Fragment-based discovery of new highly substituted 1H-pyrrolo[2,3-b]- and 3H-imidazolo[4,5-b]-pyridines as focal adhesion kinase inhibitors

Article abstract of DOI:10.1021/jm3016014

Focal adhesion kinase (FAK) is considered as an attractive target for oncology, and small-molecule inhibitors are reported to be in clinical testing. In a surface plasmon resonance (SPR)-mediated fragment screening campaign, we discovered bicyclic scaffolds like 1H-pyrazolo[3,4-d]pyrimidines binding to the hinge region of FAK. By an accelerated knowledge-based fragment growing approach, essential pharmacophores were added. The establishment of highly substituted unprecedented 1H-pyrrolo[2,3-b]pyridine derivatizations provided compounds with submicromolar cellular FAK inhibition potential. The combination of substituents on the bicyclic templates and the nature of the core structure itself have a significant impact on the compounds FAK selectivity. Structural analysis revealed that the appropriately substituted pyrrolo[2,3-b]pyridine induced a rare helical DFG-loop conformation. The discovered synthetic route to introduce three different substituents independently paves the way for versatile applications of the 7-azaindole core.

Full text of DOI:10.1021/jm3016014

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Article
Fragment-based Discovery of New Highly Substituted 1H-Pyrrolo[2,3-b]-
and 3H-Imidazolo[4,5-b]-Pyridines as Focal Adhesion Kinase Inhibitors
Timo Heinrich, Jeyaprakashnarayanan Seenisamy, Lourdusamy Emmanuvel, Santosh S. Kulkarni, Jörg
Bomke, Felix Rohdich, Hartmut Greiner, Christina Esdar, Mireille Krier, Ulrich Grädler, and Djordje Musil
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jan 2013
Downloaded from http://pubs.acs.org on January 16, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Fragment-based Discovery of New Highly Substituted 1H-Pyrrolo[2,3-b]- and 3H-
Imidazolo[4,5-b]-Pyridines as Focal Adhesion Kinase Inhibitors
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Timo Heinrich,*,† Jeyaprakashnarayanan Seenisamy,‡ Lourdusamy Emmanuvel,¥ Santosh S. Kulkarni,‡ Jörg
Bomke,† Felix Rohdich,† Hartmut Greiner,† Christina Esdar,† Mireille Krier,† Ulrich Grädler,† Djordje Musil†
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† Merck Serono Research, Merck KGaA, 64271 Darmstadt, Germany
‡ Syngene International Ltd., Biocon Park, Plot 2&3, Bommasandra-Jigani Link Road, Bangalore 560 099, India
¥
Karunya University, Dept. of Chemistry, School of science and humanities, Coimbatore 641114, India
Abstract
Focal adhesion kinase (FAK) is considered as an attractive target for oncology and small molecule inhibitors are
reported to be in clinical testing. In a surface plasmon resonance (SPR)-mediated fragment screening campaign
we discovered bicyclic scaffolds like 1H-pyrazolo[3,4-d]pyrimidines binding to the hinge-region of FAK. By an
accelerated knowledge-based fragment growing approach essential pharmacophores were added. The
establishment of highly substituted unprecedented 1H-pyrrolo[2,3-b]pyridine derivatisations provided
compounds with sub-micromolar cellular FAK inhibition potential. The combination of substituents on the
bicyclic templates and the nature of the core structure itself have a significant impact on the compounds FAK
selectivity. Structural analysis revealed that the appropriately substituted pyrrolo[2,3-b]pyridine induced a rare
helical DFG-loop conformation. The discovered synthetic route to introduce three different substituents
independently paves the way for versatile applications of the 7-azaindole core.
Introduction
FAK is a 125-kDa nonreceptor tyrosine kinase that modulates cell adhesion, migration, proliferation, and
survival in response to extracellular signals.1-4 The N-terminal FERM domain (4.1, ezrin, radixin, moesin) of
FAK is composed of three sub-domains and regulates the kinase activity of FAK.5-10 In the autoinhibited state,
the FERM domain directly binds to the kinase C-lobe, impeding access to the ATP-binding site and protecting
the activation loop from phosphorylation by Src. The inhibitory FERM–kinase interaction involves the F2 lobe
of the FERM domain and a site of the C-lobe centered at Phe596.6 The FERM domain has been shown to
associate with integrins and growth factors, implying the important role of FAK in integrating diverse cellular
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signalling pathways. In addition to being a key player in regulating normal cellular activities such as adhesion,
migration and survival, FAK is also implicated in cancer cell invasion, metastasis and survival.11-13 Accordingly,
FAK inhibition is considered as an effective antineoplastic strategy by inducing apoptosis and sensitizing tumor
cells to chemotherapy. A couple of FAK inhibitors with mono- and bi-cyclic core structures have been
described, some even in clinical phase-1 testing.14-21 Structurally very similar pyrimidines I (TAE-226),22 II (PF-
562271),23 pyridine III (PND-1186 )24 and the pyrrolopyrimidine IV25 are described as hinge-binders whereas
Chloropyramine26 and Y1527 are anticipated to target the FAK-VEGFR-3 interface and the Y397 site,
respectively.
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Cl
CF3
NH
CF3
NH
N
N
N
N
N
HN
O
N
O
HN
N
NH
O
HN
N
HN
O
O
O
NH
NH
O
N
S
N
NH
N
O
N
O
O
O
O
I (TAE226)
II (PF-562271)
III (PND-1186)
IV
Cl
O
N
O
N
S O
S O
H2N
H2N
NH2
NH2
N
N
N
N
N
N
H
N
Chloropyramine
Y15
V
VI
Figure 1. FAK inhibitors
Selective inhibition of protein kinases is a sometimes very difficult task in drug discovery research. Accordingly,
drugs like sunitinib targeting the highly conserved ATP-binding pocket are multi-kinase inhibitors.28 Allosteric
kinase inhibition has been reported as an attractive option as enzyme blockade is achieved by addressing protein
specific areas outside of the ATP-site.29,30 Selective kinase inhibition of a ligand-template binding the backbone
amides of the hinge region within the ATP-site might be achieved by shaping protein specific contacts or protein
conformations. This was demonstrated for drugs such as imatinib, which bind to an inactive conformation of Abl
kinase, adopting a particular conformation of the activation-loop.31,32 This conformation is characterized by a
rotation of the π-backbone torsion angle of the Asp in the DFG motif by approximately 180° (“DFG-out”).
Imatinib's specificity has been attributed to its recognition of the DFG-out conformation of the Abl kinase
activation loop.33 For some FAK inhibitors like II or III bearing a pyrimidine or pyridine scaffold as hinge-
binder, a remarkable FAK selectivity could be achieved and was explained by X-ray crystallography.24,34,35 The
crystal structure of FAK in complex with II revealed a helical conformation of the activation loop next to
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Gly563, that is located ahead of the DFG-motif.23 The high conformational flexibility of Gly563 and
appropriately substituted ligands can force the activation loop of FAK into a helical conformation. Binding to
this unusual kinase conformation is considered to be responsible for the inhibitor's selectivity as only 17 out of
90 tyrosine kinases have a Gly preceding the DFG motif.36 In a very recent paper novel allosteric FAK inhibitors
V and VI are described.37 These sulphonamide based compounds inhibit unphosphorylated FAK more strongly
than phosphorylated FAK by binding to the DFG-out conformation and inducing the unique HRD-loop
conformation. This binding mode implicates steric occlusion of ATP-binding by activation loop residues Phe565
and Leu567, an inhibition mechanism also described for allosteric IGF-1R inhibitors.30
In this paper we introduce for the first time novel highly substituted bicyclic FAK inhibitors which induce a
helical DFG-loop conformation of FAK.
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Results
At the beginning of the project a set of commercially available fragments38 was screened against the immobilized
kinase domain of FAK.39 The binding affinity was measured by SPR additionally enabling the determination of
binding kinetics for most of the screened fragments.40 Within a set of bicyclic screening hits (2-Fluoro-phenyl)-
(1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amine 1 was detected41 with a KD value of 43 µM.42 The superimposed X-
ray structures of FAK in complex with this fragment 1 (PDB ID: 4GU9) and with II (PDB ID: 3BZ3) are shown
in figure 2.
