Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 1

Article abstract of DOI:10.1016/j.bmcl.2006.01.053

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to target focal adhesion kinase (FAK). A number of these pyrrolopyrimides exhibited low micromolar inhibitory activities against focal adhesion kinase, and their prelimi

Full text of DOI:10.1016/j.bmcl.2006.01.053

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2173–2176
Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as
focal adhesion kinase inhibitors. Part 1
Ha-Soon Choi,a Zhicheng Wang,a Wendy Richmond,a Xiaohui He,a Kunyong Yang,a
Tao Jiang,a Taebo Sim,a Donald Karanewsky,a Xiang-ju Gu,a Vicki Zhou,a Yi Liu,a
Osamu Ohmori,b Jeremy Caldwell,a Nathanael Graya and Yun Hea,*
aGenomics Institute of the Novartis Research Foundation (GNF), 10715 John Jay Hopkins Drive, San Diego, CA 920121, USA
bNovartis Institute for BioMedical Research Tsukuba, Okubo 8, Tsukuba-shi, Ibaraki 300-2611, Japan
Received 4 December 2005; revised 12 January 2006; accepted 12 January 2006
Available online 3 February 2006
Abstract—A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to target focal adhesion kinase
(FAK). A number of these pyrrolopyrimides exhibited low micromolar inhibitory activities against focal adhesion kinase, and their
preliminary SAR was established via systematic chemical modifications. The 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines represent
a new class of kinase inhibitors.
Ó 2006 Elsevier Ltd. All rights reserved.
Focal adhesions are found at the cell membrane where
the cytoskeleton interacts with the proteins of the extra-
cellular matrix. The clustering of integrins at these sites
attracts a large complex of proteins which regulate pro-
cesses such as anchorage-dependent proliferation and
cell migration. Signal transduction mediated by interac-
tions between cells and the extracellular matrix (ECM)
at focal adhesions is an important determinant of cell
fate. Focal adhesion kinase (FAK) was first discovered
in 1992 and was implicated in integrin signaling.1–3 It
is a 125 kDa protein tyrosine kinase recruited at an early
stage to focal adhesions and is phosphorylated in
response to cell attachment and mediates focal adhesion
formation.4,5 FAK is known to promote cellular move-
ment and survival. In a variety of human epithelial and
mesenchymal tumors, such as melanoma, lymphoma,
and multiple myeloma, FAK is highly active. Moreover,
increased FAK expression correlates with increased
invasiveness and increased ability of cancer to metasta-
size. Inhibition of FAK signaling in vitro induces cell
growth arrest, reduces motility, and in certain contexts
causes cell death in cancer cell lines.6–12
In the mouse xenograft studies using human melanoma
cells, antisense oligonucleotides against FAK inhibited
the growth of the primary tumor and virtually eliminat-
ed metastases with few adverse effects to normal tis-
sues.13,14 These observations suggest that FAK
represents a promising therapeutic opportunity for both
the treatment of primary disease and the prevention of
metastatic disease.
While much of research has been performed to elucidate
the biological roles of FAK, the only documented FAK
inhibitors are described by AstraZeneca and a represen-
tative structure is shown in Figure 1 (1a).15
Despite screening historical kinase-directed medicinal
chemical libraries of inhibitors, we were unable to dis-
cover interesting starting points for lead optimization.
To circumvent this problem, we employed a rational
Br
7
N
N
2
8
NH
HN
N
9
N
HN
N
N
MeO
OMe
R2
R1
Keywords: Focal adhesion kinase; Inhibition; 7H-Pyrrolo[2,3-d]pyrim-
idines; SAR.
