Discovery of 4-azaindoles as novel inhibitors of c-Met kinase

Article abstract of DOI:10.1016/j.bmcl.2009.03.110

A series of 4-azaindole inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and series optimisation was performed on the basis of this structure. Future directions for series development are dis

Full text of DOI:10.1016/j.bmcl.2009.03.110

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2780–2784
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-azaindoles as novel inhibitors of c-Met kinase
a
a
a
a
John Porter a, , Simon Lumb , Richard J. Franklin , Jose M. Gascon-Simorte , Mark Calmiano ,
Kelly Le Riche a, Bénédicte Lallemand b, Jean Keyaerts b, Helen Edwards a, Alison Maloney a, Jean Delgado a,
Lloyd King a, Anne Foley a, Fabien Lecomte a, James Reuberson a, Christoph Meier a, Mark Batchelor a
*
a UCB Celltech, 216 Bath Road, Slough SL1 3WE, United Kingdom
b UCB Pharma, R&D, Chemistry, Chemin du Foriest, B-1420 Braine-l’Alleud, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-azaindole inhibitors of c-Met kinase is described. The postulated binding mode was con-
firmed by an X-ray crystal structure and series optimisation was performed on the basis of this structure.
Future directions for series development are discussed.
Received 10 March 2009
Revised 20 March 2009
Accepted 23 March 2009
Available online 27 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
c-Met Kinase inhibitor
Hepatocyte growth factor receptor inhibitor
Kinase inhibitor
Azaindole
Members of the receptor tyrosine kinase (RTK) family are
attractive targets for cancer therapy as inhibition can disrupt sig-
naling pathways that mediate tumour formation and growth.1
c-Met kinase is a member of this family that, together with its
ligand, hepatocyte growth factor (HGF) or scatter factor (SF), is
important for normal mammalian development. However, c-Met
has been shown to be deregulated and associated with high
tumour grade and poor prognosis in a number of human cancers.2
c-Met can become activated by a variety of mechanisms, including
gene amplification and mutation inducing motility, invasiveness
and tumourgenicity into the transformed cells.3 Activation leads
to receptor dimerisation and recruitment of several SH2 domain
containing signal transducers that activate a number of pathways
including the Raf-Mek-Erk and PI3k-Akt cascades. Targeting the
ATP binding site of c-Met is a popular strategy for inhibition of
the kinase, with a number of drug candidates reaching the clinical
trials phase.4 We now wish to report our efforts in this area.
Using our knowledge of the ATP-binding site of kinases in gen-
eral and c-Met in particular, we identified a series of scaffolds that
could potentially form either monodentate or bidentate H-bonds
with the kinase hinge residues. Consideration of docked models
of these scaffolds allowed us to determine appropriate positions
for the addition of suitable substituents to interact with key struc-
tural or functional features in the kinase. These elaborated scaf-
folds were then prioritised on the basis of synthetic tractability
and novelty. During this process we observed that the 4-azaindole
1 had moderate activity against c-Met in a biochemical assay5 (IC50
3.5
l
M) and decided to study this scaffold further.
O
O
S
7
N
6
5
1
2
4
N
3
1
The sulphonylated 4-azaindoles were prepared as shown in
Scheme 1, by treatment of the commercially available azaindoles
with the appropriate sulphonyl chloride. Where functionality was
present in the 6-position, this was protected as appropriate prior
to the sulfonylation and then elaborated.
Sulphonylated 7-azaindoles were also studied for comparative
purposes, their preparation is shown in Scheme 2, starting from
the commercially available 7-azaindole. Addition of 2 equivalents
O
O
H
N
S
R
R
i
N
N
N
2
3
* Corresponding author. Tel.: +44 1753 534655.
E-mail address: john.porter@ucb.com (J. Porter).