Figure 2. Superimposition of the FAK X-ray structures in complex with II (green, PDB ID: 3BZ3) and 1
(magenta, PDB ID: 4GU9, Monomer A) reveals hydrogen bond contacts (distances in Å) to backbone amides of
Glu500 and Cys502 of the hinge-region. A non-classical H-bond is formed between the aromatic CH of 1 and
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the carbonyl O-atom of Cyc502. In the FAK•II complex, the activation loop is partly (Asp564-Arg569) in a
4
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unique helical conformation.
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The X-ray structure analysis reveals that both pyrimidine like nitrogens, N7 of 1 and N1of II, function as
hydrogen-bond acceptors to the backbone-NH of Cys502 in the hinge region of the ATP-binding site. The
exocyclic amino-group at C2 of the PF-compound is in hydrogen-bond distance of 2.83 Å to the backbone
carbonyl-O of Cys502 whereas the hydrogen donor of fragment 1 is binding to the backbone carbonyl-O of
Glu500 (2.94 Å). In addition the exocyclic amino-functions of fragment 1 and at C4 of II are equally oriented.
Instead of the 1H-pyrazolo[3,4-d]pyrimidine scaffold of 1 the bicyclic system 1H-pyrrolo[2,3-b]pyridine was
chosen as scaffold for the fragment growing work, as it offered various options for decoration (figure 3).
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N
N
N
N
H
1H-pyrazolo[3,4-d]pyrimidine
4
3
5
2
6
N
N
H
1H-pyrrolo[2,3-b]pyridine =
7-azaindole
Figure 3. Hinge-binding scaffold 1H-pyrazolo[3,4-d]pyrimidine of fragment 1 and 1H-pyrrolo[2,3-b]pyridine
template selected for optimization as the lower nitrogen content of the later allows the introduction of more
substituents.
A significant activity gain was achieved by introduction of a N-(3-Amino-methyl-pyridin-2-yl)-N-methyl-
methanesulfonamide moiety in the 4-position of the 1H-pyrrolo[2,3-b]pyridine ring (2; KD: 4.76 µM), which was
previously described to interact with the rare helical DFG-loop conformation.23 Based on these results scaffold
modifications and residue extensions were realized to optimize the starting point 2.
Two different strategies were followed to synthesize three-fold substituted pyrrolo[2,3-b]pyrimidines, with the
objective to introduce residues as needed.43 The first approach describes the de novo synthesis of the bicyclic
scaffold structure (scheme 1) where as the second sequence was used for selective successive derivatisations of
the 7-azaindole template (scheme 2). As depicted in scheme 1 commercially available 2-amino-pyridines 3 were
applied in a Sonogashira coupling – base induced cyclisation sequence to prepare 2-substituted 7-azaindoles 4,
followed by selective halogenation of position 4 (8) and Buchwald amination (11). This Pd-catalysed last
reaction worked best after pyrrolo-NH SEM protection (10).
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Scheme 1: 7-Azaindoles by scaffold synthesisa
4
5
6
R'
7
8
R
R
I
R
b
a
9
N
3
NH2
N
4
NH2
N
5
NH2
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c
Cl
N
R
R
R
e
d
R'
R'
R'
N+
O
N
N
N
N
H
H
H
6
8
7
f
R''
N
Cl
N
R''
N
R
R
g
R
h
R'
R'
R'
N
H
N
10 SEM
N
9
SEM
11
a Reaction conditions: R = CF3, CN; (a) Ag2SO4, I2; (b) R’-phenyl-ethin, PdCl2(PPh3)2, CuI, DMF; (c) NaH,
NMP, 60 °C; (d) m-CPBA; (e) POCl3, MsCl, DMF; (f) SEM-Cl, DCM; (g) Cs2CO3, S-Phos, Pd(OAc)2, R’’-
NH2, dioxane, 150 °C; (h) 4N HCl, THF, reflux, 18 h.
This robust route suffers from the disadvantage that residues in position 2 of the final product have to be
introduced at an early stage of the sequence and have to be compatible with challenging oxidative conditions.
The alternative synthesis depicted in scheme 2 allows a more flexible approach to highly substituted 7-
azaindoles without relying on harsh chemical reaction conditions.
Scheme 2: Synthesis of 7-azaindoles by selective and successive scaffold derivatisationsa
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Cl
Cl
a
b
c
5
6
7
8
N
H
N
N+
N
N
H
N
H
N
N
d
O
TIPS
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12
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Cl
N
Cl
N
Cl
N
Cl
F3C
I
I
I
g
f
e
N
N
SEM
N
H
N
N
SEM
TIPS
18
17
16
h
Cl
N
Cl
N
R''
N
F3C
F3C
F3C
i
j
k
I
R'
R'
N
N
N
H
SEM
SEM
21
22
a Reaction conditions: (a) m-CPBA, 0 °C ꢀ RT, 77 %; (b) MsCl, DMF, 2 h, 50 °C, 72 %; (c) TIPS-Cl, NaH,
THF, 1h, 0 °C, 65 %; (d) sec-BuLi, -78 °C, I2, DMF, 60 %; (e) TBAF, THF, 1 h, 0 °C ꢀ RT, 80 %; (f) SEM-Cl,
NaH, THF, 0 °C, 2h, 88 %; (g) 2,2-Difluoro-2-(fluorosulfonyl)acetic acid, CuI, DMF, 100 °C, 3 h, 72 %; (h)
BuLi, -45 °C, I2, THF, 64 %; (i) Pd(OAc)2, S-Phos, Cs2CO3, R’-B(OH)2, dioxane, H2O, 60°C, 58 %; (j)
Pd(OAc)2, Xanthphos, Cs2CO3, R”-NH2, dioxane, H2O, 60 °C, 46 %; k. 4N HCl, THF, reflux, 65 %.
In a two-step sequence, commercially available 7-azaindole 12 is selectively chlorinated in positions 4 (14).
Iodination in position 5 works best after TIPS protection of the pyrrole-nitrogen (15). Further transformations
were only possible after protecting group exchange from TIPS (16) to SEM (18). Afterwards, the iodine
substitution with CF3 in position 5 (19) is possible, but also other residues like cyano can be introduced. Then the
N-SEM protected 4-chloro-5-CF3-7-azaindole 19 was lithiated in position 2 and quenched with iodine. The very
versatile applicable building block 20 was used in selective Suzuki- and Buchwald coupling reactions and finally
deprotected to allow access to novel highly decorated 7-azaindoles 22. The introduction of a further heteroatom
results in imidazo-pyridines 27. The synthesis of these analogues was straightforward as outlined in scheme 3.44
Scheme 3: Synthesis of imidazopyridines 47-50a
R''
N
O
R
R
N+
R
NH2
NH2
N
N
R
R
a
b
c
d
O
R'
R'
N
H
N
H
N
N
NH2
N
NH2
N
23
24
25
26
27
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a Reaction conditions: (a) conc. H2SO4/HNO3; (b) Fe, NH4Cl; (c) R‘-CHO, PTSA; (d)i: m-CPBA, POCl3; ii:
DIPEA, NMP, R‘‘-NH2.
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7
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9
Nitration of the amino-pyridine 23 derivative with appropriate substitution in position 3 followed by selective
nitro reduction afforded the 2,3-diamino-pyridine building block 25 which could be reacted with aldehydes or
acids under known conditions to the respective template 26. Final Buchwald coupling gave the desired products
27. SPR results of the imidazo- and pyrrolo-pyridines as well as results from biochemical and functional cellular
assays are shown in table 1.
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Table 1: Kinetic characterization as well as biochemical and cellular activities of the imidazo- and
pyrrolo-pyridines.
