Structure-based discovery of cellular-active allosteric inhibitors of FAK

Article abstract of DOI:10.1016/j.bmcl.2013.01.047

In order to develop potent and selective focal adhesion kinase (FAK) inhibitors, synthetic studies on pyrazolo[4,3-c][2,1]benzothiazines targeted for the FAK allosteric site were carried out. Based on the X-ray structural analysis of the co-crystal of the lead compound, 8-(4-ethylphenyl)-5-methyl-1,5- dihydropyrazolo[4,3-c][2,1]benzothiazine 4,4-dioxide 1 with FAK, we designed and prepared 1,5-dimethyl-1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin derivatives which selectively inhibited kinase activity of FAK without affecting seven other kinases. The optimized compound, N-(4-tert-butylbenzyl)-1,5-dimethyl-1,5- dihydropyrazolo[4,3-c][2,1]benzothiazin-8-amine 4,4-dioxide 30 possessed significant FAK kinase inhibitory activities both in cell-free (IC₅₀ = 0.64 μM) and in cellular assays (IC₅₀ = 7.1 μM). These results clearly demonstrated a potential of FAK allosteric inhibitors as antitumor agents.

Full text of DOI:10.1016/j.bmcl.2013.01.047

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1779–1785
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based discovery of cellular-active allosteric inhibitors of
FAK
a
a
b
a
Naoki Tomita a, , Yoko Hayashi , Shinkichi Suzuki , Yoshimasa Oomori , Yoshio Aramaki ,
Yoshihiro Matsushita a, Misa Iwatani a, Hidehisa Iwata a, Atsutoshi Okabe a, Yoshiko Awazu a,
Osamu Isono a, Robert J. Skene c, David J. Hosfield c, , Hiroshi Miki a, Tomohiro Kawamoto a,
Akira Hori a, Atsuo Baba a
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1 Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Company Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
c Takeda California, 10410 Science Center Drive, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to develop potent and selective focal adhesion kinase (FAK) inhibitors, synthetic studies on pyr-
azolo[4,3-c][2,1]benzothiazines targeted for the FAK allosteric site were carried out. Based on the X-ray
structural analysis of the co-crystal of the lead compound, 8-(4-ethylphenyl)-5-methyl-1,5-dihydropy-
razolo[4,3-c][2,1]benzothiazine 4,4-dioxide 1 with FAK, we designed and prepared 1,5-dimethyl-1,5-
dihydropyrazolo[4,3-c][2,1]benzothiazin derivatives which selectively inhibited kinase activity of FAK
without affecting seven other kinases. The optimized compound, N-(4-tert-butylbenzyl)-1,5-dimethyl-
1,5-dihydropyrazolo[4,3-c][2,1]benzothiazin-8-amine 4,4-dioxide 30 possessed significant FAK kinase
Received 22 September 2012
Revised 8 January 2013
Accepted 15 January 2013
Available online 26 January 2013
Keywords:
FAK
Kinase
Inhibitors
Allosteric
Type III
Cancer
SBDD
inhibitory activities both in cell-free (IC50 = 0.64
lM) and in cellular assays (IC50 = 7.1 lM). These results
clearly demonstrated a potential of FAK allosteric inhibitors as antitumor agents.
Ó 2013 Elsevier Ltd. All rights reserved.
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase
and plays a key role in cell proliferation, resistance to apoptosis
and anoikis, migration, and invasion.1 Over expression of FAK is ob-
served in a variety of tumors, and levels of FAK expression are cor-
related with grade of malignancy and poor prognosis.1,2 Studies
demonstrating suppression of FAK by using dominant-negative
FAK or siRNA have shown both in vitro and in vivo tumor growth
inhibitory activity.3 A small molecule FAK inhibitor is potentially
an effective anticancer agent for a broad range of tumors either
administered alone or in combination with existing chemothera-
pies. Several inhibitors of FAK have been reported4 and two of
them were extensively examined in preclinical or clinical studies:
PF-562271 from Pfizer is a dual inhibitor of FAK and Pyk2,5 and
NVP-TAE-226 from Novartis is a dual inhibitor of FAK and IGF1R
(Fig. 1).6 Both compounds were reported as Type I inhibitors
(bound to ATP site) based on the X-ray co-crystal analyses.5,6c
These inhibitors display remarkable inhibition of cancer cell
growth in vitro and in vivo,5–7 suggesting an FAK inhibitor with
improved selectivity would be a desirable option for an anticancer
therapy.