OMe
1a
1b
*
Corresponding author. Tel.: +1 858 332 4706; fax: +1 858 332
4513; e-mail: yhe@gnf.org
Figure 1.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.053

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H.-S. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2173–2176
Table 1. Inhibitory activities of pyrrolo[2,3-d]pyrimidines21
design approach where we introduced a five-membered
ring to bridge the pyrimidine 4 and 5 positions (Fig. 1)
to yield the pyrrolopyrimidine 1b. Although pyrrolopyr-
imidines have been reported as kinase inhibitors,16
2-amino-9-arylpyrrolopyrimidines have not been docu-
mented previously. The route used to prepare pyrrolo-
pyrimidine analogs is shown in Scheme 1. Starting
from commercially available 5-bromo-2,4-dichloro-
pyrimidine (2), the chlorine at the 5 position was regio-
selectively displaced by an amino group in excellent
yield. Palladium-catalyzed cross coupling of the result-
ing pyrimidine intermediate with vinyl stannane gave
the corresponding vinyl ether 4, which was cyclized to
furnish pyrrolopyrimidine intermediate 5 upon treat-
ment with hydrochloric acid.17 The 2-pyridyl moiety
was first introduced at the 9-position via copper-medi-
ated coupling,18 followed by the displacement of the
remaining chlorine with various amines to provide the
targeted pyrrolopyrimidines (7a–7ad). While many of
these analogs did not show significant inhibitory
activities against FAK in a biochemical time-resolved
fluorescence assay, several compounds exhibited submi-
cromolar IC50s (Table 1, 7e, 7h, 7n, 7o, 7x, 7y, 7aa, and
7ad). This was very encouraging as they could serve as
starting points for optimization. The preliminary data
suggested that a phenyl moiety as R2 is preferred for
inhibitory activity (Scheme 1 and Table 1).
Compound
IC50 (lM)
Compound
IC50 (lM)
1a
7a
7b
7c
7d
7e
7f
0.1
>10
>10
>10
>10
0.5
7aa
7ab
7ac
7ad
9a
0.3
>10
>10
0.6
7.9
9b
9c
>10
7.5
0.2
0.9
7g
7h
7i
>10
0.7
1.0
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
15
0.1
9.0
7j
1.2
0.7
>10
0.5
0.8
7k
7l
1.5
7.0
7m
7n
7o
7p
7q
7r
7s
7t
0.5
0.3
0.7
0.6
>10
0.8
0.8
2.0
>10
>10
>10
>10
3.0
>10
>10
0.2
16
18
7u
7v
7w
7x
7y
7z
>10
0.1
>10
>10
0.4
19
23
>10
0.2
0.2
24
25
0.2
8.0
26
>10
We next examined the effect of different substitutions at
the 9-position. The synthesis of these analogs is shown
in Scheme 2. Vinyl ether 4 was converted into aniline-
substituted pyrrolopyrimidine 8 in one pot in the pres-
ence of hydrochloric acid. Pyridine analogs (9a–9c) with
1, 2 or 3 methylene linkers were prepared from 8 via a
Mitsunobu reaction. All three analogs showed weak or
no activity. These data suggest that the distance of the
pyridine moiety from the pyrrolopyrimidine is impor-
tant for favorable interactions with the enzyme. To fur-
ther probe this region, different aryl moieties could be
introduced via copper-mediated coupling to give 10a–
10l in moderate to good yields.18 Among these com-
pounds, the simple 2- and 3-pyridyl analogs (10a and
10b) possessed IC50 of 0.2 and 0.1 lM against FAK,
respectively. As the 4-pyridyl analog 10c, exhibited sig-
nificantly diminished activities (9.0 lM), it suggested
that the position of pyridine nitrogen plays an important
role via either hydrogen bonding or electrostatic interac-
tions. Several electron-donating or -withdrawing substi-
tutions were introduced to the 3-pyridyl ring (10f–10h)
to probe the electronic preference of the 9-position.
The conclusion was that while all substitutions reduced
activity, an electron-donating group (10h) is favored
over an electron-withdrawing group (10f). Heterocycle
substitutions lead to variable results, thiophene (10d)
lost activity, while thiazole (10e) retained activity. The
activity of thiazole (10e) provided further evidence for
the importance of a basic nitrogen in this region of the
inhibitor. Two regioisomeric pyrimidine analogs were
also prepared. While 10i completely lost the activity,
its isomer 10j still showed submicromolar activities,
which again suggests the importance of the position of
the nitrogen atom in this region.