R''
kinase
R
X
SPR Results
kd
HT-29b
R'
a
assay
N
H
N
KD
IC50
IC50
(off rate)
[s-1]
No
R
R’
R’’
X
[nM]
[nM]
[nM]
2c
H
CH3
H
4760
1700
6380
554
147
44
rcod
>10000
8800
4800
910
nde
>10000
>10000
>10000
>10000
1100 700
1300
H
28c
29
0.56
0.80
4F-phenyl
CH3
0.071
0.40
30
31c
32c
33c
34
H
Phenyl
2400
195
N-(3-Amino-yl-
methyl-pyridin-2-yl)-
N-methyl-
0.022
0.019
0.12
4F-phenyl
Cy
C
603
242
430
177
nde
methanesulfonamide
CN
>10000
>10000
4-nBu-Phe
(2-Me-Pyridin-
4-yl)
0.028
nde
35
24
mtlf
220
6500
36
37c
38c
24
0.011
nde
37
51
2015 750
552 147
CF3
4F-phenyl
N-methyl-2-amino-yl-
nde
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39c
40c
pyridon-5-yl
4F-phenyl
benzamide
12
40
0.026
mtlf
57
37
>10000
CN
365 83
6
7
8
9
4-morpholin-4-
yl-phenyl
41c
42c
43
35
9
0.0026
0.0042
ddfg
45
45
83 65
107 12
nde
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
27
28
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6-morpholin-4-
yl-pyridin-3-yl
4-morpholinyl-
phenyl
3,5-difluoro-benzyl-
amino
ddfg
640
4-morpholinyl-
2-methoxy-
phenyl
CF3
2-amino-yl-
benzonitrile
1130
238
0.063
0.013
>10000
>10000
nde
nde
44
45
N-methyl-4-amino-yl-
2,3-dihydro-isoindol-
1-one
2,5-difluoro-benzyl-
amino
ddfg
127
ddfg
>10000
170
nde
46
47
4F-phenyl
N methyl-2-amino-yl-
benzamide
Br
CF3
Br
0.091
>10000
48c
49
N-(3-Amino-yl-
methyl-pyridin-2-yl)-
N-methyl-
33
0.029
0.034
130
700
>10000
>10000
N
4-Br-Phe
189
4F-phenyl
335
0.060
4500
>10000
50
methanesulfonamide
amean of three determinations, variations about 5%; bHT-29 cells possess a gene amplification of FAK and do
therefore have high level of P-Y397-FAK; cselectivity data available, see table 2 and supporting information;
dRate constants outside instrument range; enot determined; fmass transfer limited data: kinetic constants cannot
be unambiguously determined; gdata don’t fit to 1:1 model.
Discussion
Mono substituted azaindole 2 had shown micromolar affinity with transient binding kinetics (dissociation rate
constant > 1 s-1), but no biochemical inhibition below 10 µM could be measured for this compound. Attachment
of a methyl group as second substituent in the 5-position of the 7-azaindole core (28) improved the affinity by
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factor of 3 and an off-rate of 0.56 s-1 was measured.45 By introduction of an aryl moiety like 4-fluoro phenyl in
position 2 of 2, the twofold substituted azaindole 29 was accessible. However, the binding affinities of 29 in
comparison to 28 were somehow inconsistent as demonstrated by 3-fold higher KDSPR but a 2-fold lower
biochemical IC50 value: affinity decreased but biochemical activity was improved slightly. Combination of the
methyl group in position 5 and 4-fluoro-phenyl in position 2 resulted in the first example of a threefold
substituted azaindole (30). Binding affinity measured by SPR and the biochemical IC50 of 30 were increased
resulting in sub-micromolar values. As for 28 and 29, no cellular activity could be found for 30, indicating that
an off-rate of 0.071 s-1 might be not sufficiently slow to achieve measurable target inhibition in HT-29 cells.
Binding affinity could be more improved by the introduction of an electron withdrawing group in position 5 like
cyano- (31) than with the electron donating methyl-substituent (28). While the KD for 31 is 10-fold lower than
for 28, the off-rate was only slightly improved and biochemical activity was reduced by a factor of 4. The
combination with an aryl moiety in position 2 like phenyl (32) or 4-fluoro-phenyl (33) improved activity further.
KD values for 32 and 33 differ nearly by one order of magnitude, whereas the off-rates are in the same range of
about 0.02 s-1. This is reflected by comparable cellular IC50s of 1 µM for both compounds. Replacing the phenyl
ring with cyclohexyl (34) or adding a further lipophilic moiety like n-butyl (35) abolished FAK inhibition in the
enzymatic assay even though submicromolar KDSPR values of both compounds had suggested specific binding.
The off-rate of 35 even points to cellular activity. This apparent inconsistency can be understood as 34 and 35
are virtually insoluble,46 questioning biochemical and cellular data. Introduction of a heteroaryl moiety like
pyridine (36) results in biochemical and cellular IC50 values comparable to the phenyl derivative 33. Biophysical
characterization of 36 was not possible as kinetic parameters could not be unambiguously determined. Switching
from the linear cyano-function to the more bulky trifluoromethyl as residues in position 5 has a significant
influence on the biochemical activity and resulted in the first two digit nanomolar FAK inhibitor from this series
(37). The binding characterization revealed a KD of 24 nM and a dissociation rate constant of 0.011 s-1. This off-
rate is only slightly decreased compared to 33 and no major improvement on the cellular activity could be
expected. So far the aminomethyl-pyridine moiety in position 4 has been kept constant for the first optimisation
rounds of R and R’. In another approach the amino-benzamide known from I was applied. Decoration with CF3
in position 5 and 4-F-phenyl in position 2 did not alter biochemical activity but improved cellular FAK inhibition
significantly exemplified by 38 as the first derivative with submicromolar cellular activity.47 The 5-cyano
analogue 40 is equally active than 38, indicating a more pronounced influence on activity by substituents in
position 4 (pointing to the FAK-DFG-area, vide infra) than in position 5 (gate-keeper interaction, vide infra).
Binding to immobilized protein could only be interpreted partially for 40 as a KD could be determined but no off-
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rate was deducible from the data. Substitution of the aryl group in position 2 with morpholine finally resulted in
further improvement of cellular activity as 41 and 42 have cellular IC50 in the 100 nM range. This is also
reflected in the biophysical characterization, as these two derivatives have off-rates of 0.0026 s-1 and of 0.0042 s-
1, translating into residence times on the target of 6 and 4 minutes, respectively.48 As acceptor functions like the
sulphonamide or the benzamide in position 4 are beneficial for activity alternative acceptors were evaluated, too.
The application of another literature known amino-aryl moiety, 4-amino-2,3-dihydro-isoindol-1-one,49 in 45
which can be considered as a cyclic analogue of the amino-benzamide used for 38 – 42 gave very surprising
results. Compound 45 did not show any activity in the biochemical assay, but the the KDSPR is still in the low
three digit nanomolar range and the off-rate is only slightly higher than 0.01 s-1. As 45 could be profiled in
biophysical characterisation, low solubility of the derivative might be responsible for missing biochemical
activity.46 Other acceptor residues like the amino-benzonitrile (in 44) or the fluoro-benzylamine (in 43, 46) did
not improve FAK inhibition. Anyhow, surprisingly the 3,5-difluoro-benzylamine 43 is biochemically active in
contrast to the cyclic amide 45. The residues amino-benzamide and aminomethyl-pyridine which were used for
the optimization of the pyrrolo[2,3-b]pyridine scaffold were also used for imidazo-pyridines 47 - 50. As can be
seen by comparing 48 with 37 the enzymatic activity is only three fold lower for the imidazo-derivative but no
cellular activity could be found. This again is consistent with biophysical data as the off-rate is faster for 48. For
all imidazo-pyridines 47- 50 the missing cellular activity can be understood when considering their off-rates ≥
0.03 s-1.
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We assumed a favourable kinase selectivity profile for the 7-azaindole and imidazo-pyridine derivatives
described in this paper, because the decorated rigid bicyclic scaffolds might have little flexibility to interact with
different targets. For experimental confirmation of this hypothesis, an array of compounds (2, 28, 31-33, 37-42,
48) was chosen for selectivity profiling against 110 to 121 kinases. In table 2, we summarized the number of
kinases inhibited with higher and 10% lower potency than FAK-IC50 by the respective compound.
Table 2: Kinase selectivity of tested the imidazo- and pyrrolo-pyridines.a
No
Ab
Bc
2
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32
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37
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adetailed selectivity data and graphical visualisations are given in the supporting information, bthe number of
kinases inhibited by the compound with higher potency than FAK , cthe number of kinases inhibited by the
compound with 10% lower potency than FAK.