With the aim of developing novel FAK inhibitors that provide an
inhibition profile differentiated from known FAK inhibitors, we
Cl
N
CF3
SO2Me
N
N
MeN
HN
N
NH NHMe
O
HN
HN
N
O
N
H
MeO
N
O
⇑
Corresponding author. Tel.: +81 466 32 1167; fax: +81 466 29 4448.
PF-562271
FAK/Pyk2 dual inhibitor
NVP-TAE-226
FAK/IGF1R dual inhibitor
E-mail address: naoki.tomita@takeda.com (N. Tomita).
Present address: Molecular Tagging Systems Inc., 1425 Russ Boulevard, San Diego,
CA 92101, United States.
Figure 1. ATP-competitive inhibitors of FAK.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.047

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N. Tomita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1779–1785
Synthesis of pyrazolo[4,3-c][2,1]benzothiazine scaffold is de-
FAK IC50 [µM]
scribed in Scheme 1. N-Methanesulfonylanthranilic acid esters 6–
8 were prepared from commercially available methyl anthranilates
3 and 4, and methyl 2-fluoro-4-nitrobenzoate 5 in 31–78% yield.
Treatment of esters 6–8 with sodium hydride provided cyclized
products 9–11 in 83–96% yield. Biphenyl derivative 12 was ob-
tained from bromide 9 under typical Suzuki-coupling condition
in 71% yield.16 Subsequent treatment of the ketosultams 9–12 with
N,N-dimethylformamide dimethylacetal followed by cycloconden-
sation with hydrazines afforded desired pyrazolo[4,3-c][2,1]benzo-
thiazines 13–17 in a regioselective manner (80–96% yield). The
regiochemistry of 17 was confirmed by X-ray crystallographic
analysis.17
Terminal carboxamides 20–22 were prepared as described in
Scheme 2. Bromide 13 was treated with 3,4-dihydro-2H-pyran
(DHP) in the presence of TFA to give tetrahydropyran (THP)-pro-
tected pyrazolo[4,3-c][2,1]benzothiazine 18 (THP position was
not determined) in 97% yield. Subsequent palladium-catalyzed cou-
pling of 18 with 4-methoxycarbonylphenylboronic acid afforded a
coupled product, which was converted to carboxylic acid 19 by
saponification in 66% yield. Condensation of 19 with several amines
followed by deprotection of THP group provided terminal carbox-
amides 20–22 in 40–55% yield.
N
NH
pre-incubation
5 min 60 min
O
O
ATP conc.
S
N
Et
Me
0.5 µM
1000 µM
1.2
6.4
0.96
2.9
1
Figure 2. HTS hit compound 1.
carried out a systematic and exhaustive search to identify ATP-
noncompetitive compounds.8 High-throughput screening (HTS) of
our internal compound library provided more than 10 hit com-
pounds of different chemical classes. These hit compounds were
evaluated using time-dependent assay (pre-incubation time:
5 min and 60 min) under high and low concentration of ATP
(1000 and 0.5 lM). As a result, we found 1,5-dihydropyrazol-
o[4,3-c][2,1]benzothiazine 1, which inhibited FAK kinase activity
under the high concentration of ATP (Fig. 2).9 The inhibitory activ-
ity at 1000 lM of ATP was slightly increased after 60-min preincu-
bation.10 Subsequent co-crystallization and X-ray structure
determination of compound 1 in the FAK kinase domain revealed
a novel binding mode in which compound 1 bound in a newly
formed FAK allosteric site.8,11 Since several Type III kinase inhibi-
tors, such as allosteric inhibitors of MEK1/212 and Akt1/2,13 pos-
sessed remarkable selectivity against other kinases,14,15 we
decided to develop FAK allosteric inhibitors starting from the lead
compound 1. We report herein our medicinal chemistry efforts on
the discovery of novel allosteric FAK inhibitors by utilizing struc-
ture-based drug design.
