Building skeletally diverse architectures on the Indoline Scaffold: The discovery of a chemical probe of focal adhesion kinase signaling networks

Article abstract of DOI:10.1016/j.bmc.2008.09.025

Inspired by bioactive indoline alkaloid natural products, here, we report a divergent synthesis approach that led to skeletally diverse indoline alkaloid-inspired compounds. The natural product-inspired compounds obtained were then subjected to a series o

Full text of DOI:10.1016/j.bmc.2008.09.025

Bioorganic & Medicinal Chemistry 16 (2008) 9596–9602
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Building skeletally diverse architectures on the Indoline Scaffold: The discovery
of a chemical probe of focal adhesion kinase signaling networks
Michael Prakesch b, Krikor Bijian c, Valérie Campagna-Slater d, Sophie Quevillon a, Reni Joseph a,
Chang-Qing Wei a, Esther Sesmilo a, Ayub Reayi a, Rajamohan R. Poondra a, Michael L. Barnes a,
Donald M. Leek a, Bin Xu c, Caroline Lougheed c, Matthieu Schapira d,e, Moulay Alaoui-Jamali c,
,
*
Prabhat Arya a,b,
*
a Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ont., Canada K1A 0R6
b Ontario Institute for Cancer Research, MaRS Centre, South Tower, 101 College Street, Toronto, Ont., Canada M5G 0A3
c Lady Davis Institute for Medical Research, 3755 Chemin Cote-Ste-Catherine, Room E524, Montreal, Que., Canada H3T 1E2
d Structural Genomics Consortium, Banting Building, University of Toronto, 100 College Street, Toronto, Ont., Canada M5G 1L5
e Department of Pharmacology, University of Toronto, 100 College Street, Toronto, Ont., Canada M5G 1L5
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2008
Revised 5 September 2008
Accepted 9 September 2008
Available online 12 September 2008
Inspired by bioactive indoline alkaloid natural products, here, we report a divergent synthesis approach
that led to skeletally diverse indoline alkaloid-inspired compounds. The natural product-inspired com-
pounds obtained were then subjected to a series of in vitro and cellular assays to examine their properties
as modulators of focal adhesion kinase (FAK) activity. This study resulted in the identification of a prom-
ising lead inhibitor of FAK (42), which also showed activity in a wound healing and cell invasion assay.
The in silico study of the lead compound (42) was also undertaken.
Keywords:
Natural products
Ó 2008 Elsevier Ltd. All rights reserved.
Natural product-like compounds
Small-molecule chemical probes
Diversity-oriented synthesis
Focal adhesion kinase
Signaling networks
Cell migration
Anti-cancer agents
Docking
1. Introduction
certain cases, to develop into new therapeutic approaches in drug
discovery and the probes could function as starting candidates for
The post-genomics chemical biology age has brought challenges
to the biomedical research community trying to develop a better
understanding of protein–protein interaction-based signaling net-
works.1–7 This has resulted in a growing use of small molecules as
chemical probes for understanding these networks.8–10 There are
several advantages in the use of small molecules. These advantages
include the ability of the small molecules (i) to interact with the
biological targets in a reversible manner, (ii) to selectively modu-
late only one of the multiple interactions being made by the bio-
logical target, and (iii) to interfere with the cellular machinery in
a non-destructive manner. Another advantage is that, in addition
to developing useful chemical probes for understanding cellular
machinery, the study of these probes also has the potential, in
drug design.
2. Results and discussion
2.1. Synthesis of a diverse set of indoline-derived compounds
Several indole and indoline alkaloid natural products are
known to interfere with protein surfaces involved in protein–pro-
tein interactions.11 Some of the bioactive alkaloid natural products
(e.g., staurosporine, 1, vindoline, 2, and vinblastine, 3) are shown in
Figure 1. As a result, we were interested in synthesizing alkaloid-
inspired compounds having the indoline scaffold containing an
additional medium size cyclic ring to generate skeletally diverse
compounds.12,13 Indoline-based compounds are quite abundant
in nature and are considered highly privileged building blocks in
medicinal chemistry as well.14 Since natural products containing
* Corresponding author. Tel.: +1 613 993 7014; fax: +1 613 952 0068.