Br
OEt
Br
Cl
N
N
N
a
b
Cl
N
NH2
Cl
N
NH2
Cl
N
2
3
4
c
N
N
R1
N
e
N
N
d
N
R2
N
Cl
N
N
N
N
H
Cl
N
7
6
5
1
2
7a:
7b:
7c:
7d:
7e:
7f:
7g:
7h:
7i:
7j:
7k:
7l:
7m:
7n:
7o:
7p:
7q:
7r:
O
R =H, R =Bn
7s: R1=H, R2=
1
2
R =H, R =3,4,5-(OMe)3-Bn
N
1
2
R R =(CH2)2N(Me)(CH2)2
1
2
R R =(CH2)2O(CH2)2
R7
R6
R
3
1
2
R =H, R =2-Me-Ph
R1=H, R2=2-Et-Ph
R1=H, R2
=
R4
R5
1
2
i
R =H, R =2- Pr-Ph
1
2
7t: R4=R5=R7=H, R3=Me, R6=Br
7u: R4=R5=R7=H, R3=Me, R6=I
R =H, R =2-MeO-Ph
R1=H, R2=2-MeS-Ph
R1=H, R2=2-F-Ph
7v: R4=R5=R7=H, R3=Me, R6=CH2OH
7w: R4=R5=R7=H, R3=Me, R6=NO2
7x: R4=R5=R7=H, R3=Me, R6=CONH2
7y: R4=R5=R7=H, R3=Me, R6=OMe
7z: R4=R6=R7=H, R3=Me, R5=I
1
2
R =H, R =2-Cl-Ph
R1=H, R2=2-Br-Ph
1
2
R =H, R =2-CF3-Ph
7aa: R3=R4=R5=H, R6R7=(CH2)4
7ab: R3=R6=R7=H, R4R5=OCH2O
7ac: R3=R6=R7=H, R4R5=CHCHNH
1
2
R =H, R =3-Me-Ph
1
2
R =H, R =3-MeO-Ph
1
2
R =H, R =3-MeS-Ph
N
1
2
7ad: R4=R5=R7=H, R3=Me, R6=
R =H, R =3-HO-Ph
R1=H, R2=4-CF3-Ph
N
Scheme 1. Synthesis of pyrrolopyrimidines 7a–7ad. Reagents and
conditions: (a) NH3 (l), THF, 25 °C, 15 h, 94%; (b) EtOCHCHSnBu3
(1.2 equiv), Pd(PPh3)4 (0.05 equiv), toluene, 110 °C, 16 h, 58%; (c) HCl
i
(3.0 N), PrOH, reflux, 2 h, 96%; (d) 2-bromopyridine (1.5 equiv), 1,4-
dioxane, CuI (0.1 equiv), K3PO4 (20 equiv), trans-1,2-diaminocyclo-
hexane (0.1 equiv), 90 °C, 4 h, 89%; (e) R1R2NH (2.0 equiv), KOtBu,
THF, reflux, 7 h, 30–65%.

H.-S. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2173–2176
2175
OEt
N
N
N
O
N
H
N
R1
HN
N
HN
N
OEt
O
HN
N
OH
O
O
N
b
a
a
b
EtO
EtO
OEt
OEt
Cl
N
NH2
NH
O
O
MeO
OMe
MeO
OMe
MeO
OMe
HN NH
OMe
14
OMe
OMe
MeO
OMe
OMe
11
12
4
8
9
9a: R1=CH2-3-Py
13
c
c
9b: R1=(CH2)2-3-Py
9c: R1=(CH2)2-4-Py
OH
OEt
OEt
N
N
N
N
O
O
O
HN
N
NH
HN
N
NH
HN
N
Cl
f
d
N
Ar
HN
N
N
N
MeO
OMe
MeO
OMe
MeO
OMe
OMe
MeO
OMe
OMe
OMe
OMe
17
16
15
10
e
Ar=2-Py
Ar=3-Py
Ar=4-Py
Ar=2-thiophenyl
Ar=2-thiazole
Ar=3-(4-F-Py)
Ar=3-(4-Cl-Py)
10a:
g
10a
h,i
X
Y
10b:
10c:
10d:
10e:
10f:
Ar =
Z
R2
Br
j
N
N
N
N
O
X=Y=N, Z=CH, R2=Cl
X=CH, Y=Z=N, R2=Cl
X=Y=CH, Z=N, R2=Cl
X=CH, Y=N, Z=C(Me), R2=H
10i:
10j:
10k:
10l:
N
N
HN
N
HN
HN
N
NH
N
N
N
10g:
10h:Ar=3-(4-OMe-Py))
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OMe
Scheme 2. Synthesis of pyrrolopyrimidines 9a–9c and 10a–10l.