Starting from intermediate 8-bromo-pyrazolo[4,3-c][2,1]benzo-
thiazine 14, nitrogen-linked pyrazolo[4,3-c][2,1]benzothiazines 23
and 24 were prepared by transition-metal mediated coupling reac-
tions assisted with microwave irradiation (Scheme 3). Copper-
mediated C–N coupling18 of 14 with 4-fluorobenzamide provided
carboxamide 23 in 46% yield, whereas palladium-catalyzed C–N
coupling19 with 4-fluoroaniline afforded phenylamine 24 in 22%
yield.
X-ray crystallographic analysis of the co-crystal of compound 1
and FAK kinase domain revealed several polar and hydrophobic
interactions (Fig. 3 PDB ID: 4EBV). The fused pyrazole ring interacts
The ester 15 was transformed into carbon-tethered analogues
25 and 26 (Scheme 4). Basic hydrolysis of 15 followed by conden-
sation with 4-fluoroaniline provided amide 25 in 66% yield. Reduc-
tion of 15 with lithium aluminum hydride gave a corresponding
carbinol, which was converted to amine 26 by mesylation and
nucleophilic substitution in 13% yield.
8-Aminopyrazolo[4,3-c][2,1]benzothiazine derivatives 28–30
were prepared from corresponding 8-nitro intermediate 16
(Scheme 5). Compound 16 was reduced by hydrogenation to give
aniline 27 in 96% yield. Reductive alkylation of 27 with 4-substituted
benzaldehydes afforded benzylamines 28–30 in 35–77% yield.
with the cationic side chain of Arg550 via cation–
p interaction.
One of the pyrazole nitrogens serves as a hydrogen bond donor
to a water molecule. This water molecule seems to be displaceable
by R1, because its N1-ethyl analogue 2 possessed similar inhibitory
activity to 1.8 The p-ethylphenyl group might be transformed into
another hydrophobic group to optimize hydrophobic interactions.
Polar interaction with Gly563 or Asp564 also seems to be possible
by introducing substituents as R2.
Met475
Asp564
Arg550
Met607
H
N
Asp604
H2O
Phe542
His544
NH2
H2O
+
NH2
N
Phe542
His544
NH
O
O
S
N
Et
Asp564
Val552
Leu537
Me
1
Gly563
Arg550
Leu534
R1
N
N
Phe608
Met607
Met206
Asp604
O
O
S
N
R2
Me
Figure 3. Structure-based design of pyrazolo[4,3-c][2,1]benzothiazines. PDB ID: 4EBV.

N. Tomita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1779–1785
1781
N
R1
O
N
MeOOC
H2N
MeOOC
e
O
O
g, h
Ms
a, b, c
d
O
O
S
R2
N
R2
S
R2
N
R2
N
Me
Me
: R2 = Br
Me
3
: R2 = Br
6
: R2 = Br
4: R2 = COOMe
13
: R = H, R2 = Br
1
9
7: R2 = COOMe
8: R2 = NO2
10: R2 = COOMe
14: R1 = Me, R2 = Br
f
MeOOC
11: R2 = NO2
15: R1 = Me, R2 = COOMe
2
: R = Me, R2 = NO2
1
12
: R = 4-EtPh
16
F
NO2
17: R1 = Me, R2 = 4-EtPh
5
Scheme 1. Reagents and conditions: (a) MsCl, Et3N, THF; (b) K2CO3, MeOH; (c) MeI, K2CO3, DMF; (d) MsNHMe, K2CO3, DMSO; (e) NaH, DMF or DMA; (f) 4-EtPhB(OH)2,
Pd(OAc)2, PPh3, Na2CO3, DME, H2O, 150–170 °C, microwave; (g) DMFDMA, THF; (h) R1NHNH2, EtOH, reflux.