E-mail address: prabhat.arya@nrc.ca (P. Arya).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.025

M. Prakesch et al. / Bioorg. Med. Chem. 16 (2008) 9596–9602
9597
H
N
O
Me
OH
Me
N
N
Me
N
OAc
OH
H
H
H
N
N
N
O
OAc
OH
H
H
H
COOMe
N
Me
MeO
Me
MeOOC
MeO
COOMe
N
MeO
Me
Vinblastine (3)
NHMe
Staurosporine (1)
Vindoline (2)
Figure 1. Indole and indoline alkaloid natural products as modulators of protein–protein interactions.
this scaffold have led to interesting biological properties, this war-
ranted further structural modifications to its backbone.
was carried out as follows. The -OMEM ether of 3-(C-allyl)-4-nitro-
phenol 10c (see the Supporting Material) was synthesized from
2-nitro-5-hydroxybenzaldehyde in four steps. The allyl derivative
was then subjected to Sharpless dihydroxylation reaction giving
the dihydroxy derivative, 11. Surprisingly, the ee of this reaction
was low and the reason is not clear at this stage. The primary-
OH group was then protected as –OBz, followed by tosylation of
the secondary –OH group. The tosyl derivative was then hydroge-
nated over 10% Pd/C (THF) for the nitro group reduction. The cor-
responding amine was then treated with K2CO3 in DMF at 40 °C
giving the indoline derivative, 12 (after the N-Alloc protection) in
high yield. In another study, the corresponding vinyl and allyl
derivatives, 19 and 21 (see 22 and 23 to explain the stereocontrol
outcome) were easily obtained using (S)-(+)-indolinemethanol (17)
as the starting material.
Using the ring closing metathesis as the key step, the generation
of tricyclic architectures, 25, 27, 29, 31, and 33 is shown in Scheme
2. Indolinol 12 was converted to the corresponding aldehyde 13
(80%). Following this, compound 13 was then subjected to allyla-
tion reaction. Thus, treatment of 13 with allylMgBr in the presence
of ZnCl2, followed by the protection of the hydroxyl groups, gave
14 and 15 as a separable mixture of diastereomers. (Note. the ste-
reochemistry was assigned in the cyclic products obtained from
the RCM.) At this point, the stage was set to explore the olefin ring
closing metathesis reaction with different substrates. For our mod-
el studies to explore the RCM reaction, pure diastereomers, 14 and
15 were independently converted to N-acryloyl derivatives (24 and
26). With the 1st generation Grubbs’ 10 mol% catalyst, it was a de-
light to observe the cyclic enamide formation (25 and 27) in high
yields within 2–5 min at the room temperature. For other three
systems, compounds 29, 31, and 33 were independently obtained
from 16, 19, and 21. All of them successfully generated the tricyclic
products, 29, 31, and 33 that utilized the 2nd generation Grubbs’
catalyst in the ring closing metathesis.
In our synthetic planning to generate skeletally diverse indo-
line-based tricyclic compounds, the objective was to synthesize
generic compounds 5–9 from a common functionalized intermedi-
ate such as 4 as shown in Figure 2. Compound 4 as a bicyclic scaf-
fold possesses the phenolic hydroxyl group and an amino alcohol
functionality. The primary hydroxyl group on 4 could be converted
to the corresponding aldehyde and homoaldehyde derivatives and
be further subjected to allylation and vinylation reactions, produc-
ing allylic and vinylic derivatives. Through developing the ring
closing metathesis approaches, the indoline scaffold could then
lead to the formation of tricyclic compounds 5–9. The RCM reac-
tion15–17 has been extensively utilized in formation of enone/ena-
mide moieties in solution phase and several examples in the
literature further validated our approach. Alternatively, 9 could
be derived from the regioselective opening of an epoxide in 8 to
form the enamide moiety. As a result, an attractive feature to this
strategy is that indoline-based tricyclic derivatives5, 6, 7, and 9 all
contain the enamide moiety and could be further subjected to ste-
reocontrolled diversity-based reactions. In addition to the amino
alcohol moiety, the phenolic hydroxyl group on indoline scaffold,
4, could act as an anchoring site onto solid support in solid phase
reactions to generate indoline-based library members. In a skeletal
diversity-based synthesis approach, the formation of indoline-
based tricyclic derivatives would provide a novel entry to a wide
variety of complex, natural product-inspired polycyclic com-
pounds on solid support.