Reagents and conditions: (a) 3,4,5-trimethoxyaniline (1.1 equiv),
nBuOH, HCl (1.0 equiv), 110 °C, 4 h, 76%; (b) R1OH (1.3 equiv),
PPh3 (1.5 equiv), DEAD, THF, 25 °C, 12 h, 30%; (c) ArBr (1.5 equiv),
1,4-dioxane, CuI (0.1 equiv), K3PO4 (2.0 equiv), trans-1,2-diaminocy-
clohexane (0.1 equiv), 100 °C, 4 h, 40–90%.
OMe
18
19
1a
Scheme 3. Synthesis of 18 and 19. Reagents and conditions: (a)
NaOEt, Et2O, HCO2Et, 25 °C, 14 h, 54%; (b) 13 (0.7 equiv), NaOEt,
EtOH, relux, 3 h, 55%; (c) POCl3, 40 °C, 2 h, 94%; (d) 2-aminopyridine
(1.3 equiv), Pd2(dba)3 (0.1 equiv), BINAP (0.2 equiv), Cs2CO3 (1.4
equiv), toluene, 80 °C, 2 h, 70%; (e) nBu3SnCH2CO2Et (1.5 equiv),
Pd2(dba)3 (0.1 equiv), DMF, 80 °C, 16 h, 30%; (f) NaOH (1.0 N),
EtOH, 25 °C, 1 h, 60%; (g) SOCl2 (3.0 equiv), CH2Cl2, 25 °C, 2 h, 50%;
(h) LAH (4.0 equiv), THF, 25 °C, 2 h, 63%; (i) DEAD (1.5 equiv),
PPh3 (1.5 equiv), THF, 25 °C, 58%; (j) H2, Pd/C (10%), EtOAc, AcOH
(5–10 equiv), 25 °C, 12 h, 91%.
With the preliminary SAR information on the substitu-
tions in hand, we decided to explore the importance of
pyrrole moiety on the interaction with the enzyme.
The first two analogs designed to address this question
are 18 and 19 and their synthesis is shown in Scheme
3. Condensation of succinic acid diethyl ester with ethyl
formate led to aldehyde intermediate 12, which was
reacted with trimethoxyguanidine 13 to furnish the tri-
substituted pyrimidine 14 in moderate yield. The hydro-
xy group was then converted into chlorine in excellent
yield using phosphorus oxychloride. Palladium-cata-
lyzed coupling of the resulting pyrimidine chloride with
2-aminopyridine led to 2,4-diaminopyrimidine 16 in
good yield.19 Compound 16 could also be prepared in
one step from 1a through reaction with stannane acetate
under the Negishi–Reformatsky coupling conditions.20
Sponification of the ethyl ester followed by the lactam
formation using thionyl chloride gave the target mole-
cule 18. Compound 19 was prepared from 16 in two
steps: (1) reduction of the ester by LAH to the corre-
sponding alcohol and (2) Mitsunobu cyclization. Com-
pound 19 could also be synthesized in excellent yield
from 10a by palladium-catalyzed hydrogenation. Enzy-
matic assay against FAK demonstrated that 18 was
inactive, while 19 still retained similar biological activity.
These data suggest that the electronics on the pyrroli-
dine nitrogen or the conformation of the pyrrolidine,
and possibly sterics in this region, is crucial for the activ-
ity. The electron-withdrawing 8-carbonyl is detrimental
to the interaction with the enzyme.
Additional displacement of the remaining chlorine in
20 with trimethoxyaniline led to diaminopyrimidine 1a
in good yield.15 Heck coupling of 1a with ethyl acrylate
gave the corresponding olefin intermediate 21. Hydroge-
nation of the double bond followed by the treatment of
the resulting ester led to the lactam target 23. Partial
reduction of the lactam with DIBAL-H furnished the
corresponding pyrimidinol 24. The ester moiety in 22
can be completely reduced using LAH. Cyclization of
the resulting alcohol under the Mitsunobu conditions
gave 25 in moderate yield. Target 26 was obtained by
directly treating ester 21 with sodium ethoxide. Consis-
tent with the loss of FAK activity for 8-keto compound
18, neither 23 nor 26 exhibited significant FAK inhibito-
ry activity. As had been observed for the saturated pyr-
rolidinyl analog 19, the corresponding fused piperidinyl
analog 24 also exhibited an IC50 of 0.2 lM.