THP
N
THP
N
N
N
N
N
NH
NH
(1) d or e
(2) f
a
b, c
O
O
O
O
O
O
O
O
S
S
S
S
N
R3
N
Br
N
Br
N
COOH
Me
Me
Me
Me
3
13
18
19
20
21
: R = CONH2
3
: R = CONH(CH2)2OH
O
OH
3
22
N
: R
=
Scheme 2. Reagents and conditions: (a) DHP, TFA, THF, reflux; (b) 4-MeO2CPhB(OH)2, PdCl2(dppf), Et3N, DME, H2O, 150 °C, microwave; (c) NaOH, H2O, MeOH, THF, DMA; (d)
HOBtꢀNH3, EDC, DMF; (e) amine, DMT-MM, THF, 2-propanol; (f) HCl, MeOH.
F
N
N
N
Me
Me
Me
N
N
N
b
a
O
O
O
O
O
O
O
S
F
S
S
N
NH
N
Br
N
NH
Me
Me
Me
14
23
24
Scheme 3. Reagents and conditions: (a) 4-FPhCONH2, CuI, N,N0-dimethylcyclohexane-1,2-diamine, K2CO3, DMA, 150–180 °C, microwave; (b) 4-FPhNH2, Pd2(dba)3, X-phos,
tBuONa, DMA, 150 °C, microwave.
N
N
N
Me
Me
Me
N
N
N
c, d, e
a, b
O
O
O
O
O
O
S
HN
O
F
HN
F
S
S
N
N
COOMe
N
Me
Me
Me
25
15
26
Scheme 4. Reagents and conditions: (a) NaOH aq, MeOH; (b) 4-FPhNH2, EDC, HOBt, DMF; (c) LiAlH4, THF; (d) MsCl, Et3N, THF; (e) 4-FPhNH2, Et3N, THF.
N
N
N
Me
Me
Me
N
N
N
a
b
O
O
O
O
O
O
S
S
S
R5
N
NO2
N
NH2
N
NH
Me
Me
Me
5
16
27
28
29
: R = F
5
: R = Br
30: R5 = tBu
Scheme 5. Reagents and conditions: (a) Pd-C, H2, MeOH, MeCN; (b) 4-R5PhCHO, AcOH, MeCN, DMA, then NaBH4.
Methylpyrazole regioisomer 31 was synthesized as described in
identical to the compound 17 prepared by a different method as
Scheme 6. Suzuki coupling of bromide 18 with 4-ethylbenzenebo-
ronic acid and subsequent deprotection of THP group gave pyraz-
olo[4,3-c][2,1]benzothiazine 1 in 60% yield. Alkylation of 1 with
iodomethane gave a mixture of regioisomers 31 and 17, both of
which were isolated by column chromatography (81% and 14% iso-
lated yield, respectively). Analytical data of the minor product was
described in Scheme 1.
FAK kinase inhibitory activities of pyrazolo[4,3-c][2,1]benzothi-
azines 20–22 bearing 4-substituted phenyl groups were evaluated
as shown in Table 1. Since some compounds showed slow off-rate
properties, enzymatic activity was measured after 60 min of
pre-incubation to observe clearer response of time-dependent

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N. Tomita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1779–1785
Me
THP
N
N
N
N
N
Me
N
NH
N
O
O
O
O
O
O
O
O
c
a, b
S
S
S
S
+
N
Br
N
Et
N
Et
N
Et
Me
Me
Me
Me
18
31
1
17
81%
14%
Scheme 6. Reagents and conditions: (a) 4-EtPhB(OH)2, Pd(OAc)2, PPh3, Na2CO3, H2O, DME, 150–170 °C, microwave; (b) HCl, MeOH; (c) MeI, K2CO3, DMF.
Table 1
in the ATP binding site. The N-free pyrazole of 22 interacts with
SAR of substituents on the benzene ring
Glu500 and Cys502 in the hinge region by two hydrogen bonds.