Scheme 1. Preparation of sulphonylated 4-azaindoles. Reagents and conditions: (i)
PhSO2Cl, Et3N, THF.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.110

J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2780–2784
2781
Table 1
NO2
NO2
SAR for 4-azaindoles and pyrrolopyrazines
O
S
SO2R
N
O
S
X
N
R
R
R
i
ii
N
H
N
N
H
N
N
N
H
Compd
R
X
c-Met IC50 (nM)
4
5
6
15
16
17
18
19
20
21
22
23
24
25
26
12
Phenyl
3-Chlorophenyl
2-Chlorophenyl
2,3-Dichlorophenyl
2,5-Dichlorophenyl
2,6-Dichlorophenyl
3-Nitrophenyl
2-Nitrophenyl
4-Nitrophenyl
3-Methoxyphenyl
3-Aminophenyl
3-Methylsulphonyl phenyl
3-Nitrophenyl
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
3500
5670
900
100
2030
480
70
iii
NO2
NO2
O
S
S
O
8
R
R
ii
20
16% @ 20
4600
lM
N
N
N
N
7
4400
25,200
1730
Scheme 2. Preparation of sulphonylated 7-azaindoles. Reagents and conditions: (i)
NaH, DMF, bis(3-nitrophenyl)disulphide; (ii) mCPBA, CH2Cl2; (iii) NaH, DMF, MeI.
Interestingly, 2,3-disubstitution, for example, 18, gives an even
greater increase. However, it was with the incorporation of a nitro
group, 21 and 22, that we observed the greatest enhancement in
potency, though similar substitution in the 4-position, 23, abol-
ished activity.
of base followed by the appropriate disulphide gave selective addi-
tion at the 3-position.
The sulphonylated pyrrolopyrazine 12 was prepared as shown
in Scheme 3 using a modification of a reported route6 with a palla-
dium catalysed heteroannulation of the pyrazine 9, followed by
deprotection and sulphonylation. Alkylated 4-azaindoles were pre-
pared as shown in Scheme 4 by deprotonation of the azaindole fol-
lowed by addition of the appropriate bromides.
Initial efforts aimed at exploring the SAR focused on the phenyl-
sulphonyl group; activities for a representative selection of com-
pounds are shown in Table 1. Substitution in the 3-position
generally leads to a loss in potency, however, substitution in the
2-position resulted in an improvement (compare 16 with 17).
A limited study of the scaffold showed that the introduction of a
second nitrogen atom in the pyridine ring (i.e., 12) reduced po-
tency, presumably because of reduced H-bonding to the hinge res-
idues. Modeling studies had showed that the 3-sulphonylated 7-
azaindole would be expected to form a bidentate H-bond with
the residues in the hinge region of the kinase with a consequent
improvement in potency. This regioisomer would also avoid the
potential for the sulphonylaryl group to act as a leaving group.
However, the expected improvement in activity was not observed,
Table 2. Interestingly, N-methylation of the indole, to give 30, did
not abolish activity suggesting that the pyridine N atom-hinge H-
bond was a major contributor to binding potency. As the 7-azain-
dole scaffold had been used quite extensively in a number of kinase
inhibitors,7 we returned to the 4-azaindole series with the belief
that optimisation of the substitution pattern would improve
potency.
A systematic study of the 4-azaindole scaffold revealed that the
6-position would be suitable for further potency optimisation
studies. It was found that a range of substituents could be accom-
modated at this position but the amine functionality, especially
piperazine groups appeared to be preferred, Table 3. In an attempt
to rationalise these observations we solved the X-ray crystal struc-
ture of the c-Met-32 complex, Figure 1.8 The binding mode was re-
vealed to be as we had predicted with the 4-N atom of the
azaindole forming an H-bond to the hinge Met1160, the 2-nitro-
O
S
NH
O
S
N
O
O
H
N
N
N
N
N
N
N
ii
i
Cl
iii
11
10
9
NO2
O
S
N
O
N
N
12
phenyl ring
p-stacking to Tyr1230, the nitro group forming an H-
Scheme 3. Preparation of pyrrolopyrazines. Reagents and conditions: (i) TMS-
acetylene, LiCl, PdCl2(dppf), Na2CO3, DMF; 100 °C; (ii) Cs2CO3, THF, MeOH;
(iii) 3-nitrophenylsulphonyl chloride, Et3N, THF.
bond to the backbone NH of Asp1222 and the carboxamide group
pointing toward solvent. The same conformational change to
Table 2
SAR for 7-azaindoles
SO2R1
O
N
N
H
N
N
R
R2
N
R
i
N
Compd
R1
R2
c-Met IC50 (nM)
N
N
27
28
29
30
Phenyl
H
H
H
Me
1175
200
75
13
14
3-Nitrophenyl
2-Nitrophenyl
3-Nitrophenyl
Scheme 4. Preparation of non-sulphonylated 4-azaindoles. Reagents and condi-
tions: (i) NaH, DMF, THF, 5-bromomethyl-benz[1,2,5]oxadiazole.