Central to this idea is the development of an efficient solution
method to obtain an indolinol derivative (12, Scheme 1). This
derivative is a versatile building block and can be utilized in the
synthesis of indoline-based, natural product-inspired polycyclic
derivatives. This building block has three orthogonal functional
groups including a phenolic hydroxyl moiety that is protected as
–OMEM. Upon deprotection, this functional group could serve as
an anchoring site in solid phase synthesis. These derivatives are
easy to synthesize on a large scale (i.e., 10–20 g) and the synthesis
The synthesis plans leading to tricyclic architectures, 38 and 41
are shown in Scheme 3. To achieve these goals, compound 13 was
converted into an olefin through a Wittig reaction (96%). The N-al-
O
O
N
H
N
R
R
H
HO
P2
N
2
OH
6
5
OP1
R
O
O
4
O
N
H
N
N
R
R
H
7
R
H
OH
8
9
Figure 2. Skeletally diverse tricyclic architectures derived from an indolinol scaffold.

9598
M. Prakesch et al. / Bioorg. Med. Chem. 16 (2008) 9596–9602
NO2
NO2
Alloc
a
b
OH
N
OH
OH
HO
CHO
MEMO
d
MEMO
10
11
12
Alloc
CHO
Alloc
Alloc
c
N
N
N
H
12
and
MEMO
MEMO
MEMO
H
OTBS
OTBS
13
14
15
Felkin-Anh model
Alloc
Alloc
N
e
N
H
O
R
CHO
R
22
N
NH
OAc
f
13 R=OMEM
18 R=H
16 R=OMEM
19 R=H
Alloc
Nu-
OH
H
H
R
RO
g
17 R = H
R = H
O
(S)-(+)-2-Indolinemethanol
Alloc
N
H
H
Alloc
h
N
N
H
OTBS
Nu-
O
CHO
re-face
attack
23
20
21
Scheme 1. Synthesis of the indoline skeleton. Reagents and conditions: (a) i—Ph3PHC(OMe)Cl, t-BuOK, 0 °C, 96%; ii—1.5 HCl, 60 °C, 98%; iii—NaHMDS, Ph3PMeBr, THF, 0 °C,
86%; iv—MEMCl, DIPEA, rt, 89%; v—sharpless dihydroxylation reaction-AD mix-b, 99%; (b) i—BzCl, pyridine, À5 °C, 24h, 89%; ii—p-TsCl, DMAP, 35 °C, 96%; iii—H2, 10% Pd/C,
K2CO3, rt; iv—K2CO3, DMF, 40 °C, 80% for 2 steps; v—K2CO3, MeOH, rt, 83%; vi—AllocCl, DIPEA, À78 °C, 94%; (c) SO3 pyridine, 80%; (d) i—AllylMgBr, ZnCl2, À78 °C to rt, 41% for
13a (syn) and 47% for 13b (anti); ii—TBDMSCl, imidazole, 83% for 14 (syn) and 88% for 15 (anti); (e) i—vinylMgBr, À78 °C to rt; ii—Ac2O, DMAP, 16: 37% and 19: 70% for 2 steps;
(f) i—AllocCl, DIPEA, À78 °C; ii—SO3 pyridine, 91%; (g) i—Ph3PHC(OMe)Cl, t-BuOK, 0 °C, 96%; ii—pTSA, 40 °C, 94%; (h) i—AllylMgBr, À78 to À15 °C (ii) TBDMSCl, imidazole, 65%
for 2 steps.
