marked
diffuse erythema and swelling, or =4 digit joints affected);
5 = severe diffuse erythema and severe swelling entire paw,
unable to flex digits). On day 26, mice were euthanized and
paws were fixed in buffered formalin and prepared for
histopathology. Inflammation, cartilage damage, pannus
invasion, and bone erosion were scored using a 4-point
scale.
10. FMS, FLT-3, KIT, Axl, TrkA and IRKb 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, Axl, TrkA, and IRKb were purchased from
ProQinase, Upstate, Invitrogen, and BioMol, respectively.
FMS 555–568 peptide (SYEGNSYTFIDPTQ) was syn-
thesized and purified by AnaSpec. FMS was assayed using
a fluorescence polarization (FP) competition immunoas-
say 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 tempera-
ture, and quenched by the addition of 5.4 mM EDTA. Ten
microliters of FP buffer/tracer/phospho-Y antibody mix