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Pyrrolopyrimidine 2 which is decorated with the literature known sulphonamide bearing ligand is quite
unselective and more active on non-FAK kinases. This can be understood as the hinge binder is small and might
interact with altered orientations in different kinases. The derivatives 28-33 show increased FAK activity but no
improved FAK selectivity. Extension of the core by aryl addition in position 2(32 and 33) was not sufficient to
induce more selective FAK inhibition. Changing CN (33) to CF3 (37) in position 5 of the 7-azaindole core
improved on the inhibition of FAK activity as well as selectivity. Derivative 38 confirms the tendency within
this compound class: 2-(4-F-phenyl)-5-CF3-7-azaindole scaffold bears FAK selectivity tolerating different small
residues in position 4. This is corroborated by 39 with slightly decreased selectivity by replacement of the 4-F-
phenyl in position 2 with a pyridinone ring. The importance of 4-F-phenyl for FAK-selectivity is strengthened by
40 as in this derivative the CN functionality is reintroduced without loss of preference for FAK. Also 41 and 42
confirm the 4-F-phenyl trend. These compounds bear the CF3 in position 5 and amino-phenyl-carboxamide in
position 4, known from 38 to be beneficial for selectivity, but have 4-morpholino-phenyl in position 2 and are
less selective than 38. As pointed out, the effect of 4-F-phenyl on selectivity should not be overestimated as 33 is
not a FAK specific inhibitor, indicating that selective target inhibition requires a well balanced presentation of all
pharmacophores. The effect of the template structure for selectivity considerations is insinuated by 48. This
imidazo-pyridine core is substituted with the same moieties (CF3, 4-F-phenyl, aryl-sulfonamide) as pyrrolo-
pyridine compound 37 but is much less FAK selective.
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The fragment growing initiative of 1 was accompanied by X-ray structure analyses and figure 3 depicts the X-
ray structure of 32 binding to the hinge-region of FAK (PDB ID: 4GU6).50-55
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Figure 4: X-ray structure of FAK in complex with 32 indicates H-bond interactions to the hinge region (Cys502)
and a rarely observed helical conformation of the activation-loop (Phe565-Tyr575; the helix is shown only partly
until Tyr570 for clarity). Weaker H-bond contacts are formed between the cyano-nitrogen and the sulphonamide-
oxygen of 32 and the Asp564 backbone-NH (distances in Å).
It is known that fragments can bind in different orientations to kinases and that binding mode can change in the
growing process.56,57 In a HCV dedicated approach for example, a benzoic acid fragment has even been
described to flip by 180° after amide coupling.58 In this work we also observed an inverted binding mode for the
grown fragment. We were not surprised to see this difference in binding mode as the selected growing vector in
1 was pointing to the gatekeeper amino acid Met499, leaving not much space between protein and ligand. The
anticipated hinge-interaction of N7 of 32 was confirmed by H-bond contacts to the backbone N-atom of Cys502.
In contrast to the hydrogen donation of 1 to backbone carbonyl-O of Glu500, the 1H-NH of 32 bind to backbone
carbonyl-O of Cys502. The CN-group of the 7-azaindole core is pointing towards gatekeeper residue Met499
and interacting with the backbone-NH of Asp564 from the DFG-motif. Also, one oxygen atom of the SO2 group
of 32 is in hydrogen-bond distance to the same backbone-NH of Asp564. Although both contacts are quite
extended (3.32 & 3.58 Å) they might be responsible for the induction of the unusual activation loop
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conformation, as described for II. Even though the phenyl residue in position 2 of the 7-azaindole is not
interacting with lipophilic amino acid side chains the IC50 comparison of 32 with 31 proves that the aryl residue
in 32 contributes to activity.
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Conclusion
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In an accelerated knowledge-based fragment-growing approach we decorated bicyclic hinge binding pyrrolo[3,4-
d]pyridine in an unprecedented fashion. New chemical routes were discovered to enable selective and variable
scaffold decorations even in late stages of the syntheses. Kinetic characterization and cellular testing of the
selected compounds show that an off-rate of around 0.02 s-1 seems to be necessary for moderate cellular activity
in HT-29 cells, while submicromolar cellular activity was only achieved with compounds having off-rates lower
than 0.01 s-1. Additionally, solubility and protein binding can disturb biochemical and cellular assays results
even of compounds with confirmed FAK-binding by SPR. Here we have shown that the kinetic characterization
of the compound-target interaction can deliver reliable data to ascertain the target activity of considered
derivatives. In our fast knowledge-based fragment growing approach, starting from a hinge-binder it became
obvious, that it was easier to improve kinase inhibition than kinase selectivity. We could show that the 4-F-
phenyl substituent at the pyrrolopyridine is important for FAK selectivity. The described hit-series is a qualified
starting point for a FAK dedicated hit-optimization program. Beyond the utilization of the described compounds
in this targeted approach the unprecedented synthetic route can be used to get systematically highly substituted
7-azaindoles for alternative targets in medicinal chemistry.
Experimental Section.
Biology
FAK – Kinase Assay (autophosphorylation)
The Focal Adhesion Kinase (FAK) assay is performed as 384-well Flashplate assay. 2 nM FAK, 400 nM
biotinylated substrate (His-TEV-hsFAK (31 – 686)(K454R) x Biotin) and 1 µM ATP (spiked with 0.25 µCi 33P-
ATP/well) are incubated in a total volume of 50 µl (60 mM Hepes, 10 mM MgCl2, 1.2 mM Dithiothreitol, 0.02
% Brij35, 0.1 % BSA, pH 7.5) with or without test compound for 2 hours at 30 °C. The reaction is stopped with
25µl 200 mM EDTA. After 30 min at 30 °C the liquid is removed and each well washed thrice with 100 µl 0.9 %
sodium chloride solution. Non-specific reaction is determined in presence of 1 µM PF-431396. Radioactivity is
measured with a Topcount Microplate Scintillation Counter (Perkin Elmer). Results are calculated with Symyx
Assay Explorer.
P-Y397-FAK Cellular Assay
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HT29 cells which have gene amplification of FAK were plated at 30,000 cells/well in a 96-well microtiter plate
and allowed to adhere overnight. Inhibitor compounds were added to each well in a 3-fold serial dilution (range
from 30 µM to 0.03 µM) in triplicates for 45 min. After compound treatment, cells were lysed and cleared by
centrifugation through a 96-well filter plate. FAK was captured from total lysates by incubation with a mouse-
anti-FAK antibody (Merck Millipore, #05-537) coupled to Luminex microspheres overnight. The level of P-
Y397-FAK was then detected by applying a rabbit-anti-P-Y397-FAK antibody (Sigma, #F7926) and an anti-
rabbit-PE secondary antibody in a Luminex100 machine according to the manufacturer’s instruction. Samples
treated with DMSO vehicle were set as maximal phosphorylation and inhibitor treated samples were calculated
as percent inhibition. Non-linear regression analysis (variable slope) was applied for determination of IC50 values
(Accelry Assay Explorer).
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Protein Crystallography
The construct FAK (411-686) was expressed and purified as described in the literature.53 The protein was stored
in a buffer containing 50 mM Tris/HCl, 250 mM NaCl, 1 mM EDTA, 1 mM DTT pH 7.6 and was concentrated
to 6.5 mg/ml. FAK (410-689) P410G mutant was expressed and purified as described in the literature.23 This
protein construct was stored in 10 mM HEPES, 200 mM ammonium sulfate, 0.1 mM TCEP, pH 7.5 and was
concentrated to 5.9 mg/ml. Crystals for the complex between 1 and the kinase domain (aa 410-686) of human
recombinant FAK were prepared in the following way: FAK kinase domain was crystallized at 20 °C by hanging
drop vapour diffusion against 0.1 M sodium citrate, pH 6.0, 18% PEG MME 2000. The protein was mixed 1:1
with the reservoir solution. For complex formation with the inhibitor, the crystals were transferred to a
stabilizing solution (0.1 M sodium citrate, pH 6.0, 40 % PEG MME 2000) containing 5 mM 1, 5 % DMSO and
were soaked for 24 hours.