In addition, one of the oxygens of sulfonamide binds to the termi-
nal amino group of Lys454. The terminal hydroxyl group of 22
forms direct and/or water-mediated hydrogen bonds with
Glu506, Ser509, and Arg514. Considering the fact that the lead
compound 1 could also bind to the allosteric site,8 we supposed
that N-free pyrazolo[4,3-c][2,1]benzothiazine analogues poten-
tially possess two different binding modes: allosteric binding and
hinge binding. This hypothesis was also supported by the fact that
FAK inhibitory potential of compound 22 under the high concen-
N
NH
O
S
O
N
R3
Me
a
Compound
R3
FAK IC50 (lM)
1
20
Et
0.96
3.5
CONH2
tration of ATP was much weaker (IC50 >30
preincubation time 60 min).
lM at ATP 1000 lM,
O
OH
21
22
0.99
0.50
N
H
In order to facilitate pure allosteric inhibition of FAK, the poten-
tial hinge-binding motif of this chemotype should be modified.
Guided by the crystallographic analysis and our previous studies,8
we introduced a substituent on one pyrazole nitrogen to directly
interfere with hydrogen bond formation at the hinge region. 1-
Methyl and 2-methyl substituted pyrazolo[4,3-c][2,1]benzothia-
zines were evaluated as shown in Table 2. Introduction of methyl
group on the 2-position of pyrazole resulted in drop of activity
O
.
OH
N
a
ATP 0.5 lM, pre-incubation time 60 min.
inhibitors. Although lead compound 1 could also inhibit FAK in the
presence of 1 mM of ATP, ATP concentration was fixed at 0.5
for higher sensitivity of the enzymatic assays.
lM
(31, IC50 >30
ited significant inhibition (IC50 0.077
times potent activity than those of 1 (IC50 0.96
2.1 M).
In addition, compound 17 showed excellent kinase selectivity
l
M). On the other hand, 1-methyl analogue 17 exhib-
M), showing more than 10
M) and 2 (IC50
l
4-Aminocarbonyl substitution resulted in a decrease of activity
(20, IC50 3.5 M) compared with that of 4-ethyl analogue (1, IC50
0.96 M), but the activity was recovered by introduction of 2-
hydroxyethyl group (21, IC50 0.99 M). Since further elongation
of the alcohol side chain increased inhibition activity (22, IC50
0.50 M), it was suggested that some polar interactions might be
acquired.
For the purpose of analyzing interactions between the carbox-
amide 22 and FAK, the crystal structure of 22 in the FAK kinase do-
main was accomplished (Fig. 4, PDB ID: 4I4E). The X-ray crystal
structure revealed unambiguously that compound 22 did not in-
duce formation of the FAK allosteric site, and was observed bound
l
l
l
l
l
over a panel of seven kinases, including three kinases (Pyk2,
Aurora B, and MEK1) which were weakly inhibited by N-free
pyrazole 1 (Table 3). These results clearly demonstrated that
introduction of the 1-methyl group on the pyrazolo[4,3-
c][2,1]benzothiazine effectively altered the binding mode from
dual to single (genuine allosteric), by disrupting hydrogen bond
formation with hinge residues of kinases together with enhanc-
ing affinity to the allosteric site.
l
Lys 454
Leu 553
Met 499
ATP site
Cys 502
Glu 500
allosteric
site
Glu506 HOH
HOH
Ser 509
Cys 502
Val 436
Ile 428
Arg 514
Arg 426
Figure 4. Co-crystal structure of 22 with FAK: hinge binding mode. PDB ID: 4I4E.

N. Tomita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1779–1785
1783
Table 2
Table 4
SAR of substituents on the pyrazole ring
SAR of linker structure
R1
N 1
2 N
N
Me
N
O
O
S
O
S
N
L
F
O
N
Et
Me
Me
a
Compound
L
FAK IC50
(lM)
Cell pFAKb (%inh.)
a
Compound
R1
FAK IC50 (lM)
25
26
23
28
17
CONH
CH2NH
NHCO
>30
2.0
2.4
0.32
0.077
Not tested
1
17
2
H
0.96
0.077
2.1
4.8
8.8
85
1-Me
1-Et
2-Me
NHCH2
31
>30
31
a
ATP 0.5
l
M, pre-incubation time 60 min.
a
ATP 0.5
l
M, pre-incubation time 60 min.
b
At 30
lM.