O
O
b
b
N
H
a
N
H
14
MEMO
MEMO
TBSO
TBSO
24
25
O
O
N
H
N
a
15
MEMO
MEMO
H
TBSO
TBSO
27
26
O
O
N
H
c
N
H
a
16
MEMO
MEMO
OAc
O
OAc
28
29
O
N
c
a
N
H
19
21
H
OAc
OAc
30
31
O
O
c
a
N
N
H
H
OTBS
OTBS
32
33
Scheme 2. Synthesis of tricyclic architectures by RCM. Reagents and conditions: (a) i—Pd(PPh3)4, acetic acid, 4-methylmorpholine, rt; ii—acryloyl chloride, DIPEA, rt, 74–95%
for 2 steps; (b) 10 mol% 1st generation Grubbs’ catalyst, rt, 90–97%; (c) 10 mol% 2nd generation Grubbs’ catalyst, rt, 85–92%.

M. Prakesch et al. / Bioorg. Med. Chem. 16 (2008) 9596–9602
9599
loc group was then removed and substituted by coupling with vi-
R1
R2
nyl acetic acid on the cyclic amine to afford 34. Upon treatment
with Grubbs’ 1st generation catalyst, 34 was converted into 35 in
a RCM reaction. Compound 35 was then subjected to epoxidation
conditions using DMDO and 36 was the only product observed
by 1H NMR. Treatment with LiHMDS followed by coupling with
phenylacetic acid using DIC produced 37. Reaction of 37 with ben-
zenethiol afforded 38 in a stereocontrolled manner containing two
diversity sites. In a similar manner, the series of reactions that in-
cluded (i) Wittig reaction, (ii) N-alloc removal, and (iii) N-acryloy-
lation (77%), on 13 yielded the product 39. Indoline-based tricyclic
derivative 40 was obtained from 39 in 75% yield by RCM reaction
with the 1st generation Grubbs’ catalyst. As a test study, 40 on
reaction with PhSH/Et3N gave the Michael-type product, 41, as
the major isomer (ratio 5.5:1). Compounds shown in Figure 3
(see, 42 and 43) were also synthesized as a part of the study lead-
ing to the development of the solid phase synthesis program (not
shown in this paper). The details of the synthesis procedure are
provided in the Supporting Material.
diastereomeric mixture
*
O
O
O
OAc
Alloc
R2
(42)
N
O
OH
R1
(43)
OAc
Figure 3. Two other indoline derivatives (see the Supporting Material for the
synthesis details).
2.3. In vitro kinase assay
All the indoline-derived compounds were tested in a search for
small molecule inhibitors of FAK. In a typical study, human recom-
binant full-length FAK (60 ng) was incubated in kinase buffer con-
taining ATP (2
temperature with or without the presence of the indoline-derived
compounds at 30 M final concentration. Remaining ATP in solu-
tion was then quantified utilizing the Kinase-Glo-luminescence
kit (Promega). This study led to the discovery of a novel indoline-
derived compound, compound 42, which successfully inhibited
lM) and substrate (20 lg/ml) for 4 h at room
2.2. Testing of indoline-derived, chemical modulators of FAK
l
With the goal of finding small molecule modulators of focal
adhesion kinase (FAK), the diverse collection of indoline-derived
compounds was then subjected to several biological assays.
Belonging to the family of non-receptor tyrosine kinase, FAK plays
a crucial role in signal transduction pathways that are initiated by
integrin-mediated cell adhesions and growth factor receptors.18–24
As an adapter protein,25–27 FAK is involved in regulating several
cellular processes including cell survival, cell proliferation, and cell
migration and invasion in the development and progression of can-
cer.24,28–31 It is well documented that an over expression of FAK is
commonly seen in a wide variety of human cancers.32 This further
confirms the role of FAK in tumorigenesis, metastasis, and survival
signaling.23,28,32 Due to these reasons, there is a growing interest in
identifying small molecules that could serve as chemical probes to
enhance our current understanding of signaling networks involv-
ing FAK.33–39 In addition to this, some of these probes could also
become an excellent starting point in developing new therapeutic
agents for cancer.
FAK kinase activity (30% inhibition at 30 lM) (Fig. 4A). Additional
studies examining the structure–activity relationship of compound
42 and FAK will be necessary in order determine if this compound
is functioning as an ATP competitor or allosteric inhibitor.