32 was co-crystallized with the kinase domain (aa 410-689) of the P410G mutant of human recombinant FAK at
20 °C by hanging drop vapour diffusion against 0.1 M Tris, pH 8.6 18 % PEG 3350, 200 µM 32. The protein
was mixed 1:1 with the reservoir solution.
X-ray diffraction data were collected on X06SA-PX beamline at Swiss Light Source (SLS) synchrotron radiation
source using a Pilatus detector50 and the images were indexed, integrated and scaled using XDS program
package.51 The data were collected using a cryo-cooled crystals at 90 K. The structure was solved by molecular
replacement using the program MOLREP from the CCP4 program suite.53 The structure 1mp8 from the Protein
Data Bank (PDB)59 was used as the search model. Subsequently, several cycles of refinement using the program
Buster54 and crystallographic model building using the graphic package COOT55 were applied. The data
collection and refinement statistics can be found in Table 3S.
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Surface Plasmon Resonance (SPR)
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The kinase domain of FAK was immobilized in the presence of an ATP-site specific inhibitor onto CM5 (series
S) sensor chips using standard amine coupling. FAK inhibitor compounds (stored as 10 mM stock solutions in
100 % DMSO) were diluted in running buffer (10 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT,
0.05 % Tween 20, 2 % DMSO pH 7,4) and analyzed with a Biacore 4000 or Biacore S51 (Biacore AB, GE
Healthcare Life Sciences, Uppsala, Sweden) using a 2-fold dilution series. The highest compound concentration
varied according to the expected dissociation constant, but all compounds were tested at 10 different
concentrations. Interaction analysis cycles were run at 30 µL/min and consisted of a 180 s sample injection
followed by 240 s of buffer flow (dissociation phase). All sensorgrams were evaluated by first subtracting the
binding response recorded from the control surface (reference spot), followed by subtracting a buffer blank
injection. To determine kinetic rate constants, data sets were fitted to a simple 1:1 interaction model including a
term for mass transport using numerical integration and nonlinear curve fitting. Equilibrium analysis was
performed by fitting the response at the end of the association phase to a single-site binding isotherm.
Chemistry. General Information: All reactions were carried out under nitrogen atmosphere or in sealed vials
unless noted otherwise. Dry solvents and reagents were of commercial quality and were used as purchased.
Reactions were magnetically stirred and monitored by thin-layer chromatography using Merck silica gel 60 F254
by fluorescence quenching under UV light or by LCMS detection, except if indicated otherwise. LCMS-analyses
were run on Agilent 1100/1200 series) according to the following method: A-0.1 % TFA in H2O, B-0.1 % TFA
in ACN: Flow- 2.0 mL/min. on XBridge C8 (50 x 4.6mm, 3.5ꢀm), +ve mode or Chromolith Performance
RP18e. Retention times (Rt) are given in minutes. In addition, TLC plates were stained using phosphomolybdic
acid or potassium permanganate stain. Chromatographic purification of products (flash chromatography) was
performed on Isco Combiflash systems using Redisep columns and ethyl acetate/heptanes gradients.
Concentration under reduced pressure was performed by rotary evaporation at 40 °C at the appropriate pressure
unless otherwise stated. The purity of the compounds reported in the manuscript was established through HPLC-
MS methodology. HPLC-analyses were run according to the LCMS method. 1H-NMR (in DMSO-d6) and mass
spectra are in agreement with the structures and were recorded on a Bruker AMX 400 MHz NMR spectrometer
(TMS as an internal standard), and Vaccum Generators VG 70-70 or 70-250 at 70 eV, respectively. Elemental
analyses (obtained with a Perkin-Elmer 240 BCHN analyser) for the final products were within 0.4 % of
calculated values if not stated otherwise. All the compounds reported in the manuscript have a purity ≥ 95 %
unless noted otherwise.
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Syntheses as outlined in scheme 1 (R = CF3; for others see supporting information)
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3-Iodo-5-(trifluoromethyl)pyridin-2-amine (4)
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5-(trifluoromethyl)pyridin-2-amine 3 (5.0 g, 30.8 mmol) was dissolved in 1,2-dichloroethane(50 mL) and silver
trifluoroacetate (6.81 g, 30.8 mmol) was added. The suspension was refluxed for 7 h, after cooling to room
temperature, iodine (7.8 g, 30.8 mmol) was added. The mixture was heated again for 12 h. After completion of
the reaction, the reaction mixture was cooled to RT and the salt was removed by filtration. The filtrate was
treated with water, extracted with DCM and the combined organic layer was dried over sodium sulphate and
evaporated. The crude was purified by (60-120) silica gel chromatography to obtain 4 as brown solid in 56 %
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yield. H-NMR: δ 8.26 (d, J = 1.12 Hz, 1H), 8.15 (d, J = 2.00 Hz, 1H), 6.88 (br s, 2H). LCMS: 289.0 (M+H),
RT. 3.29 min, 91.93 % (Max), 93.21 % (254 nm).
3-((4-Fluorophenyl)ethynyl)-5-(trifluoromethyl)pyridin-2-amine (5)
To a degassed solution of 4 (2 g, 6.9 mmol) in dry DMF (35 mL), 4-fluoro phenyl acetylene (1.1 g, 8.7 mmol),
copper iodide (66 mg, 0.3 mmol), diisopropyl amine (2.9 mL) and dichlorobis(triphenylphospine)palladium (II)
(0.243 g, 0.3 mmol) were added to a sealed tube and heated at 80 °C for 10 min. The reaction mixture was
passed through celite and washed with 30 % methanol in dichloromethane (30 mL), the filtrate was concentrated
1
and purified by column chromatography to get 5 as brown solid in 93 % yield. HNMR: δ 8.28 (d, J = 1.44 Hz,
1H), 7.90 (d, J = 2.36 Hz, 1H), 7.73-7.77 (m, 2H), 7.26-7.31 (m, 2H), 7.17 (br s, 2H). LCMS: 281.0 (M+H), RT.
4.73 min, 99.17% (Max), 99.44 % (254 nm).
2-(4-Fluorophenyl)-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine (6)
To a solution of potassium tert-butoxide (1.45 g, 12.8 mmol) in NMP under nitrogen, a solution of 5 (1.8 g, 6.4
mmol) in NMP (15 mL) was added drop wise at RT and the reaction mixture was heated at 120 °C for 12 h. The
reaction was then quenched with water (50 mL) and extracted with ethyl acetate (50 mL) 3 times. The combined
organic layer was washed with water, brine and was dried over anhydrous Na2SO4 to get 6 as yellow solid in 66
% yield. 1H-NMR: δ 12.68 (br s, 1H), 8.55 (d, J = 0.68 Hz, 1H), 8.44 (d, J = 77.76 Hz, 1H), 8.01-8.04 (m, 2H),
7.33-7.38 (m, 2H), 7.06 (s, 1H). LCMS: 281.0 (M+H), RT. 5.03 min, 92.56 % (Max), 97.40 % (254 nm).
2-(4-Fluorophenyl)-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine 7-oxide (7)
6 (1.2 g, 4.2 mmol) was suspended in acetone and cooled to 5 °C, mCPBA (0.83 g, 4.7 mmol) was added and the
reaction mixture was stirred at RT for 12 h. The precipitate formed was then filtered and dried to get 7 as pale
1
yellow solid in 39 % yield. H-NMR: δ 13.45 (br s, 1H), 8.61 (d, J = 0.72 Hz, 1H), 8.10-8.13 (m, 2H), 8.05 (s,
1H), 7.31-7.35 (m, 2H), 7.16 (s, 1H). LCMS: 297.0 (M+H), RT. 3.95 min, 99.48 % (Max), 99.59 % (254 nm).