With the genuine FAK allosteric inhibitor in hand, we evaluated
Table 5
SAR of amino substituents
its inhibitory activity of intracellular FAK autophosphorylation
(pFAK) by cell-based ELISA in PC3 M-luc cells.20 Although com-
pound 17 showed excellent enzymatic activity, its cellular activity
N
Me
N
was not sufficient (Table 4, pFAK 31% inhibition at 30 lM). We sup-
O
O
posed that rigid biphenyl structure of 17 might limit its physico-
chemical properties and cellular potential. Thus, linker insertion
between the 1-methylpyrazolo[4,3-c][2,1]benzothiazine core and
the terminal phenyl group was examined (Table 4). Fortunately,
three linkers were tolerated in the enzymatic assay, except for
S
H
N
N
R4
Me
a
Compound
R4
FAK IC50
(lM)
Cell pFAK IC50
>30
(lM)
amide linker of 25 (FAK IC50 >30
l
M). Among them, benzylamine
24
F
4.3
28 (FAK IC50 0.32 M, pFAK 85% inhibition at 30
l
lM) was selected
F
as a new lead for further optimization because of its high potential
of cellular activity.
28
29
0.32
1.4
19
Next, the aromatic side chain was optimized (Table 5). Although
Br
aniline 24 (FAK IC50 4.3
stitution on the benzyl unit 29 (FAK IC50 1.4
>30 M) reduced activities, introduction of bulky tert-butyl group
led to enhancement of cellular activity (30, FAK IC50 0.64 M, pFAK
IC50 7.1 M). In addition, kinase selectivity profiles of 30 suggested
lM, pFAK IC50 >30
lM) and p-bromo sub-
>30
lM, pFAK IC50
l
Me
Me
Me
l
30
0.64
7.1
l
that compound 30 was also a genuine allosteric inhibitor of FAK
(Table 3). These results clearly demonstrated that inhibition of
autophosphorylation of FAK with a small molecule by inducing
FAK into an inactive conformation could be a novel strategy of
modulating FAK signaling pathways. It is also noteworthy that this
allosteric inhibition mode is highly specific to FAK. Unlike MEK1/2
and Akt1/2, FAK does not seem to share its allosteric site with the
same subfamily kinase, Pyk2.21
Interaction of the optimized compound 30 with FAK kinase do-
main was examined in detail by X-ray crystallographic analysis
(Fig. 5, PDB ID: 4I4F). The overall structure of the FAK kinase do-
main and binding site of 30 were similar to those observed in
the complex of the lead compound 1. The methyl substituent on
the pyrazole ring is inserted in the hydrophilic space surrounded
by Asp604 and His544, avoiding disruption of the hydrogen bond
a
ATP 0.5 lM, pre-incubation time 60 min.
bond with the backbone carbonyl of Gly563, while inducing a
slight rotation of its side chain. The terminal tert-butyl group occu-
pies a hydrophobic pocket formed by Met475, Leu486, Met499
(gatekeeper of FAK) and Val484. These interactions resulting from
the induced structural rearrangement of the HRD-loop in FAK,
facilitate the acquired slow-dissociative and ATP-noncompetitive-
like properties observed with these compounds,8 resulting in the
intracellular inhibition of FAK autophosphorylation.
In summary, we have identified novel allosteric inhibitors of
FAK, guided by the structural analyses of FAK-inhibitor complexes.
Introduction of methyl group on the pyrazole ring effectively
blocked potential hinge-binding mode and enhanced inhibition
activity. Kinase inhibition profiles of 1-methyl derivatives showed
excellent FAK selectivity over other kinases including Pyk2, which
network of water molecules and presumably enhancing cation–
p
interaction between Arg550 and pyrazole ring by increasing elec-
tron density of the pyrazole. The 8-amino group gains a hydrogen
indicated this allosteric site was highly specific to FAK.
A
Table 3
Kinase selectivity profiles of representative compounds
Compound
FAK IC50
(
lM)
Tyrosine kinases IC50
Src
(
lM)
Serine threonine kinases IC50 (lM)
Pyk2
HER2
Akt1
Aurora B
MEK1
p38a
1
17
30
1.2a/0.96b
9.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.1
>10
>10
8.6
>10
>10
>10
>10
>10
0.42a/0.077b
2.2a/0.64b
a
ATP 0.5
ATP 0.5
l
l
M, pre-incubation time 5 min.