2.4. Cell proliferation assay
Exponentially growing cells (1 Â 103 cells) were seeded in 96-
well plates. After 18 h cells were continuously treated with com-
pound 42 dissolved in DMSO. The final concentration of DMSO
was less than 0.05%. Following this, after 96 h, cell survival was
evaluated using the 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) metabolic assay as previously de-
scribed.40 The inhibitory activity (IC50) of compound 42 was
determined to be 38.7 lM (Fig. 4B).
O
O
O
a
c
b
N
N
H
N
MEMO
MEMO
MEMO
O
H
35
36
34
O
O
N
d
N
H
e
13
MEMO
SPh
OCOBn
MEMO
H
OCOBn
38
37
O
O
O
e
N
H
b
N
N
f
MEMO
MEMO
MEMO
H
SPh
39
40
41
Scheme 3. Exploration of functional group diversity. Reagents and conditions: (a) i—NaHMDS, Ph3PMeBr, THF, 0 °C, 96%; ii—Pd(PPh3)4, acetic acid, 4-methylmorpholine, rt,
90%; iii—vinyl acetic acid, DIC, HOBt, DMF, rt, 61%; (b) 25 mol% 1st generation Grubbs’ catalyst, dichloromethane, for 35: rt, 1 h, 77%, for 40: reflux, 48 h, 75%; (c) DMDO,
acetone; (d) i—LiHMDS, À78 °C, 95%; ii—phenylacetic acid, DMAP, DIC, 96%; (e) benzenethiol, triethylamine, 0 °C, 97% for 38 and 95% for 41; (f) repeat step (a) (i) and (ii), (iii)
acryloyl chloride, pyridine, 0 °C, 77%.

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M. Prakesch et al. / Bioorg. Med. Chem. 16 (2008) 9596–9602
Figure 4. Effect of compound 42 on FAK kinase activity (A and C) and cell proliferation (B). (A) In vitro kinase assay of full-length FAK with 30
lM of indicated compounds. (B)
MTT assay of MDA231-M cells treated with increasing concentrations of compound 42. IC50 = 38.7 M. (C) Expression level of pY397-FAK (P-FAK), FAK, and actin in MDA231-
l
M cells pretreated with or without compound 42 (25
lM, 1 h) examined by Western blot. Compound 42 pretreatment led to a significant decrease in phosphorylated tyrosine
397-FAK as compared to control (DMSO).
2.5. Cellular FAK activity
2.7. Cell motility assay (Fig. 6)
The level of phosphorylated tyrosine 397 (the major autophos-
phorylation site of FAK) in MDA231-M cells was evaluated in cells
pretreated with or without compound 42 (Fig. 4C). Compound 42
treated MDA231-M cells demonstrated reduced levels of phos-
phorylated tyrosine 397, indicative of the reduced FAK activity.
The effect of compound 42 on cell motility was investi-
gated using the wound healing assay as described earlier.43
In this study, cells were grown on sterile coverslips for 24 h
and were then wounded by cell scraping using a micropipette
tip. Cultures were washed and then incubated with fresh cul-
ture media at 37 °C with or without the presence of com-
2.6. Docking simulation
pound 42 (25 lM) for the indicated time periods. Cells were
allowed to migrate and heal the wound. Photomicrographs
were taken at each time point in order to examine the
wound healing areas (Fig. 6).
Compound 42 was docked with Glide (Schrödinger, NY)41 to a
grid representation of the catalytic site of human FAK co-crystal-
lized with a sub-micromolar pyrrolopyrimidine inhibitor (PDB
code 2ETM, http://www.pdb.org). A constraint was used during
docking that forced hydrogen bonding of compound 42 to the
backbone nitrogen of Cys502, since a similar interaction was ob-
served in all FAK co-crystal structures available. The resulting com-
plex was then locally minimized with flexible receptor side chains
in the internal coordinates space with ICM (Molsoft LLC, CA).42 Two
hydrogen bonds are observed between 42 and FAK, the first one
between the acyl carbonyl of Alloc and the backbone nitrogen of
Cys502, the other between the piperidine oxygen of compound
42 and the backbone nitrogen of Asp564 (Fig. 5).