4-Chloro-2-(4-fluorophenyl)-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine (8)
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7 (0.5 g, 2.1 mmol) was taken in phosphorus oxychloride (10mL) and heated at 85 °C for 3 h. After the
completion of reaction, excess phosphorus oxychloride was removed under high vacuum and the residue was
diluted with cold water (5 mL) and basified with NaHCO3 solution. The aqueous layer was extracted with ethyl
acetate (25 mL), the combined organic layer was washed with water and followed by brine solution, and was
dried over anhydrous Na2SO4, concentrated under reduced pressure to obtain 8 as a pale yellow solid which was
taken to the next step without further purification (70 %). 1H-NMR: δ 8.58 (s, 1H), 8.08-8.12 (m, 2H), 7.20-7.39
(m, 2H), 7.10 (s, 1H). LCMS: 315.0 (M+H), RT. 5.51 min, 97.70 % (Max), 97.12 % (254 nm).
4-Chloro-2-(4-fluorophenyl)-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine
(9)
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A pre oven-dried two neck round bottom flask was charged with 8 (520 mg, 2 mmol) and dry THF (20 mL) at
0°C. Sodium hydride (60 % dispersion in mineral oil, 88 mg, 2.2 mmol) was added portion wise keeping the
temperature below 5 °C and the resulting suspension was stirred at 0 °C for another 30 minutes followed by
addition of SEM-Cl (365 mg, 2.2 mmol) drop wise. After the completion of the reaction, it was quenched with
saturated ammonium chloride solution (5 mL) diluted with ethyl acetate (50 mL) washed with water, brine and
dried over anhydrous Na2SO4. Concentration of the organic layer gave pale yellow oil which was purified by
column chromatography to get 9 as colorless solid in 75 % yield. 1H-NMR: δ 8.68 (s, 1H), 7.86-7.89 (m, 2H),
7.37-7.42 (m, 2H), 6.96 (s, 1H), 5.65 (s, 2H), 3.57 (t, J = 8.12 Hz, 2H), 0.81 (t, J = 7.96 Hz, 2H), (s, 9H). LCMS:
445.3 (M+H), RT. 7.342 min, 96.16 % (Max), 96.97 % (254 nm).
7-azaindole N-Oxide (13)
To a stirred solution of 7-azaindole 12 (50.0 g, 0.42 mol) in ethyl acetate (1.5 L) at 0 °C was added mCPBA (60
%) (144 g, 0.60 mol) portion wise over a period of 10 minutes and the reaction mixture was stirred for another 1
h. After the completion of the reaction the solid was filtered and dissolved in chloroform/methanol = 9:1 and
neutralized with saturated Na2CO3 solution. The layers were separated and the organic layer was dried over
1
anhydrous Na2SO4, concentrated under reduced pressure to get 13 as white solid in 60 % yield (34 g). H-NMR:
δ 12.47 (s, 1H), 8.10 (d, J = 6.12 Hz, 1H), 7.62 (dd, J = 1.80, 6.88 Hz, 1H), 7.44 (d, J = 3.28 Hz, 1H), 6.56 (d, J
= 3.28 Hz, 1H).
4-Chloro-1H-pyrrolo[2,3-b]pyridine (14)
To a stirred solution of 13 (18.5 g, 0.14 mol) in DMF (150 mL) at 52°C was added methane sulfonyl chloride
(32 mL, 0.41 mol) drop wise and the reaction mixture was heated to 72 °C for 2 h. After the completion of the
reaction, the reaction mixture was poured over crushed ice and neutralized with 5M NaOH solution. The solid
1
obtained was filtered and dried under vacuum to get 14 as orange color solid in 75 % yield (16 g). H-NMR: δ
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12.03 (s, H), 8.16 (d, J = 5.12 Hz, H), 7.58 (t, J = 3.00 Hz, H), 7.18 (d, J = 5.16 Hz, H), 6.49 (dd, J = 1.96, 3.44
Hz, H). LCMS: 153.0 (M+H), RT. 2.10 min, 93.4 % (Max), 93.6 % (254 nm).
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4-Chloro-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridine (15)
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To a stirred solution of 14 (16 g, 0.10 mol) in THF (300 mL) at 0 °C was added NaH (60 % in mineral oil, 5 g,
0.14 mol) portion wise and the mixture was stirred at the same temperature for 20 minutes, then triisopropyl silyl
chloride (22.3 g, 0.16 mol) was added drop wise maintaining the temperature at 0 °C. After the reaction was
complete, the reaction mixture was quenched with saturated NH4Cl solution (10 mL) diluted with water and
extracted with petroleum ether (3 × 200 mL). The crude compound was purified by column chromatography
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giving 15 as colorless liquid in 92 % yield (30 g). H-NMR: δ 8.18 (d, J = 5.16 Hz, 1H), 7.59 (d, J = 3.80 Hz,
1H), 7.23 (d, J = 5.16 Hz, 1H), 6.67 (d, J = 3.52 Hz, 1H), 1.82-1.90 (m, 3H), 1.05 (d, J = 7.52 Hz, 18H). LCMS:
309.2 (M+H), RT. 7.56 min, 84.8 % (Max).
4-Chloro-5-iodo-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridine (16)
To a solution of 15 (11 g, 35.7 mmol) in THF (100 mL) at -78 °C was added sec-BuLi (53 mL, 78.5 mmol) drop
wise over a period of 30 minutes and the reaction mixture was stirred for another 1h at the given temperature. A
solution of iodine (18 g, 71.1 mmol) in THF (50 mL) was then added drop wise over a period of 30 minutes at
the same temperature and the resulting suspension was stirred for 1 h and slowly brought to 0 °C. The reaction
was quenched with saturated NH4Cl solution, extracted with ethyl acetate, washed with water, brine, dried over
Na2SO4 and concentrated under reduced pressure to get crude compound which was purified using petroleum
ether by chromatography over silica gel to get 16 as a colorless liquid in 53 % yield (8.25 g). 1H-NMR: δ 8.52 (s,
1H), 7.57 (d, J = 3.52 Hz, 1H), 6.67 (d, J = 3.48 Hz, 1H), 1.80-1.88 (m, 3H), 1.04 (d, J = 7.52 Hz, 18H). LCMS:
435.0 (M+H), RT. 7.98 min, 96.9 % (Max).
4-chloro 5-iodo-1H-pyrrolo [2,3-b]pyridine (17)
To a stirred solution of 16 (11 g, 25.3 mmol) in dry THF (200 mL) at 0 °C was added TBAF (1M solution in
THF; 27 mL, 27 mmol) and allowed to stir for 30 minutes. After the completion of the reaction, the solvent was
evaporated, diluted with ethyl acetate, washed with water, brine and dried over anhydrous Na2SO4.
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Concentration of the organic layer gave 17 as pale yellow solid in 99 % yield (7 g). H-NMR: δ 12.15 (s, 1H),
8.50 (s, 1H), 7.58 (d, J = 3.40 Hz, 1H), 6.49 (d, J = 3.44 Hz, 1H), 5.09 (s, 1H). LCMS: 279.0 (M+H), RT. 3.97
min, 63.5 % (Max).
4-chloro 5-iodo-1-(2-trimethylsilanyl-ethoxymethyl) -1H-pyrrolo [2,3-b]pyridine (18)
A pre oven-dried two neck round bottom flask was charged with 17 (7 g, 25.2 mmol) and dry THF (75 mL) at 0
°C. Sodium hydride (60 % dispersion in mineral oil, 1.2 g, 32.5 mmol) was added portion wise keeping the
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temperature below 5 °C and the resulting suspension was stirred at 0 °C for another 30 minutes followed by
addition of SEM-Cl (5.03 g, 30.3 mmol) drop wise over a period of 15 minutes. After the completion of the
reaction, it was quenched with saturated ammonium chloride solution (15 mL) diluted with ethyl acetate (100
mL) washed with water, brine and dried over anhydrous Na2SO4. Concentration of the organic layer gave 18 as
pale yellow oil in 88 % yield 9 g). LCMS: 409.0 (M+H), RT. 6.51 min, 57.4 % (Max).
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4-chloro 5-trifluoromethyl-1-(2-trimethylsilanyl-ethoxymethyl) -1H-pyrrolo [2,3-b]pyridine (19)
An oven dried two neck flask was charged with 18 (9 g, 22.1 mmol), cuprous iodide (4.2 g, 22.1 mmol), DMF
(45 ml) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (8.5 mL, 66.4 mmol). The resulting suspension was
heated at 100°C for 2h. After the completion of the reaction, the copper residue was filtered off and the filtrate
was extracted with ethyl acetate (100 mL) washed with water, brine and dried over anhydrous Na2SO4.