In summary, we have discovered that 2-amino-9-aryl-
7H-pyrrolo[2,3-d]pyrimidines can be elaborated into
moderately potent FAK inhibitors based on rational de-
sign. Efficient and flexible chemistries have been devel-
oped to synthesize pyrrolopyrimidine analogs with
various substitutions at the 2 and 9 positions. These com-
pounds are different from the previously reported pyrrol-
opyrimidines-based kinase inhibitors, which possess
characteristic 3,8, or 3,9 disubstitutions. Thus, 2-ami-
no-9-aryl-7H-pyrrolo[2,3-d]pyrimidines represent a new
class of kinase inhibitors. Preliminary SAR studies sug-
Several additional analogs (23–26) were synthesized to
further explore the SAR in this region (Scheme 4). Selec-
tive displacement of 5-bromo-2,4-dichloro-pyrimidine
(2) with 2-aminopyridine gave 20 in moderate yield.

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H.-S. Choi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2173–2176
Br
6. Gilmore, A. P.; Romer, L. H. Mol. Biol. Cell 1996, 7,
1209.
7. Hauck, C. R.; Sieg, D. J.; Hsia, D. A.; Loftus, J. C.;
Gaarde, W. A.; Monia, B. P.; Schlaepfer, D. D. Cancer
Res. 2001, 61, 7079.
N
Br
N
Br
Cl
HN
N
NH
N
a
b
Cl
N
NH
N
Cl
N
N
MeO
OMe
8. Ridyard, M. S.; Sanders, E. J. Cell Biol. Int. 2001, 25, 215.
9. Maung, K.; Easty, D. J.; Hill, S. P.; Bennett, D. C.
Oncogene 1999, 18, 6824.
OMe
1a
2
20
c
10. Hungerford, J. E.; Compton, M. T.; Matter, M. L.;
Hoffsstrom, B. G.; Otey, C. A. J. Cell Biol. 1996, 135,
1383.
11. Ilic, D.; Furuta, Y.; Kanazawa, S.; Takeda, N.; Sobue, K.;
Nakatsuji, N.; Nomura, S.; Fujimoto, J.; Okada, M.;
Yamamoto, T.; Aizawa, S. Nature (London) 1995, 377,
539.
12. McLean, G. W.; Carragher, N. O.; Avizienyte, E.; Evans,
J.; Brunton, V. G.; Frame, M. C. Nat. Rev. Cancer 2005,
5, 505.
CO2Et
CO2Et
d
N
N
N
HN
N
NH
HN
N
N
O
HN
N
NH
e
N
N
N
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OMe
23
OMe
22
21
f
g,h
i
13. Rovin, J. D.; Ledinh, W.; Parsons, J. T.; Adams, R. B.
Surg. Forum 2001, 52, 272.
14. Monia, B. P.; Gaarde, W. A.; Nero, P. S. U.S. Patent
20010034329, 2001.
N
N
N
N
HN
N
N
OH
HN
N
HN
N
N
O
N
N
N
15. Bradbury, R. H.; Breault, G. A.; Jewsbury, P. J.; Pease, J.
E. PCT WO 2000039101, 2000.
MeO
OMe
MeO
OMe
MeO
OMe
OMe
24
OMe
25
OMe
26
16. (a) Nishioka, H.; Sawa, T.; Nakamura, H.; Iinuma, H.;
Ikeda, D.; Sawa, R.; Naganawa, H.; Hayashi, C.;
Hamada, M. J. Nat. Prod. 1991, 54, 1321; (b) Showalter,
H. D. H.; Bridges, A. J.; Zhou, H.; Sercel, A. D.;
McMichael, A.; Fry, D. W. J. Med. Chem. 1999, 42, 5464;
(c) Gangjee, A.; Yang, J.; Ihnat, M. A.; Kamat, S. Bioorg.
Med. Chem. 2003, 11, 5155.
17. Cheung, M.; Harris, P. A.; Lackey, K. E. Tetrahedron
Lett. 2001, 42, 999.