8100

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J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2780–2784
Table 3
Table 4
SAR of 2-nitrophenyl analogues
SAR of benzofurazan analogues
O2N
O
N
N
O
O
S
N
O
S
N
O
R
R
N
N
Compd
R
c-Met IC50 (nM)
Compd
R
c-Met
MKN-45
A549
31
CO2H
310
82
IC50 (nM)
IC50 (nM)
IC50 (nM)
32
CONH2
36
37
H
22
nd
nd
MeN
MeN
N
33
300
N
N
15
23
820
1310
O
t-Boc
HN
N
34
35
20
20
HN
38
nd
nd
N
N
N
N
39
40
41
9
200
130
HN
N
Tyr1230 that we had observed with our quinoxaline inhibitors was
also evident.9
NH2
16
11
1300
480
2200
430
O
Attention now turned to stabilising the arylsulphonamide. Re-
sults from hepatocyte stability studies and observations during
synthesis suggested that the arylsulphonamide was relatively la-
bile, and that this was enhanced by the electron withdrawing prop-
erties of the nitro group making the sulphonyl group prone to
nucleophilic attack. Attempts to sterically block access to the sul-
phonyl group by introducing a methyl group at the 2-position of
the azaindole gave compounds with a substantial drop in potency.
Incorporation of benzofurazan as a nitrophenyl isostere, a modifi-
cation that had proved successful in our quinoxaline series,9 gave
a compound that was equipotent (compare 36, Table 4 with 22,
Table 1). A search for other suitable 2-nitrophenyl replacements
was conducted by constructing a 76-membered library of sulph-
onamides prepared by the reaction between the 4-azaindole and
readily available sulphonyl chlorides. This library identified the
imidazo[2.1.b]thiazole group as a potential replacement, based
on their potency profile in the biochemical assay, Table 5. Docking
N
N
N
O
42
12
14
370
570
500
570
Me
N
N
N
O
MeHN
43
N
nd: no data.
We also investigated potential replacements for the sulphona-
mide, Table 6. Although the methylene and carbonyl linked ana-
logues were inactive for the nitrophenyl analogues, the
methylene linked benzofurazans 55 and 57 did show activity.
Interestingly, the two carbon atom linked 2-nitrophenyl analogue
54 also showed promising activity, suggesting that these com-
pounds could form the basis of a new series of c-Met inhibitors.
Compounds with sufficient potency in the biochemical assay
were screened in functional cell-based assays using two different
cell lines, MKN45 and A549. MKN45 cells are derived from a poorly
differentiated gastric adenocarcinoma and have constitutive c-Met
activity due to amplification of the c-Met gene locus.10 These cells
represent a ligand-independent cell line in which activation of
suggests that the thiazole ring can p-stack with Tyr1230, the 7-N
imidazothiazole atom can H-bond with the backbone N-H of
Asp1222, see Figure 1, and the chlorine atom enters a hydrophobic
pocket formed by Leu1157, Ala1226 and Leu1140. Combination of
the imidazothiazole and a solubilising group at the 6-position gave
46 that had promising activity, Table 5. The imidazothiophene also
appeared to stabilise the sulphonamide. For example, whereas 35
had an hepatocyte clearance rate that was too rapid to measure,
45 and 46 had rates of 1.0 and 0.3 sÀ1, respectively.
Figure 1. (A) X-ray crystal structure of the c-Met (green)/compound 32 (off-white) complex showing key interactions; (B) docking of a model of compound 46 (off-white)
with c-Met (green), showing postulated interaction of the imidazothiazole with Asp1222 and Tyr1230. The positions of Met1229-Tyr1230 (from PDB:1ROP) are shown in pink
for comparison.