Concentration of the organic layer gave the title compound as colorless solid which was purified by column
1
chromatography using ether giving 19 in 59 % yield (4.6 g). H-NMR: δ 8.64 (s, 1H), 7.98 (d, J = 3.6 Hz, 1H),
6.78 (d, J = 3.64 Hz, 1H), 5.67 (s, 2H), 3.49-3.53 (m, 2H), 0.78-0.82 (m, 2H), -0.14- -0.12 (m, 9H). LCMS:
351.0 (M+H), RT. 6.75 min, 43.3 % (Max).
4-chloro-2-iodo- 5-trifluoromethyl-1-(2-trimethylsilanyl-ethoxymethyl) -1H-pyrrolo [2,3-b]pyridine (20)
An oven dried two neck flask was charged with 19 (4.6 g, 13.1 mmol) and THF (50 mL) at -45 °C BuLi (1.6M
stock solution in THF, 13.1 mmol) was added drop wise to the reaction mixture maintaining the temperature
below -40 °C. The reaction mixture was stirred for another 30 minutes at the same temperature and I2 (8.32 g,
32.7 mmol) solution in THF (25 mL) was then added drop wise. The reaction mixture was slowly brought to RT.
After the completion of the reaction, it was quenched with saturated ammonium chloride solution, diluted with
ethyl acetate (100 mL), washed with water, brine and dried over anhydrous Na2SO4. Concentration of the
1
organic layer gave 20 as pale brown solid in 64 % yield (4 g) which was used without further purification. H-
NMR: δ 8.62 (s, 1H), 7.20 (s, 1H), 5.66 (s, 1H), 3.53 (t, J = 7.84 Hz, 2H), 0.81 (t, J = 7.9 Hz, 2H),-0.11 (s, 9H).
LCMS: 477.0 (M+H), RT. 7.1 min, 74.6 % (Max).
Synthesis of N-Methyl-2-[2-(6-morpholin-4-yl-pyridin-3-yl)-5-trifluoromethyl-1H-pyrrolo[2,3-b]pyridin-4-
ylamino]-benzamide (42):
4-Chloro-2-(6-morpholin-4-yl-pyridin-3-yl)-5-trifluoromethyl-1-(2-trimethylsilanyl-ethoxy
pyrrolo[2,3-b]pyridine
methyl)-1H-
To a 50 ml round bottom flask was added 20 (400 mg, 0.84 mmol), 6-(morpholin-4-yl) pyridine 3-boronic acid
pinacol ester (290 mg, 1 mmol), Palladium acetate (19 mg, 0.084 mmol), S-Phos (34 mg, 0.084 mmol) and
cesium carbonate (800 mg, 2.5 mmol). This mixture was suspended in dioxane (2.7 mL) and water (0.3 mL) and
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heated at 60 °C for 12 h. The reaction was cooled to room temperature diluted with water (10 mL) and ethyl
acetate (20 mL) and extracted with ethyl acetate (20 mL). The combined organic layer was washed with water
(100 mL) and brine solution, then dried over anhydrous Na2SO4 and evaporated. The residue was purified by
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column chromatography to get title compound in 58 % yield (250 mg) as pale brown oil. H-NMR: δ 8.65 (s,
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1H), 8.57 (d, J = 2.3 Hz, 1H), 8.00-8.03 (m, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.94 (s, 1H), 5.66 (s, 2H), 3.70-3.72
(m, 4H), 3.56-3.63 (m, 6H), 0.82-0.86 (m, 2H),-0.11 (s, 9H).
N-Methyl-2-[2-(6-morpholin-4-yl-pyridin-3-yl)-5-trifluoromethyl-1-(2-trimethylsilanyl-ethoxymethyl)-1H-
pyrrolo[2,3-b]pyridin-4-ylamino]-benzamide
To a 50 mL round bottom flask with stir bar was added 4-chloro-2-(6-morpholin-4-yl-pyridin-3-yl)-5-
trifluoromethyl-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrrolo[2,3-b]pyridine (80 mg, 0.156 mmol), 2-amino-
N-methyl-benzamide (23 mg, 0.156 mmol), cesium carbonate (148 mg, 0.468) and dioxane (2 mL). The
suspension was degassed using argon. XanthPhos (18 mg, 0.031 mmol) and Palladium acetate (7 mg, 0.031
mmol) were added and the reaction mixture was heated at 100 °C for 12 h. The reaction was then cooled to room
temperature diluted with ethyl acetate (30 mL) and filtered through celite pad. Water (20 mL) was added,
filtrated and extracted with ethyl acetate (60 mL). The combined organic layer was washed with water (50 mL)
and brine solution, then dried over anhydrous Na2SO4 and evaporated. The residue was purified by column
chromatography to get the title compound as viscous yellow liquid in 46 % yield (45 mg). LCMS: 626.8 (M+H),
RT. 5.24 min, 74.7 %
N-Methyl-2-[2-(6-morpholin-4-yl-pyridin-3-yl)-5-trifluoromethyl-1H-pyrrolo[2,3-b]pyridin-4-ylamino]-
benzamide (42)
To
a
solution of N-Methyl-2-[2-(6-morpholin-4-yl-pyridin-3-yl)-5-trifluoromethyl-1-(2-trimethylsilanyl-
ethoxymethyl)-1H-pyrrolo[2,3-b]pyridin-4-ylamino]-benzamide (30 mg, 0.047 mmol) in THF (2 mL) was added
HCl solution in dioxane (4N, 0.25 mL) and heated at reflux for 5 h. After completion of the reaction, the solvent
was evaporated; the residue was dissolved in ethyl acetate (15 mL) and neutralized with saturated Na2CO3
solution. The organic layer was separated, washed with water, brine and dried over anhydrous Na2SO4 and
evaporated. The residue was purified by column chromatography to get 42 as off white solid in 65 % yield (12
1
mg). H-NMR: δ 12.40 (d, J = 2.00 Hz, 1H), 10.57 (s, 1H), 8.69 (d, J = 3.60 Hz, 1H), 8.51 (d, J = 3.0 Hz, 1H),
8.37 (s, 1H), 7.83-7.85 (m, 1H), 7.72-7.74 (m, 1H), 7.33-7.37 (m, 1H), 7.03 (t, J = 6.80 Hz, 1H), 6.97 (d, J =
8.28 Hz, 1H), 6.88 (d, J = 8.88 Hz, 1H), 5.99 (d, J = 2.20 Hz, 1H), 3.67-3.69 (m, 4H), 3.47-3.50 (m, 4H), 2.79
(d, J = 4.52 Hz, 3H). LCMS: 497.2 (M+H), RT. 2.85 min, 93.2 % (Max), 92.4 % (254 nm). HPLC: RT 2.79 min,
94.2 % (Max), 95.1 % (254 nm).
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ASSOCIATED CONTENT
Supporting Information
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Synthesis and characterization of intermediates from schemes 1, 2 and 3 & from table 1. Crystallographic data,
refinement parameters, electron density maps for both respective monomers of 1 and 32; selectivity data for 2,
28, 31-33, 37-42, 48 are given also visualization of II binding to FAK and kinetic data for II. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Accession codes
X-ray structures of 1 (4GU9) and 32 (4GU6) bound to FAK have been deposited in the PDB (www.pdb.org).59
AUTHOR INFORMATION
Corresponding author
Email: timo.heinrich@merckgroup.com; phone: +49 6151 79 85 89; fax: +49 6151 72 31 29; URL:
http://www.merckgroup.com
Notes:
The authors declare no competing financial interest
ACKNOWLEDGEMENTS
We acknowledge Michael Krug how helped in the visualization of the selectivity data
ABBREVIATIONS USED
BuLi, n-Butyllithium; DCM, Dichloromethane; DIPEA, Diisopropylethylamine; DMA, Dimethylacetamide;
DMF, Dimethylformamide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EtOAc, Ethyl acetate; EtOH,
Ethanol; FAK, Focal adhesion kinase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LCMS:
High-pressure liquid chromatography with subsequent mass detection; mCPBA, meta-Chloroperoxybenzoic
acid; MeOH, Methanol; MsCl, Methanesulfonyl chloride; NMP, N-Nethylpyrrolidinone; PDB, protein data base;
PTSA, para-Toluenesulfonic acid; RT, Roomtemperature; Rt, Retention time; SEM,
Trimethylsilylmethoxymethyl; S-Phos, 2-Dicyclohexylphosphino-2’,6’-dimethoxybiphenyl; SPR, Surface
plasmon resonance; TFA, Trifluoroacetic acid; THF, Tetrahydrofurane; TIPS, Triisopropylsilyl; TLC, Thin layer
chromatography; Xanthphos, 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene
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domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res.