This first generation hit confirms that our indoline-based chem-
ical series is suitable for FAK inhibition. Docking studies suggest
opportunities to optimize 42. For instance, the side chains of
E506 and K454 are less than 5 Å away from docked 42, but are
not making polar interaction. Additionally, another structure of
FAK (PDB code 2J0L)26 shows an alternate conformation of the acti-
vation loop with a larger catalytic site that may accommodate
more potent derivatives.
2.8. Cell invasion assay (Fig. 7)
Cell invasion experiments were performed with 8 lm porous
chambers coated with Matrigel (Becton–Dickinson) as described
earlier.43 Briefly, serum starved cells were placed into the upper
compartment (30,000 cells) of a 24-well Boyden chamber with
or without compound 42 (25
lM) and the chambers were then
placed into 24-well culture dishes containing 400
ll of DMEM
0.2% BSA with 10% serum (lower compartment) as chemoattrac-
tant. Invasive cells are able to invade through the Matrigel,
infiltrating through the pores and attaching themselves to the
underside of the membrane. The invasive cells underneath the
membrane were fixed and stained after 48 h. Filters were
viewed under bright-field 40Â objective and the counting was
performed for three fields in each sample. As shown in Figure
7, reduced invasive activity of MDA231-M cells was observed
in the presence of compound 42, as compared to DMSO treated
control.
Figure 5. Docked conformation of compound 42 complexed to the ATP site of FAK. Compound 42 is deeply buried in the pocket at the interface of the FAK N- and C-terminal
lobes (A) and makes hydrogen bonds with the backbone amide groups of Cys502 and Asp564 (B).

M. Prakesch et al. / Bioorg. Med. Chem. 16 (2008) 9596–9602
9601
compound 42 was also able to demonstrate its inhibitory activity
in the Boyden chamber invasion assay, indicative of its ability to
inhibit cell movement on a three-dimensional scale and/or by
reducing the cells’ ability to produce extracellular matrix degrad-
ing enzymes, such as matrix metalloproteinases. Although the pre-
cise mechanism of action of 42 is not clear at this stage, it certainly
serves as a good starting point in developing next generation deriv-
atives and warrants further investigation.
Acknowledgments
This study was conducted with the support of the NRC Genom-
ics and Health Initiative, Canadian Cancer Society (CCS), National
Cancer Institute of Canada (NCIC), Canadian Institutes of Health Re-
search (CIHR), and Ontario Institute for Cancer Research (OICR)
through funding provided by the Government of Ontario. The
Structural Genomics Consortium is
a registered charity (No.
1097737) that receives funds from the Canadian Institutes for
Health Research, the Canadian Foundation for Innovation, Genome
Canada through the Ontario Genomics Institute, GlaxoSmithKline,
Karolinska Institutet, the Knut and Alice Wallenberg Foundation,
the Ontario Innovation Trust, the Ontario Ministry for Research
and Innovation, Merck, the Novartis Research Foundation, the
Swedish Agency for Innovation Systems, the Swedish Foundation
for Strategic Research, and the Wellcome Trust.
Figure 6. The effect of compound 42 on the MDA231-M cell motility. Wound
healing assay of MDA231-M cells treated with or without the presence of 42
(25 lM). Arrows indicative of remaining wound area.
3. Summary
With the goal of obtaining diverse architectures from the indo-
line scaffold, several modular approaches were developed. This al-
lowed us to generate a wide variety of indoline-based bicyclic and
tricyclic compounds. To extend the scope of these methods on solid
phase for high-throughput generation of indoline-based libraries,
further work is in progress. The highly diverse collection obtained
in this program (through the solution phase synthesis) was then
examined in a search of chemical modulators of FAK activity. To-
ward this objective, the collection was subjected to full-length
FAK inhibition assay and this led to the discovery of a novel class
of moderate small molecule inhibitor of FAK (compound 42). To
explore the scope of 42 further, it was also investigated as a mod-
ulator of cell migration through the wound healing assay and
showed a significant inhibitory activity of cell migration. Moreover,
Supplementary data
Experimental details and full characterization data for all new
compounds are provided. This material is available free of charge
via the internet. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmc.2008.09.025.
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