M, pre-incubation time 60 min.
b

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N. Tomita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1779–1785
Met 475
Asp 564
Asp 604
His 544
HOH
HOH
Leu 486
Lys 454
Arg 550
Gly 563
Met 499
Met 607
Val 484
Phe 608
Val 552
Cys 611
Orange: compound 1
Purple: compound 30
Figure 5. Co-crystal structure of 30 with FAK: allosteric binding mode. PDB ID: 4EBV (compound 1), 4I4F (compound 30).
4. (a) Slack-Davis, J. K.; Martin, K. H.; Tilghman, R. W.; Iwanicki, M.; Ung, E. J.;
Autry, C.; Luzzio, M. J.; Cooper, B.; Kath, J. C.; Roberts, W. G.; Parsons, J. T. J. Biol.
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benzylamine analogue 30 showed significant decrease of auto-
phosphorylation of FAK in PC3M-luc cells, demonstrating that allo-
steric inhibitors could effectively block a key event of FAK signaling
pathways in living cells. Further optimization of related com-
pounds would provide allosteric inhibitors of FAK with enhanced
activities and selectivity, which could be an option for novel
molecular-targeted antitumor therapy.
Acknowledgments
We thank Clifford D. Mol and Gyorgy Snell for analyses of crys-
tal structures and X-ray data collections. We thank Keiko Higashik-
awa and Mitsuyoshi Nishitani for analysis of X-ray single crystal
analysis of compound 17.
Supplementary data
Supplementary data (synthetic procedures of new compounds
and X-ray single crystal analysis of compound 17) associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2013.01.047. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
7. (a) Bagi, C. M.; Roberts, G. W.; Andresen, C. J. Cancer 2008, 112, 2313; (b)
Watanabe, N.; Takaoka, M.; Sakurama, K.; Tomono, Y.; Hatakeyama, S.; Ohmori,
O.; Motoki, T.; Shirakawa, Y.; Yamatsuji, T.; Haisa, M.; Matsuoka, J.; Beer, D. G.;
Nagatsuka, H.; Tanaka, N.; Naomoto, Y. Clin. Cancer Res. 2008, 14, 4631.
8. Iwatani, M.; Iwata, H.; Okabe, A.; Skene, R. J.; Mol, C. D.; Tomita, N.; Hayashi, Y.;
Hosfield, D. J.; Hori, A.; Baba, A.; Miki, H. Eur. J. Med. Chem. 2012, In press.
9. For the measurement of enzymatic activity of FAK, we used HTRF detection
system (Cisbio, France) with anti-phosphotyrosine antibody. Kinase buffer
consisted of 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, 2 mM DTT,
0.01% Tween20 and 0.01% BSA. 30 ng/mL of unphosphorylated FAK kinase
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20. Human prostate cancer PC3M-luc cells were purchased from Xenogen
(Alameda, CA), and cultured in minimum essential medium supplemented
with 10% fetal bovine serum in an incubator containing 5% CO2. Cells
(5 ꢁ 104 cells/100
lL) were seeded into low-attachment 96-well plate
(Corning, Cat#3363) in culture medium with serially diluted compounds.
After 30 min incubation, the cell suspension was transferred to the fibronectin-
coated 96-well plate (BD, Cat#354402) and incubated for 2 h. After removing
culture medium, cells were lysed in 50 lL of RIPA buffer (SIGMA, Cat#R0278).
Lysates were transferred to the rabbit anti-FAK antibody immobilized plate
(STAR phospho-FAKTyr397 ELISA kit, Upstate, Cat#17-480), and incubated with
anti-phospho FAKTyr397 antibody for 2 h. After washing,
a horseradish
peroxidase (HRP)-labeled anti-rabbit IgG was added and plates were
incubated for 30 min. After washing, HRP substrate was added and the plate
was incubated for further 30 min. Then, the HRP reaction was measured in the
absorbance at 450 nm.
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