18. Antila, J. C.; Klapars, A.; Buchwald, S. L. J. Am. Chem.
Soc. 2002, 124, 11684.
Scheme 4. Synthesis of compounds 23–26. Reagents and conditions:
(a) 2-aminopyridine (1.2 equiv), DIEPA, MeOH, 100 °C, 16 h, 40%;
(b) 3,4,5-trimethoxyaniline (2.0 equiv), nBuOH, 16 h, reflux, 85%; (c)
CH2CHCO2Et (10.0 equiv), Pd(OAc)2 (0.01 equiv), CH3CN, reflux,
16 h, 75%; (d) H2, Pd/C (10%), EtOH, 25 °C, 8 h, 85%; (e) NaOEt (4.0
equiv), EtOH, 80 °C, 16 h, 30%; (f) DIBAL-H (3.0 equiv), THF, 25 °C,
2 h, 55%; (g) LAH (4.0 equiv), THF, 25 °C, 2 h, 60%; (h) DEAD (1.5
equiv), PPh3 (1.5 equiv), THF, 25 °C, 40%; (i) NaOEt (4.0 equiv),
EtOH, 80 °C, 16 h, 60%.
19. (a) Wagaw, S.; Buchwald, S. L. J. Org. Chem. 1996, 61,
7240; (b) Iwaki, T.; Yasuhara, A.; Sakamoto, T.
J. Chem. Soc., Perkin Trans. 1 1999, 1505; (c) Khan,
M. M.; Ali, H.; van Lier, J. E. Tetrahedron Lett. 2001,
42, 1615; (d) Yang, J.-S.; Lin, Y.-H.; Yang, C.-S. Org.
Lett. 2002, 4, 777.
gested that a combination of an electron-rich arylamino
group at the 2-position and pyridinyl group at the 9-po-
sition resulted in moderately potent FAK inhibitors
(7aa, 10a, 10b, 10h, 19, 24 and 25). It appears that the
correct positioning of the substituents at the 9-N and
the electronics on the N are important for the inhibitory
activities against FAK, and the oxo-analogs (18 and 26)
exhibited significantly reduced activities. Interestingly,
the aromaticity of the pyrrole moiety does not have big
impact on the activity (10a, 10b vs 19, 24, and 25).
20. Kousgi, M.; Negishi, Y.; Kaneyama, M.; Migita, T. Bull.
Chem. Soc. Jpn. 1985, 58, 3383.
21. A TR-FRET-based FAK kinase assay was used to
measure the potency of the FAK inhibitors. Briefly,
15 lL of assay mixture containing 133 nM of the FAK
substrate peptide (Biotin-Ahx-SETDDYAEIID) and
2.4 lg/mL of FAK in assay buffer (20 mM Hepes, pH
7.4, 5 mM MgCl2, 2 mM MnCl2, 50 lM Na3VO4, 0.01%
BSA, and 0.05% Tween 20) was added into a 384-well
plate, followed by the addition of 0.5 lL of compounds in
DMSO. After incubation at room temperature for 20 min,
the kinase reaction was initiated by the addition of 5 lL of
40 lM ATP. The kinase reaction was performed at 37 °C
for 2 h and then stopped by the addition of the stop
solution mixture containing 0.15 nM Eu-PT66 (Perkin-
Elmer), 1.5 lg/mL SA-APC in detection buffer (10 mM
Tris–HCl, pH 7.4, 6.25 mM EDTA, 0.01% BSA, and
0.05% Tween 20). The plate was incubated at room
temperature for 1 h and the TR-FRET signal was detected
with an Acquest plate reader.
References and notes
1. Schaller, M. D.; Borgman, C. A.; Cobb, B. S.; Vines, R. R.;
Reynolds, A. B.; Parsons, J. T. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 5192.
2. Hanks, S. K.; Calalb, M. B.; Harper, M. C.; Patel, S. K.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8487.
3. Guan, J. L.; Shalloway, D. Nature (London) 1992, 358, 690.
4. Parsons, J. T. J. Cell Sci. 2003, 116, 1409.
5. Mitra, S. K.; Hanson, D. A.; Schlaepfer, D. D. Nat. Rev.
Mol. Cell Biol. 2005, 6, 56.