J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2780–2784
2783
Table 5
SAR of imidazo[2.1.b]thiazole analogues
Cl
N
O
S
N
O
N
S
R
N
Compd
R
c-Met IC50
(nM)
MKN-45 IC50
(nM)
A549 IC50
(nM)
44
45
H
35
24
HN
1700
110
1710
90
N
N
46
9
HN
N
47
48
NH2
H
10
35
490
nd
580
nd
49
OH
15
3600
2810
Figure 2. Western blot of MKN-45 cells after treatment with compound 39
showing absence of p-Erk and p-Akt.
t-Boc
N
50
27
1946
110
N
features in the potent c-Met inhibitor PF-234106613) appeared to
enhance potency. Also noteable is the effect of the piperidinyl pyr-
azole group on the cell-based activity, as shown by comparison of
46 with the piperazine analogue 45.
t-Boc
N
N
51
19
147
450
N
In order to exert its cellular functions c-Met signals through
multiple downstream signaling pathways, including the mitogen
activated protein kinase (ERK1/2) and mediated signaling PI3K/
AKT pathways. The Western blot in Figure 2 shows that 39 inhib-
ited c-Met autophosphorylation in MKN-45 cells in a dose depen-
dent manner and, significantly, inhibition of phospho-AKT and
phospho-ERK1/2 was also observed.14 No activity was observed
for 39 against AKT and ERK 1/2 in a screen against a panel of 60
kinases.15
nd: no data.
Table 6
Non-sulphonylated 4-azaindoles analogues
R1
N
R2
In conclusion we have identified a novel series of 4-azaindole
inhibitors of c-Met kinase. Guided by an X-ray crystal structure
we have been able to rationalise the SAR and identified positions
where further optimisation would prove profitable.
N
Compd R1
R2
c-Met
IC50
(nM)
MKN-45
IC50
(nM)
A549
IC50
(nM)
References and notes
52
53
54
Benzyl
2-Nitro benzoyl
2-Nitro
H
H
H
i/a
12100
250
nd
nd
nd
nd
nd
nd
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5. IC50 values for inhibitors of c-Met were determined using an IMAP Time
Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay. 50 nM 6
His-tagged recombinant human c-Met residues 974-end (Millipore) was
incubated in 20 mM Tris, 10 mM MgCl2, 2.5 mM MnCl2, 0.01% Tween 20 and
phenylethyl
O
N
N
N
N
HN
N
55
56
57
19
1800
nd
1880
nd
N
O
N
HN
1040
33
N
2 mM DTT with 5
volume of 25 l for 60 min at ambient temperature. Inhibitors were tested at
10 concentrations starting from 20 M at a final concentration of 1% DMSO.
The reaction was stopped by addition of 50 l of IMAP Stop solution containing
lM ATP and 200 nM 5FAM-KKKSPGEYVNIGFG-NH2 in a total
t-Boc
l
N
O
N
l
l
N
3080
3280
N
60%Buffer A:40%Buffer B and a 1 in 400 dilution of beads and Terbium reagent.
Plates were read after an overnight incubation at 4 °C on an Analyst HT reader.
Reported IC50s are from a minimum of 2 experiments (n = 2). Data analysis was
carried out using a four parameter curve fit. The standard errors of the mean
were calculated and expressed as a percentage of the mean IC50. The average
for this value was 12%.
nd: no data.
6. Hopkins, C. R.; Cohen, N. Tetrahedron Lett. 2004, 45, 8087.
7. For example see: Graczyk, P. P.; Dimopoulos, P.; Khan, A.; Bhatia, G. S.; Farthing,
C. N. Patent application WO2008095944.; Ibrahim, P. N.; Artis, D. R.; Bremer, R.;
Mamo, S.; Nespi, M.; Zhang, C.; Zhang, J.; Zhu, Y-L.; Tsai, J.; Hirth, K-P.; Bollag,
G.; Spevak, W.; Cho, H.; Gillette, S. J.; Wu, G.; Zhu, H.; Shi, S. Patent application
WO2007002325.
c-Met and subsequent downstream signaling are consequences of
receptor overexpression. A549 cells are human derived non-small
cell lung cancer cells (NSCLC) and in contrast to MKN45 cells, c-
Met activation is dependent upon engagement with ligand-inde-
pendent c-Met phosphorylation11). In this assay12 addition of a sol-
ubilising group such as the piperidinyl pyrazole 39 (a group that
8. Crystallisation and solution of structure performed by Proteros Biostructures
GmbH,
Am
Klopferspitz
19,
D-82152,
Martinsried,
Germany,
www.proteros.com. Crystallographic data for the structure in this paper have
been deposited with the PDB (pdb code 2wd1).