2002, 62, 4236–4243.
(32) Nagar, B.; Hantschel, O.; Young, M. A.; Scheffzek, K.; Veach, D. Structural basis for the autoinhibition of
c-Abl tyrosine kinase. Cell 2003, 112, 859–871.
(33) Seeliger, M. A.; Nagar, B.; Frank, F.; Cao, X.; Henderson, M. N. c-Src binds to the cancer drug imatinib
with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty. Structure 2007, 15, 299–311.
(34) Lietha, D.; Eck, M.J. Crystal Structures of the FAK Kinase in Complex with TAE226 and Related Bis-
Anilino Pyrimidine Inhibitors Reveal a Helical DFG Conformation. PLoS ONE 2008, 3, e3800, 1-7.
(35) Heinrich, T.; Brugger, N.; Rohdich, F.; Esdar, C.; Greiner, H.; Krier, M.; Seenisamy, J.; Jayadevan, J.;
Sundararaman, V.-r. New Highly Selective FAK Inhibitors. P421, ISMC Berlin 2012.
(36) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. The protein kinase complement of the
human genome. Science 2002, 298, 1912–1934.
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A.; Baba, A.; Miki, H. Discovery and characterization of novel allosteric FAK inhibitors. Eur. J. Med. Chem.
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(38) Fragment definition, compound attributes: MW <200; solubility >1µg/ml, number of hydrogen bond donors
and acceptors ≤ 3.
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(39) Erlanson, D. A.; McDowell, R. S.; O’Brien, T. Fragment-Based Drug Discovery. J. Med. Chem. 2004, 47,
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(40) The complete FAK-fragment screening will be described elsewhere, manuscript in preparation.
(41) Unpublished results. 43 screening hits could be crystallized successfully and characterized by x-ray
analysis.
(42) Initial fragment decorations gave N-Methyl-N-{3-[(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-methyl]-
pyridin-2-yl}-methanesulfonamide with an affinity of KD: 3.1 µM and a surprising biochemical IC50 of 8.9 µM.
The addition of methyl in ‘position 2’ gave N-Methyl-N-{3-[(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-
methyl]-pyridin-2-yl}-methanesulfonamide with a significant loss of affinity (KD: 57 µM). So for IP reasons and
technical feasibility neither the pyrazolo-pyrimdine nor the pyrrolo-pyrimidine core were considered for further
fragment growing initiatives. It was anticipated that the 1H-N and the 7N are the hinge-binding elements and
hence the pyrrolo[2,3-b]pyridine (7-azaindole) should be sufficient to work as hinge-binder.
(43) Popowycz, F.; Routier, S.; Joseph, B.; Mérour, J.-Y. Synthesis and reactivity of 7-azaindole (1H-
pyrrolo[2,3-b]pyridine). Tetrahedron 2007, 63, 1031-1064.
(44) Depending on the availability of starting material slightly modified schemes were followed. 32 and 46 were
prepared as outlined in Scheme 3; 34 and 38 were prepared as described in the experimental section.
(45) We had observed in other projects that the cellular activity often depends on the off-rate of the ligand.
Accordingly it was anticipated that low micromolar affinity and a koff = 0.4 s-1 is sufficient to show cellular
activity. In the subsequent work we decided to analyse in detail those derivatives with respect to solubility,
plasma-protein-binding and permeability where a slow off-rate was determined but no cellular activity could be
measured.46
(46) Data not shown
(47) Stock of this compound was depleted and re-synthesis not possible so that biophysical characterisation
could not be done.
(48) Copeland, R. A.; Pompliano, D. L.; Meek, T.D. Drug-taget residence time and its implications for lead
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(49) Stadt-Müller, H.; Betzemeier, B.; Sapountzis, I. WO 2010/058030.
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(50) Henrich, B., Bergamaschi, A., Broennimann, C., Dinapoli, R., Eikenberry, E. F., Johnson, I., Kobas, M.,
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(52) Collaborative Computational Project, Number 4. "The CCP4 Suite: Programs for Protein Crystallography".
Acta Cryst. 1994, D50, 760–763.
(53) Nowakowski, J., Cronin, C.N., McRee, D.E., Knuth, M.W., Nelson, C.G., Pavletich, N.P., Rodgers, J.,
Sang, B.-C., Scheibe, D.N., Swanson, R.V. & Thompson, D.A. Structures of the cancer-related Aurora-A, FAK,
and EphA2 protein kinases from nanovolume crystallography. Structure 2002, 10, 1659–1667.
(54) Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart,
O.S., Vonrhein, C. & Womack, T.O. 2011. BUSTER version 2.11.1. Cambridge, United Kingdom: Global
Phasing Ltd.
(55) Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Cryst. 2004, D60, 2126–
2132.
(56) Koolman, H.; Musil, D.; Heinrich, T.; Krier, M.; Reggelin, M. Co-crystal Structures of FAK with a Novel
Pyrrolo[2,3-d]thiazole. Acta Cryst. D 2012 submitted.
(57) de Kloe, G. E.; Bailey, D.; Leurs, R.; de Esch, I. J. P. Transforming fragments into candidates: small
becomes big in medicinal chemistry. Drug Discov. Today 2009, 14, 630-646.
(58) Antonysamy, S. S.; Aubol, B.; Blaney,J.; Browner, M. F.; Giannetti, A. M.; Harris, S. F.; Hébert, N.;
Hendle, J.; Hopkins, S.; Jefferson, E.; Kissinger, C.; Leveque, V.; Marciano, D.; McGee, E.; Nájera, I.; Nolan,
B.; Tomimoto, M.; Torres, E.; Wright, T. Fragment-based discovery of hepatitis C virus NS5b RNA polymerase
inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 2990-2995.
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P.E. The Protein Data Bank. Nucleic Acids Research, 2000, 28, 235-242.
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a Reaction conditions: R = CF3, CN; (a) Ag2SO4, I2; (b) R’-phenyl-ethin, PdCl2(PPh3)2, CuI, DMF; (c) NaH,
NMP, 60 °C; (d) m-CPBA; (e) POCl3, MsCl, DMF; (f) SEM-Cl, DCM; (g) Cs2CO3, S-Phos, Pd(OAc)2, R’’-NH2,
dioxane, 150 °C; (h) 4N HCl, THF, reflux, 18 h.
203x162mm (96 x 96 DPI)
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a Reaction conditions: (a) m-CPBA, 0 °C ∀ RT, 77 %; (b) MsCl, DMF, 2 h, 50 °C, 72 %; (c) TIPS-Cl, NaH,
THF, 1h, 0 °C, 65 %; (d) sec-BuLi, -78 °C, I2, DMF, 60 %; (e) TBAF, THF, 1 h, 0 °C ∀ RT, 80 %; (f) SEM-Cl,
NaH, THF, 0 °C, 2h, 88 %; (g) 2,2-Difluoro-2-(fluorosulfonyl)acetic acid, CuI, DMF, 100 °C, 3 h, 72 %; (h)
BuLi, -45 °C, I2, THF, 64 %; (i) Pd(OAc)2, S-Phos, Cs2CO3, R’-B(OH)2, dioxane, H2O, 60°C, 58 %; (j)
Pd(OAc)2, Xanthphos, Cs2CO3, R”-NH2, dioxane, H2O, 60 °C, 46 %; k. 4N HCl, THF, reflux, 65 %.
203x162mm (96 x 96 DPI)
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