2784
J. Porter et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2780–2784
9. Porter, J.; Lumb, S.; Lecomte, F.; Reuberson, J.; Foley, A.; Calmiano, M.; Le Riche,
K.; Edwards, H.; Delgado, J.; Franklin, R. J.; Gascon-Simorte, J. M.; Maloney, A.;
Meier, C.; Batchelor, M. Bioorg. Med. Chem. Lett. 2009, 19, 397.
10. Nakamura, T.; Matsumoto, K.; Kiritoshi, A.; Tano, Y.; Nakamura, T. Cancer Res.
1997, 57, 3305.
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Koudriakova, T. B.; Alton, G.; Cui, J. J.; Kung, P.-P.; Nambu, M. D.; Los, G.;
Bender, S. L.; Mroczkowski, B.; Christensen, J. G. Cancer Res. 2007, 67, 4408; Cui,
J. J.; Botrous, I.; Shen, H.; Tran-Dube, M.; Nambu, M.; Kung, P. P.; Funk, L.; Jia, L.;
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S.; Zou, H.; Christensen, J.; Mroczkowski, B. Abstracts of Papers, 235th ACS
National Meeting, New Orleans, LA, United States, April 6–10, 2008, MEDI-177.
14. MKN-45 cells were cultured in RPMI 1640 medium containing 20% serum and
pretreated for 1 h at 37 °C with increasing concentrations of compound or DMSO,
cells were then lysed in RIPA buffer containing appropriate inhibitors. Western
blotting was carried out following standard procedures and probed with the
following antibodies: anti-phospho c-Met (Tyr -234/5), anti-c-Met, anti-phospho
AKT (Ser 473), anti-AKT, anti-phospho ERK1/2 (Thr 202/Tyr 204), anti ERK1/2 (Cell
Signaling Technologies Inc.), following the manufacturer’s protocol. Total cellular
protein loadings were semi-quantified by probing with anti-GAPDH.
11. Rege-Cambrin, G.; Scaravaglio, P.; Carozzi, F.; Giordano, S.; Ponzetto, C.;
Comoglio, P. M.; Saglio, G. Cancer Genet. Cytogenet. 1992, 64, 170.
12. All cells were seeded at 20,000 cells/well in a 96-well microplate in RPMI/20%
FBS/2 mM glutamine. Compound treatments at 6 dilutions were performed for
1 h at 37 °C followed by immediate 30 min lysis on ice with MSD lysis buffer
and frequent vortexing. Lysates were centrifuged at 2500 rpm for 10 min at
4 °C. All washes were performed four times using MSD wash buffer. MSD plate
blocking and all other incubations were carried out for 1 h at room
temperature with rocking. MSD c-Met (pY1349) 96-well plates were blocked
with 3% BSA/MSD wash buffer followed by washing. Twenty-five microlitres of
supernatant were added to each well and incubated as previously described,
15. 39 was screened against a 60 member kinase panel by Millipore Bioscience
Division, Millipore UK Ltd, Gemini Crescent, Dundee Technology Park, Dundee,
DD2 1SW, United Kingdom. www.millipore.com. Results were expressed as
followed by washing. Each well was incubated with 25
ll of detection antibody
(anti-total Met)/1% BSA/MSD wash buffer followed by washing. Read buffer
TM
(150
ll) was added to each well and the plates were read on a MSD SECTOR
percentage of activity remaining @ 20
lM: both AKT and ERK1 having 88%
6000 Instrument. Background BSA signals were subtracted from pY1349 signal
for each well and IC50s were determined using XLfit 4.2 software.
activity remaining. This screen did show that 39 had some activity against KDR,
Mer, TrkA and Ron (3%, 19%, 18% and 29% activity remaining respectively).